Registration Dossier
Registration Dossier
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EC number: 208-534-8 | CAS number: 532-32-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
No data on reproductive effects and effects on fertility of the test substance are available. From the studies that are available on repeated dose and carcinogenicity no report on effects on the reproductive organs/tissues in both male and female laboratory animals are reported.
In a 4-generation study with the structural analogue benzoic acid (Kieckebusch and Lang, 1960), no effects on reproductive performance and off-spring were reported at 1% benzoic acid in feed (500 mg/kg bw). A key study evaluating effects on fertility (Kieckebusch and Lang, 1960), evaluated the effects of benzoic acid over 4 generations in rats via feeding. While this study does have some limitations when compared to the current OECD 443 EOGRTS, when supplemented by information on reproductive organs/tissues (sperm parameters, including epididymis/cauda epididimys/testis weights, sperm motility/density/abnormal sperm; Estrus cyclicity in females) from a 13 -week repeated dose study of benzyl acetate (a substances that is metabolized completely to benzoic acid), the apparent gaps in data from the current OECD 443 study design are filled.
Link to relevant study records
- Endpoint:
- reproductive toxicity, other
- Remarks:
- Evaluation of Rodent Sperm, Vaginal Cytology, and Reproductive Organ Weight Data from National Toxicology Program 13-Week Studies
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Remarks:
- In a chronic feeding study, male and female rats were exposed to up to 1% benzoic acid in the diet through four generations.
- Adequacy of study:
- key study
- Study period:
- Publication compiles reproductive parameters from 13-week repeated dose studies from 50 different substances.
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Data are derived from National Toxicology Program (NTP) testing program.
- Justification for type of information:
- See Read Across Justification document in Section 13.2 of IUCLID
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The studies were designed as standard repeated-dose (13-week) and carcinogenicity (2-year) assays, commensurate with accepted standards at the time.
Data are derived from National Toxicology Program series of 13-week repeated dose studies. The publication states that "Sperm morphology and vaginal cytology examinations (SMVCEs), which include evaluations of motility, concentration and head morphology of sperm from the cauda epididymis, and male reproductive organ weight data, were developed by the National Toxicology Program as a screening system for reproductive toxicants. - GLP compliance:
- yes
- Remarks:
- GLP Compliance confirmed in NTP TR 431
- Limit test:
- no
- Justification for study design:
- The studies were designed as standard repeated-dose (13-week) and carcinