Calcium dibenzoate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

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 test item related effects on reproductive performance (decreased gestation index) and general toxicity to the male and female parental animals, the no observed adverse effect level (NOAEL) for reproduction toxicity as well as general parental toxicity is 300 mg/kg bw/day.

 

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 reproductive function (oestrous cycle and sperm measures) and reproductive performance or general parental toxicity up to a dose level of 1000 mg/kg bw/day. Therefore, the NOAEL for reproductive toxicity as well as general parental toxicity is considered to be greater than 1000 mg/kg bw/day for both sexes.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

extended one-generation reproductive toxicity – with F2 generation and both developmental neuro- and immunotoxicity (Cohorts 1A, 1B with extension, 2A, 2B, and 3)

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.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Justification for study design:

not specified

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 (P0 generation): approximately 10 weeks
- Weight at study initiation (estimated; (P0 generation)): 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 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 P generation and F1 generation 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. Similar weekly adjustments were also made in the dietary benzoic acid concentrations administered to F1 and F2 pups following weaning (postnatal day (PND) 21 - 70) because animals at that stage of growth also show substantially greater food intake per kg of body weight than do adults.

Details on mating procedure:

NOTE: mating procedure for P0 generation and F1 generation (cohort 1B; producing F2 generation)

- M/F ratio per cage: 1 male with 1 non-sibling female of the same dose group in the home-cage of the male

- Length of cohabitation: overnight for mating until positive evidence of mating or three oestrous cycles had elapsed (a maximum of 14 days)

- Proof of pregnancy: copulatory plug or sperm in vaginal smear referred to as gestation day 0

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:

P 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 generation (males and females):
- cohort 1A: beginning at weaning and continuing until euthanasia (postnatal day (PND): 91, 13 weeks)
- cohort 1B: beginning at weaning and continuing until euthanasia following reproductive assessment following weaning
- cohort 1B (selected for F2 generation): beginning at weaning and continuing until euthanasia (PND 91, 13 weeks)
- cohort 2A: beginning at weaning and continuing until euthanasia (PND: 78, 8 weeks)
- cohort 2B: beginning at weaning and continuing until euthanasia (PND: 22, 1 day)
- cohort 3: beginning at weaning and continuing until euthanasia (PND: 59, 5.5 weeks)

Frequency of treatment:

continuously

Details on study schedule:

P generation females were allowed to rear their litters normally until weaning on postnatal day (PND) 21, when randomly selected F1 pups were assigned to Cohorts 1, 2 and 3 such that not more than 1 pup/sex/litter was assigned to each cohort. F1 males and females assigned to Cohort 1B (optional reproductive assessments) were bred in a similar manner as the P generation adults, and females were allowed to rear their litters normally until weaning on PND 21, when 2 pups/sex/litter were randomly selected to constitute the F2 generation.

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:

P generation: 30 males/ 30 females
F1 generation (cohort 1A): 20 males / 20 females
F1 generation (cohort 1B): 25 males / 25 females
F1 generation (cohort 2A, adult): 12 males / 12 females
F1 generation (cohort 2B, weanling): 12 males / 12 females
F1 generation (cohort 3): 10 males / 10 females
F1 generation (cohort 3A, positive control): 10 males / 10 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.

Types of cohorts employed in the current study:
Cohort 1A: Reproductive assessment
Cohort 1B: Reproductive assessment (F1 breeding)
Cohort 2A (adult): neurotoxicity assessment (neurobehaviour, neuropathology and brain morphometry)
Cohort 2B (weanling): neurottoxicity assessments
Cohort 3: immunotoxicity assessments
Cohort 3A: immunotoxicity assessments (positive control group)

Positive control:

F1 generation (cohort 3A):
Control animals of cohort 3A were exposed to cyclophosphamide, a known immunosuppressant, and served as positive controls for the T-cell antibody response assay (TDAR).

Parental animals: Observations and examinations:

NOTE: the following parameters were assessed for the P0 generation and the P1 generation

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

NEUROBEHAVIOURAL EXAMINATION: not specified
IMMUNOLOGY: not specified

FURTHER OBSERVATIONS
- females were allowed to deliver naturally, and the day parturition was initiated was identified as lactation day 0.
- individual gestation lengths were calculated using the date delivery was first observed.

Oestrous cyclicity (parental animals):

1) P0- GENERATION
Beginning with the first day of test item administration, female oestrous cycles were monitored based on daily vaginal lavages (2 weeks prior to mating) and throughout the mating period, until evidence of mating (by the presence of a vaginal copulatory plug or sperm in a vaginal lavage).

A terminal vaginal lavage sample was collected for all females to determine the stage of the oestrous cycle at the time of necropsy.

2) P1-Generation
Cohort 1A: animals were evaluated for onset of oestrous and oestrous cyclicity during the in-life period (postnatal day (PND) 75–91). Onset of oestrous was monitored based on daily vaginal lavages beginning on the day of attainment of vaginal patency until cornified cells were observed in a lavage specimen.

Cohort 1B: beginning with the first day of test item administration, female oestrous cycles were monitored based on daily vaginal lavages (2 weeks prior to mating) and throughout the mating period, until evidence of mating (by the presence of a vaginal copulatory plug or sperm in a vaginal lavage). A terminal vaginal lavage sample was collected for all females to determine the stage of the oestrous cycle at the time of necropsy.

Sperm parameters (parental animals):

Parameters examined in P0 and P1 male parental generations (cohort 1A):
Sperm parameters (sperm motility, spermatid count, cauda epididymis sperm count, sperm production rate, sperm morphology and cauda epidymis weight (P1 males only)) were evaluated for all males.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes, to reduce variability among litters, ten pups from each litter, of equal sex distribution (where possible) were randomly selected on postnatal day 4.

PARAMETERS EXAMINED
1) F1 generation
The following parameters were examined in F1 offspring:
- number of live and stillborn pups
- clincial signs and survival on postnatal day (PND) 1, 4, 7, 14 and 21.
- body weights of pups were recorded on PND 0, 4, 14 and 21
- sex of pups were recorded on PND 0, 4, 14 and 21
- anogenital distance was measured for all pups on PND 1
- all male pups were examined for the presence of nipples/areolae on PND 13

- all selected pups were examined for the attainment of post-weaning developmental landmarks (vaginal patency (PND 25 onwards) and balanopreputial separation (PND 35 onwards).
- body weights were recorded for individual pups on the day of attainment of the developmental landmarks.
- thyroid hormones (T4 and TSH) were measured on PND 4 and 21 (n = 10 pups/sex/group).

2) F2 generation
The following parameters were examined in F2 offspring:
- clinical signs, body weights and food consumption were measured weekly throughout the generation until termination (PND 91)
- number of live and stillborn pups
- clincial signs and survival on postnatal day (PND) 1, 4, 7, 14 and 21
- body weights of pups were recorded
- sex of pups were recorded on PND 0, 4, 14 and 21

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
F1 animals designated for developmental neurotoxicity assessments were assigned to Cohorts 2A and 2B. In Cohort 2A, animals were evaluated for neurobehavior (auditory startle response, functional observational battery [FOB], locomotor activity and learning and memory [Biel maze] assessment) during the in-life period, and at termination (PND 78), brain measurements (weight, length, width; n = 12/sex/group)) and neuropathology, including microscopic morphometric measurements on homologous stained sections of the brain. Brain morphometric measurements (n = 12/sex/group) were performed on samples from control and high-dose animals; examination of low- and mid-dose animals was not conducted or warranted based on the lack of positive findings in the high-dose animals.

Auditory startle response was assessed on PND 24 using the MED Associates Startle Reflex System; sessions consisted of 50 trials each, with each trial consisting of 110–120 dB mixed-frequency noise burst stimulus, approximately 20 ms in duration. The intertrial interval was 8 s. Startle response measurements obtained were PEAK (peak response amplitude) and Tpeak (latency to PEAK). An FOB was conducted on PND 65, prior to the locomotor activity assessments, by trained technicians, blind to treatment group, and was counterbalanced across testing time, sex, and group. Animals were evaluated in the home cage, during handling, in an open field arena and observations included sensory, neuromuscular and physiological observations. Locomotor activity was monitored immediately following the FOB using the Kinder Scientific Motor Monitor System; sessions were 60 min in duration. Total motor activity was evaluated as a combination of fine motor skills (i.e., grooming; interruption of 1 photobeam) and ambulatory motor activity (e.g., interruption of two or more consecutive photobeams). Lastly, a learning and memory assessment of the type normally conducted during the OECD (2007) Test Guideline 426 developmental neurotoxicity study was conducted for the same animals beginning on PND 66. Learning and memory was assessed using a water-filled 8-unit T-maze (Biel, 1940)*. Animals were placed in the maze and were required to traverse the maze and escape by locating a submerged platform. The time to escape the maze and the number of errors were evaluated.

Animals assigned to Cohort 2B were used for brain measurements (weight, length, width) and neuropathological evaluation of brain on PND 22.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
F1 animals designated for developmental immunotoxicity assessments were assigned to Cohort 3. Animals were evaluated for T-Cell Dependent Antibody Response (TDAR) at termination. In addition, 10 additional control rats/sex were assigned to Cohort 3A - a subset of Cohort 3 - which served as the positive control group. All animals in Cohort 3 were immunized with Sheep Red Blood Cells (sRBC) on PND 54. The primary IgM antibody response to sRBC, a T-dependent antigen, is an evaluation of humoral immunity, and cyclophosphamide, a known immune suppressant, was administered to all animals in the positive control group for 5 consecutive days (2.5 mg/kg bw/day, PND 54–58) to demonstrate that the assay could detect suppression of the humoral immune response. The TDAR was conducted using a Rat Anti-sRBC IgM ELISA kit (Life Diagnostics) for the quantitation of rat anti-sRBC levels in serum samples.

*Reference:
- Biel, W.C., 1940. Early age differences in maze performance in the albino rat. J. Genet. Psychol. 56 (2), 439–453.

Postmortem examinations (parental animals):

1) P0-GENERATION
GROSS NECROPSY / HISTOPATHOLOGY / ORGAN WEIGHTS
Following necropsy, tissues and organs were collected for organ weights and full histopathological evaluation.

2) P1- GENERATION
GROSS NECROPSY / HISTOPATHOLOGY / ORGAN WEIGHTS
Following necropsy, tissues and organs were collected for organ weights and full histopathological evaluation on postnatal day (PND) 91

In Cohort 1A, animals were evaluated for splenic lymphocyte immunophenotyping on PND 91. Spleens were processed for enumeration of live cell concentrations of Total, Helper and Cytotoxic T lymphocytes, B lymphocytes, NK and NK-T lymphocytes using Luna-FL™ cell counter and immunophenotyping using a BD FACSCanto™ II Flow Cytometer.

Postmortem examinations (offspring):

HISTOPATHOLOGY / ORGAN WEIGTHS
F2 generation:
- at PND 91 all animals were subjected to a gross necropsy, determination of organ weights and brains were collected, weighed and preserved for histopathological evaluation.

Statistics:

Please refer to the field "Any other information on materials and methods incl. tables".

Reproductive indices:

P0 generation and P1 generation:
Reproductive performance was summarized in terms of mating, fertility, conception and copulation indices. Precoital index was also determined.

Offspring viability indices:

not specified

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

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:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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 P0 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.

REPRODUCTIVE FUNCTION: OESTROUS CYCLE
- 500, 750 and 1000 mg/kg bw/day: there was no statistically significant effect of dietary benzoic acid adminsitration on female oestrous cycle. The female oestrous cycle of the dose groups was not different from the control group.

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

REPRODUCTIVE FUNCTION: SPERM MEASURES
- 500, 750 and 1000 mg/kg bw/day: there was no statistically significant effect of dietary benzoic acid adminsitration on sperm parameters (sperm motility, spermatid count, cauda epididymis sperm count, sperm production rate and sperm morphology). The sperm parameters of the dose groups were not different from the control group.

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

REPRODUCTIVE PERFORMANCE
- 500, 750 and 1000 mg/kg bw/day: there was no statistically significant effect of dietary benzoic acid adminsitration on reproductive performance (mating index (%), fertility index (%), copulation/conception index (%) and precoital index (days)). The reproductive performance of the dose groups was not different from the control group. Furthermore, no statistically significant effect was observed for gestation length for the dose groups.

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

Key result

Dose descriptor:

NOEL

Remarks:

highest dose tested

Effect level:

1 000 mg/kg bw/day (nominal)

Remarks on result:

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

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

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:

not specified

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

FOOD CONSUMPTION AND 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. Exceptions for P1 males were noted during the post-mating period where mean food consumption, and consequently test substance intake was 27 – 31 % 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.

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.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

REPRODUCTIVE FUNCTION: OESTROUS CYCLE
- 500, 750 and 1000 mg/kg bw/day: there was no statistically significant effect of dietary benzoic acid adminsitration on female oestrous cycle, time to first oestrus and primordial follicle count.

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

REPRODUCTIVE FUNCTION: SPERM MEASURES
- 500, 750 and 1000 mg/kg bw/day: there was no statistically significant effect of dietary benzoic acid administration on sperm parameters (sperm motility, spermatid count, cauda epididymis sperm count, cauda epididymis weight, sperm production rate and sperm morphology).

Higher mean cauda epididymal weights were noted in all benzoic acid-treated groups compared to controls (p < 0.01 for mid and high dose group). Correspondingly, statistically significantly (p < 0.05 or p < 0.01) lower mean cauda epididymal sperm concentrations (millions/gram tissue) were noted in all benzoic acid-treated groups compared to the control group; these differences were not considered benzoic acid-related but were ascribed to the atypically low mean cauda epididymal weight and consequently higher mean cauda epididymal sperm concentration observed for the control group, which was attributed to 3 (of 20) males in the control group with atypically low values for cauda epididymal weight (0.0795–0.1502 g versus a historical control mean ± SD of 0.2942 ± 0.032 g).

Cauda epididymis sperm count
Control: 530.1 ± 133.8 M/g
500 mg/kg bw/day: 420.5 ± 94.8 M/g (p < 0.01)
750 mg/kg bw/day: 457.1 ± 96.9 M/g (p < 0.05)
1000 mg/kg bw/day: 438.9 ± 96.4 M/g (p < 0.01)
Historical control data: 458.5 M/g (range: 381.8 M/g - 624.7 M/g)

Cauda epididymis weight
Control: 0.2251 ± 0.055 g
500 mg/kg bw/day: 0.2486 ± 0.027 g
750 mg/kg bw/day: 0.2628 ± 0.025 g (p < 0.01)
1000 mg/kg bw/day: 0.2695 ± 0.030 g (p < 0.01)
Historical control data: 0.2942 g (range: 0.2438 g - 0.3675 g)

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

REPRODUCTIVE PERFORMANCE
- 500, 750 and 1000 mg/kg bw/day: there was no statistically significant effect of dietary benzoic acid administration on reproductive performance (mating index (%), fertility index (%), copulation/conception index (%) and precoital index (days)). The reproductive performance of the dose groups was not different from the control group. Furthermore, no statistically significant effect was observed for gestation length for the dose groups.

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

Key result

Dose descriptor:

NOEL

Remarks:

highest dose tested

Effect level:

1 000 mg/kg bw/day (nominal)

Remarks on result:

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

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality / viability:

mortality observed, non-treatment-related

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:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

no effects observed

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

no effects observed

CLINICAL SIGNS
- 500, 750 and 1000 mg/kg bw/day: there were no benzoic acid-related clinical signs in the offspring.

MORTALITY
- 500, 750 and 1000 mg/kg bw/day: the postnatal survival and live litter size were unaffected by the test item administration.

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

BODY WEIGHT AND WEIGHT CHANGES
- 500, 750 and 1000 mg/kg bw/day: pup body weights and body weight gains across all groups were comparable to the control group during postnatal days (PND) 1 – 14. Although lower pup body weight gains were noted for both males and females during PND 14 – 21, the reductions in absolute mean body weights were within the range of historical control data for reproduction studies
and were non-dose responsive, therefore considered unrelated to benzoic acid administration.

Male birth weight (g, PND 1):
Control: 7.4 ± 0.57 g
500 mg/kg bw/day: 7.4 ± 0.87 g
750 mg/kg bw/day: 7.5 ± 0.87 g
1000 mg/kg bw/day: 7.0 ± 0.65 g
Historical control data: 7.3 g (range: 6.8 g - 7.7 g)

Male birth weight (g, PND 21):
Control: 50.9 ± 5.05 g
500 mg/kg bw/day: 46.1 ± 6.66 g (p < 0.05)
750 mg/kg bw/day: 46.7 ± 7.15 g (p < 0.05)
1000 mg/kg bw/day: 46.2 ± 4.61 g (p < 0.05)
Historical control data: 53.7 g (range: 42.5 g - 62.8 g)

Female birth weight (g, PND 1):
Control: 7.0 ± 0.61 g
500 mg/kg bw/day: 6.9 ± 0.82 g
750 mg/kg bw/day: 7.1 ± 0.81 g
1000 mg/kg bw/day: 6.6 ± 0.65 g
Historical control data: 6.8 g (range: 6.4 g - 7.4 g)

Female birth weight (g, PND 21):
Control: 49.1 ± 4.41 g
500 mg/kg bw/day: 43.9 ± 6.75 g (p < 0.01)
750 mg/kg bw/day: 45.2 ± 6.17 g (p < 0.05)
1000 mg/kg bw/day: 44.6 ± 4.89 g (p < 0.05)
Historical control data: 51.6 g (range: 42.5 g - 59.1 g)

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: there were no significant effects on food consumption

2) Compund intake:
- 500, 750 and 1000 mg/kg bw/day: the adjustments for F1 pups during the post-weaning period successfully allowed for maintenance of target dose levels.

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

SEX RATIO
- 500, 750 and 1000 mg/kg bw/day: the offspring sex ratio was unaffected by the test item administration.

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

SEXUAL MATURATION
- 500, 750 and 1000 mg/kg bw/day: exposure to benzoic acid had no effect on balanopreputial separation and vaginal patency.

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

ANOGENITAL DISTANCE
- 500, 750 and 1000 mg/kg bw/day: exposure to benzoic acid had no effect on anogenital distance of male and female offspring.

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

NIPPLE RETENTION IN MALES
- 500, 750 and 1000 mg/kg bw/day: exposure to benzoic acid had no effect on nipple retention in male offspring.

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

NUMBER OF PUPS
- 500, 750 and 1000 mg/kg bw/day: the mean number of pups born was unaffected by the test item administration.

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

ENDOCRINE FINDINGS
- 500, 750 and 1000 mg/kg bw/day: exposure to benzoic acid had no effect on neonatal or weanling thyroid hormone levels. Minor variations in T4 and TSH levels were not considered treatment related because they were not noted in a dose responsive manner, were not generally statistically significant, or were observed in a direction that would be generally not be considered toxicologically relevant. These minor variations also fell within the range of levels noted for historical controls.

1) F1 litters (PND 4)
Total T4 hormone
- control: 15360 ± 2792 pg/mL
- 500 mg/kg bw/day: 15352 ± 4594 pg/mL
- 750 mg/kg bw/day: 15867 ± 3942 pg/mL
- 1000 mg/kg bw/day: 15229 ± 3690 pg/mL
Historical control data: 22161 pg/mL (mean (± 2 SD): 15693 pg/mL - 28630 pg/mL)

TSH hormone
- control: 1.3 ± 1.38 ng/mL
- 500 mg/kg bw/day: 0.6 ± 0.97 ng/mL
- 750 mg/kg bw/day: 0.0 ± 0.0 ng/mL (p < 0.05)
- 1000 mg/kg bw/day: 1.4 ± 1.08 ng/mL
Historical control data: 4.37 ng/mL (mean (± 2 SD): 0.32 ng/mL - 8.4 ng/mL)

2) F1 males (PND 21)
Total T4 hormone
- control: 35420 ± 7728 pg/mL
- 500 mg/kg bw/day: 36850 ± 12886 pg/mL
- 750 mg/kg bw/day: 39970 ± 8914 pg/mL
- 1000 mg/kg bw/day: 45180 ± 9328 pg/mL
Historical control data: 47030 pg/mL (mean (± 2 SD): 16477 pg/mL - 77584 pg/mL)

TSH hormone (PND 21)
- control: 3.9 ± 1.22 ng/mL
- 500 mg/kg bw/day: 3.4 ± 1.72 ng/mL
- 750 mg/kg bw/day: 2.2 ± 1.77 ng/mL
- 1000 mg/kg bw/day: 3.0 ± 1.94 ng/mL
Historical control data: 4.91 ng/mL (mean (± 2 SD): 0.99 ng/mL - 8.8 ng/mL)

3) F1 females (PND 21)
Total T4 hormone
- control: 30280 ± 7638 pg/mL
- 500 mg/kg bw/day: 39550 ± 6342 pg/mL (p < 0.05)
- 750 mg/kg bw/day: 38390 ± 7481 pg/mL
- 1000 mg/kg bw/day: 43030 ± 10125 pg/mL (p < 0.01)
Historical control data: 45236 pg/mL (mean (± 2 SD): 11484 pg/mL - 78989 pg/mL)

TSH hormone (PND 21)
- control: 2.6 ± 1.63 ng/mL
- 500 mg/kg bw/day: 3.9 ± 1.85 ng/mL
- 750 mg/kg bw/day: 2.3 ± 1.37 ng/mL
- 1000 mg/kg bw/day: 3.1 ± 2.01 ng/mL
Historical control data: 4.62 ng/mL (mean (± 2 SD): 1.21 ng/mL - 8.0 ng/mL)

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

DEVELOPMENTAL NEUROTOXICITY (Cohort 2A and 2B)
1) Brain measurements
- 500, 750 and 1000 mg/kg bw/day: there were no test item-related macroscopic findings or effects on gross brain measurements (weight, width, length) in F1 male and female pups.

2) Brain morphometric measurements
- 1000 mg/kg bw/day: there were also no test item-related effects on microscopic morphometric measurements S1 – S6 in the brains of young adult male and female pups (S1 = thickness frontal cortex; S2 = thickness parietal cortex; S3 = width caudate-putamen; S4 = thickness corpus callosum; S5 = thickness hippocampus; S6 = height cerebellum). A single measurement (mean S2, females – thickness of the parietal cortex) was significantly lower in 1000 mg/kg bw/day group females than in control group females (control group: 2391 ± 86 µm; treatment group: 2307 ± 93 (p < 0.05)); however, the value was within the range of the historical control data (2243 µm (range: 2054 µm - 2412 µm)) and thus, in the absence of any correlating changes in males, or changes in gross brain measurements, or alterations in neurobehavioral responses, this single change was considered to be an incidental statistical flag.

3) Neurobehaviour assessment
- 500, 750 and 1000 mg/kg bw/day: in the neurobehavioral assessment of male and female offspring, there were no effects of benzoic acid on auditory startle responsiveness (peak response or time to peak response on postnatal day (PND) 24), functional observational battery (FOB) and locomotor activity (ambulatory counts or total counts on PND 65) or learning and memory assessments on PND 66.

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

DEVELOPMENTAL IMMUNOTOXICITY (Cohort 3 and 3A (positive control))
- 500, 750 and 1000 mg/kg bw/day: there were no benzoic acid-related effects on the humoral immune system as indicated by lack of any changes in the sRBC-IgM responses in the TDAR Assay. The positive control, cyclophosphamide (CPS) produced the expected significant decrease in anti-sRBC-IgM in response to immunization with sRBC. Individual responses for all benzoic acid-treated animals were within the range of control values with the exception of two 1000 mg/kg bw/day females that were identified as high responders. The mean value for the 1000 mg/kg bw/day group was not statistically significantly different from the control group. Furthermore, the TDAR is designed to assess a decrease in the T cell-mediated response to the antigen, sRBC, by detecting anti-sRBC-IgM. Because a higher response to immunization is not toxicologically relevant, the isolated increase in 2 females was considered incidental. In addition, in cohort 1A there was no effect on splenic lymphocyte populations (Total T, helper T and cytotoxic T lymphocytes, B lymphocytes, NK and NK-T cells) following exposure to benzoic acid at the doses administered.

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

Key result

Dose descriptor:

NOEL

Remarks:

highest dose tested

Effect level:

1 000 mg/kg bw/day (nominal)

Remarks on result:

other: lack of any adverse effects on any of the parameters evaluated including neurotoxicity (neurobehaviour, neuropathology and brain morphometry) and immunotoxicity assessments

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality / viability:

mortality observed, non-treatment-related

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:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

no effects observed

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

CLINICAL SIGNS
- 500, 750 and 1000 mg/kg bw/day: for pups selected to constitute the F2 generation, there were no benzoic acid-related clinical signs

MORTALITY
- 500, 750 and 1000 mg/kg bw/day: the postnatal survival and live litter size were unaffected by the test item administration.

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

BODY WEIGHT AND WEIGHT CHANGES
- 500, 750 and 1000 mg/kg bw/day: pup body weights and body weight gains were unaffected by benzoic acid during the preweaning period. For pups
selected to constitute the F2 generation, there were 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: for pups selected to constitute the F2 generation, there were no significant effects on male or female food consumption,

2) Compound intake
- 500, 750 and 1000 mg/kg bw/day: the adjustments for F2 pups during the post-weaning period successfully allowed for maintenance of target dose levels.

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

ORGAN WEIGHTS
- 500, 750 and 1000 mg/kg bw/day: there were no benzoic acid-related effects on weanling organ weights.

- 500, 750 and 1000 mg/kg bw/day: exposure to benzoic acid had no effect on absolute or relative (to final body weight) brain weights in any group of males or females in the F2 generation.

SEX RATIO
- 500, 750 and 1000 mg/kg bw/day: the offspring sex ratio was unaffected by the test item administration.

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

NUMBER OF PUPS
- 500, 750 and 1000 mg/kg bw/day: the mean number of pups born was unaffected by the test item administration.

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

Key result

Dose descriptor:

NOEL

Remarks:

highest dose tested

Effect level:

1 000 mg/kg bw/day (nominal)

Remarks on result:

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

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

In an extended one generation reproductive toxicity study according to OECD 443 (2018), benzoic acid was administered via diet to male and 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. Besides the parental generation (P0; n = 30 rats/sex/group), the study included cohorts of F1 offspring to evaluate potential effects of the test item on reproduction (cohort 1A (n = 20 rats/sex/group) and 1B (n = 25 rats/sex/group) with extension to F2 generation), the developing immune system (cohort 3; n = 10 rats/sex/group) and the developing neurological system with the inclusion of learning and memory assessments (cohort 2A and 2B; n = 12 rats/sex/group). A group of rats (cohort 3A; n= 10 rats/sex/group) receiving cyclophosphamide served as positive control group for cohort 3.
No test item-related influence was noted on general toxicity (clinical signs, mortality, body weights food consumption, haematology, clinical chemistry, thyroid hormones, urinalysis, organ weights, gross pathology, and histopathology) and reproductive function (oestrous cycle and sperm parameters) as well as reproductive performance (mating index, fertility index, copulation/conception index and precoital index) of the P0 generation. In addition, the F1 generation used for reproductive assessment (cohort 1A/1B; considered to be P1 generation), showed no test item related effect in general toxicity (food consumption, haematology, clinical chemistry, thyroid hormones, urinalysis, organ weights, gross pathology, and histopathology) and reproductive function (oestrous cycle and sperm parameters) as well as reproductive performance (mating index, fertility index, copulation/conception index and precoital index).
Furthermore, no test item-related effects were observed on clinical signs, mortality, food consumption, sexual maturation, anogenital distance, nipple retention in male pups, number of pups born and thyroid hormones in the F1 offspring generation (cohort 1A and 1B). Also, no influence of the test item was observed on the neurotoxicity and neurobehavioural parameters such as auditory startle response, locomotor activity, learning and memory assessment investigated in cohorts 2A and 2B. In cohort 3, the immunotoxicity assessment did not reveal any effect of the test item. Lastly, no effect was observed on the general toxicity (clinical signs, mortality, body weight, food consumption, and organ weights) of the F2 generation. No effect on sex ratio or pups born were observed in the F2 generation.
In conclusion, the following NOAELs were determined for benzoic acid:
P0 generation:
NOAEL (general toxicity) > 1000 mg/kg bw/day
NOAEL (reproductive toxicity) > 1000 mg/kg bw/day
F1 generation
Reproductive and developmental toxicity (cohorts 1A and 1B) > 1000 mg/kg bw/day
Developmental neurotoxicity (cohorts 2A and 2B) > 1000 mg/kg bw/day
Developmental immunotoxicity (cohort 3) > 1000 mg/kg bw/day

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

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

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_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

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_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

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 (-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 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 (-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) comapred 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 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 (+281.4 %, 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). In this pup, no milk was seen in the stomach, it was hypothermic on postnatal day 0 and was found to be annibalized on postnatal day 1. Furthermore, one pup died and found to be intact on postnatal day 0. No gross necropsy findings could be observed in this animal. In addition another seven pups were found to be cannibalized on postnatal day 1 in this litter. Totally 13 out of 50 live born pups were found dead dead between postnatal days 0 and 4 in the high dose group. 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 in male pups on nipple retention in any of the treated groups on postnatal day 13.

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 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 (-41.9 %; p˂0.05) and 13 (-41.1 %; 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: except one pup in the high dose group, evidence of suckling on postnatal 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). In this pup, no milk was seen in the stomach, it was hypothermic on postnatal day 0 and was found to be annibalized on postnatal day 1. Furthermore, one pup died and found to be intact on postnatal day 0. No gross necropsy findings could be observed in this animal. In addition another seven pups were found to be cannibalized on postnatal day 1 in this litter. Totally 13 out of 50 live born pups were found dead dead between postnatal days 0 and 4 in the high dose group. No pups died from postnatal day 5 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

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Calcium

Calcium plays an essential role in male and female fertility. It is involved in normal spermatogenesis, sperm maturation and sperm motility as well as in maintaining optimal conditions for acrosome reaction, which is necessary for the fertilization process. All these processes indicate the pivotal role of calcium in human fertility in men and women. It is therefore concluded that calcium itself does not show any effects on the impairment of male or female fertility. Additional studies in animals are not believed to contribute to the existing evidence for a lack of reproductive toxicity of calcium. Thus, further studies in animals are considered to be scientifically unjustified

 

 

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 male and 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. Besides the parental generation (P0; n = 30 rats/sex/group), the study included cohorts of F1 offspring to evaluate potential effects of the test item on reproduction (cohort 1A (n = 20 rats/sex/group) and 1B (n = 25 rats/sex/group) with extension to F2 generation), the developing immune system (cohort 3; n = 10 rats/sex/group) and the developing neurological system with the inclusion of learning and memory assessments (cohort 2A and 2B; n = 12 rats/sex/group). A group of rats (cohort 3A; n= 10 rats/sex/group) receiving cyclophosphamide served as positive control group for cohort 3.

 

No test item-related influence was noted on reproductive function (oestrous cycle and sperm parameters) or reproductive performance (mating index, fertility index, copulation/conception index and precoital index) or general parental toxicity of the P0 generation as well as the P1 generation. The no-observed-adverse effect level (NOAEL) for reproductive toxicity as well as general parental toxicity is greater than 1000 mg/kg bw/day.

 

In a 4-generation rat study (Kieckebusch, 1960), no effects on general toxicity, fertility, reproductive performance and offspring were reported at up to and including 1% the test substance in feed equivalent to 500 mg/kg bw/d (highest dose tested). In this pre-guideline study, the first generation was exposed for 8 weeks and then allowed to mate (1/1 for a period of 14 days). Treatment with the substance was continued and mating was repeated in week 48 to raise a second litter. Survival of the first and second generation was measured. The third generation was terminated after 16 weeks and examined histopathologically. In this generation weights of brain, heart, liver, spleen, kidneys and testes were determined. The fourth generation was terminated after weaning of the pups. Body weights were determined in week 4, 8 and 12 weeks of each generation (week 12 males only). Feeding efficiency was measured in all generations after 2,4, 6 and 8 weeks. Reproduction parameters assessed included percentage of infertility, delayed sexual maturation, litter size, total pups, surviving pups. These parameters were assessed for all generations (summed) and for the first two generations separately. The study was used as supporting information only.

 

 

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 effect was determined in the parental animals for reproductive function (oestrous cycle and sperm measures). The no observed adverse effect level (NOAEL) for general toxicity to the parental animals is 300 mg/kg bw/day for males and females 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. In this dose level, the substance also 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).

 

In conclusion, the No-observed-adverse-effect-level (NOAEL) for parental toxicity and reproductive toxicity is 300 mg/kg bw/day. 

 

 

 

Conclusion

 

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD 422 (Rigo, 2022), the results indicated the reproductive function of males and females was not affected by calcium dibenzoate up to 1000 mg/kg bw/day. However, the reproductive performance was influenced by the substance. The gestation index was decreased at a dose level of 1000 mg/kg bw/day. Therefore, the NOAEL for reproductive toxicity is 300 mg/kg bw/day. The no observed adverse effect level (NOAEL) for general toxicity to the parental animals is 300 mg/kg bw/day for males and females 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.

 

In the gastrointestinal tract calcium dibenzoate will dissociate into the two moieties calcium and benzoic acid respectively benzoate (EFSA, 2016) and these moieties are not considered to have an adverse effect on the reproduction. Calcium is not known to cause any adverse health effects and is even essential for a proper reproductive performance. Thus, the moiety of concern if at all is the benzoate anion. In an extended one generation reproductive toxicity study with benzoic acid according to OECD 443 (2018), no test item-related influence was noted on reproductive function (oestrous cycle and sperm parameters) or reproductive performance (mating index, fertility index, copulation/conception index and precoital index) or general parental toxicity of the P0 generation as well as the P1 generation. The no-observed-adverse effect level (NOAEL) for reproductive toxicity as well as general parental toxicity is greater than 1000 mg/kg bw/day.

 

 

 

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 for calcium dibenzoate. Calcium dibenzoate will dissociate into the moiety benzoic acid that showed no adverse effects on fertility 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 EOGRT study on benzoic acid is more relevant for assessing toxicity to reproduction of calcium benzoate than a screening test on this substance. The NOAEL for benzoic acid is 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.

 

In conclusion, calcium dibenzoate will not be classified and labelled for toxicity of reproduction 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.

Effects on developmental toxicity

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 test item related effects on number of live born pups, prenatal mortality, total mortality of pups, and pup body weight/body weight gain, the no observed adverse effect level (NOAEL) for developmental toxicity of this study is 300 mg/kg bw/day. The no observed adverse effect level (NOAEL) for general toxicity to the parental animals is 300 mg/kg bw/day for males and females 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.

 

In addition, 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 in the offspring (F1 and F2 generations) up to a dose level of 1000 mg/kg bw/day. Therefore, the NOAEL for developmental toxicity is considered to be greater than 1000 mg/kg bw/day. No test item-related influence was noted on general parental toxicity of the P0 generation as well as the P1 generation. The no-observed-adverse effect level (NOAEL) for general parental toxicity is greater than 1000 mg/kg bw/day.

Furthermore, a developmental toxicity study with sodium benzoate was conducted in rats. No substance related effects were observed on the foetuses up to a dose level of 2% in feed (1310 mg/kg bw/day based on food intake). The study is used as supporting information.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

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). The development of the F1 offspring was observed until 13 days post-partum.

 

No test item related effects were observed in maternal animals for clinical signs, mortality, detailed clinical examination, body weight, food consumption, haematology, clinical chemistry, endocrine findings, behavioural examination, and gross pathology. However, increased kidney weight and histopathological findings (liver) were observed in maternal animals at the 1000 mg/kg bw/day dose level. The maternal NOAEL was 300 mg/kg bw/day.

 

No test item related effects were observed in the pups for anogenital distance, nipple retention (male pups only), thyroid weights, sex ratio, T4 level (pups on postnatal day 13 only) and suckling behaviour. 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.

 

Furthermore, 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). In this pup, no milk was seen in the stomach, it was hypothermic on postnatal day 0 and was found to be annibalized on postnatal day 1. Furthermore, one pup died and found to be intact on postnatal day 0. No gross necropsy findings could be observed in this animal. In addition another seven pups were found to be cannibalized on postnatal day 1 in this litter. Totally 13 out of 50 live born pups were found dead dead between postnatal days 0 and 4 in the high dose group. No pups died from postnatal day 5 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.

 

In conclusion, the No-observed-adverse-effect-level (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. Based on increased kidney weight and histopathological findings (liver) at the 1000 mg/kg bw/day dose level, the maternal NOAEL was 300 mg/kg bw/day.

 

 

 

Sodium benzoate

 

In a study by Onodera et al. (1978), sodium benzoate was administered via diet to groups of pregnant female Wistar rats (n = 27 – 30) at concentrations of 1, 2, 4 and 8 % (equivalent to 700, 1310, 1875 and 965 mg/kg bw/day based on total food intake). The rats were dosed daily from gestation day 1 up to gestation day 20. A control group receiving plain diet was run concurrently. Dams were sacrificed on gestation day 20 (n = 22- 25/rats/group) and foetuses were examined. Some dams (5 rats/group) were allowed to littered and the pups were investigating up to 8 weeks of age. Examinations comprised dead/resorbed, viable foetuses, average number of implants/female as well as external, soft tissue and skeletal alterations. Some dams (5 rats/group) were allowed to litter and the pups were investigating up to 8 weeks of age. Number of pups, survival rate and body weight of the pups, presence and absence of external abnormalities were recorded. Three weeks after birth, all surviving pups were weaned and examined for gross abnormalities, and one-half of the pups were necropsied. The remaining pups were necropsied at 8 weeks of age, body weight and food intake being measured weekly until necropsy. In addition, the survival and lactation rate were determined. Lastly, the organ weights were recorded at 8 weeks of age.

 

No test item-related effects were observed for maternal body weight, body weight gain and feed intake in maternal animals at the concentration levels 1 and 2 %. However, mortality was observed in maternal animals at the 4 and 8 % concentration level. Two dams at 4 % and three dams at 8 % concentration level died after convulsions and depressed motor activity. Furthermore, maternal animals at the 4 % level did not gain weight and those at 8 % lost weight (statistical comparisons not presented) and feed intake was markedly reduced in the dams at both concentration levels.

No test item-related effects were observed on the number of dead/resorbed or viable foetuses or average number of implants/female at the 1 and 2 % concentration levels. However, the number of dead or resorbed foetuses was significantly increased compared to the controls at the 4 and 8 % concentration levels.

 

In addition, no test item-related effects were observed on the average weight of viable foetuses as well as on pathological findings of foetuses at the 1 and 2 % concentration level. On the other hand, the average body weight of viable foetuses was significantly lower than that of controls and significant abnormalities finding (mild systemic oedema) were observed in the foetuses at the 4 and 8 % concentration levels. Also, delayed ossification, lumbar or cervical ribs, and varied sternebrae were reported in foetuses in both control and treated groups (4 and 8 %). The percentage of animals with these findings was not comparable to that in controls among animals at 4 % and 8 %. The percentage of animals was increased in the groups at 4 % (96.5 %) and 8 % (100 %). Additional anomalies seen in the treated groups included a higher incidence of wavy ribs and abnormal vertebrae in the rats at 4 %, but not at 8 %. Significant abnormalities and pathological findings (anophthalmia, microphthalmia, hydrocephalus, pyelectasis, renal hydroplasia and cerebral hypoplasia) were observed in the foetuses at the two highest concentration levels.

 

Furthermore, among pups that were delivered naturally, no differences in the delivery rate, number of perinatal deaths, lactation rate or survival up to week 8 were reported at the 1% and 2% dietary levels, but the groups at 4% and 8% were reported to have delivery rates reduced by 50% and 8.2%, respectively, with complete loss of litters after parturition. The surviving pups in the control group and at 1% and 2% showed no significant differences in birth weight, weight at week 3 or week 8, incidence of abnormalities at week 3 or 8, or organ weights at week 8.

 

The EFSA (2016) stated that the authors assumed that the effects on the maternal animals as well as on the foetuses at the 4 and 8 % concentration levels were due to reduced food consumption by the dams, which causes malnutrition in the animals. The compound intake of the 2 % concentration group was higher (1310 mg/kg bw/day) as the intake by the 8 % concentration group (965 mg/kg bw/day). However, no effects were observed at the 2 % concentration level. Therefore, it was concluded that the no-observed-adverse-effect level (NOAEL) is 1310 mg/kg bw/day. The original publication was in Japanese language only with an abstract in English language, however the study was assessed and peer-reviewed within e.g. the OECD SIDS initial assessment report (2001) and EFSA (2016). Thus, the study is considered useful as supporting information (RL4).

 

References:

• OECD (2001): SIDS Initial Assessment Report for 13th SIAM (Bern, 7th – 9th November 2001). OECD SIDS, UNEP Publications


• 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.

 

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 male and 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. Besides the parental generation (P0; n = 30 rats/sex/group), the study included cohorts of F1 offspring to evaluate potential effects of the test item on reproduction (cohort 1A (n = 20 rats/sex/group) and 1B (n = 25 rats/sex/group) with extension to F2 generation), the developing immune system (cohort 3; n = 10 rats/sex/group) and the developing neurological system with the inclusion of learning and memory assessments (cohort 2A and 2B; n = 12 rats/sex/group). A group of rats (cohort 3A; n= 10 rats/sex/group) receiving cyclophosphamide served as positive control group for cohort 3.

 

No test item-related influence was noted on clinical signs, mortality, body weight, food consumption, sexual maturation, anogenital distance, and nipple retention (male pups) in the F1 generation. Developmental neurotoxicity or developmental immunotoxicity was not observed in the F1 generation. Furthermore, no substance-related effects were observed for clinical signs, mortality, body weight, food consumption and organ weights in the F2 generation. Therefore, the NOAEL is greater than 1000 mg/kg bw/day for the F1 and F2 generations.

 

Conclusion

 

The results of a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD 422 (Rigo, 2022), seems to indicate that calcium dibenzoate causes developmental toxicity as the survival/mortality and body weight/body weight gain of the offspring of treated dams were influenced by the substance at a dose level of 1000 mg/kg bw/day. A NOAEL of 300 mg/kg bw/day was determined for developmental toxicity. Increased kidney weight and histopathological findings (liver) were observed in maternal animals at the 1000 mg/kg bw/day dose level. The maternal NOAEL was 300 mg/kg bw/day.

 

 

In the gastrointestinal tract calcium dibenzoate will dissociate into the two moieties calcium and benzoic acid respectively benzoate (EFSA, 2016). Calcium is not known to cause any adverse health effects and is even essential for a proper reproductive performance. Thus, the moiety of concern if at all is the benzoate anion. An extended- one generation reproduction toxicity (EOGRT) study with benzoic acid showed no substance-related effects on offspring (F1 and F2 generations). Therefore, a NOAEL greater than 1000 mg/kg bw/day is derived for F1 and F2 generations. Also, the parental animals were free of any adverse effect and the NOAEL for the P generation was also set to greater than 1000 mg/kg bw/day.

 

In addition, a study with sodium benzoate (Onodera et al. 1978) did not indicate any effect on the offspring up to a dose level of 1310 mg/kg bw/day, which is above the limit dose level as foreseen by OECD guidelines.

 

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 for calcium dibenzoate. Calcium dibenzoate will dissociate into the moiety benzoic acid that showed no adverse effects on offsprings up to a dose level of 1000 mg/kg bw/day in the EOGRT 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 developmental toxicity of calcium benzoate than a screening test on this substance. The NOAEL for benzoic acid is 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.

 

In conclusion, calcium dibenzoate will not be classified and labelled for developmental toxicity 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.

 

Reference:

• Regulation (EC) No 1272/2008 of the European parliament and of the council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006 (consolidated version of 1 May 2020)


• 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

Fertility

 

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD 422 (2016), calcium dibenzoate caused adverse effects on reproductive performance (decreased gestation index) at a dose level of 1000 mg/kg bw/day. Therefore, the no-observed adverse effect level (NOAEL) for reproductive toxicity is 300 mg/kg bw/day. 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 on fertility up to a dose level of 1000 mg/kg bw/day.

 

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 toxicity to reproduction of calcium benzoate than a screening test on this substance. The NOAEL for benzoic acid is 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.

 

 

Developmental toxicity

 

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD 422 (2016), calcium dibenzoate caused adverse effects on survival/mortality and body weight/body weight gain of the offspring of treated dams at a dose level of 1000 mg/kg bw/day. Based on these findings a NOAEL of 300 mg/kg bw/day is derived for developmental toxicity. 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 on developmental toxicity up to a dose level of 1000 mg/kg bw/day.

 

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 developmental toxicity of calcium benzoate than a screening test on this substance. The NOAEL for benzoic acid is 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.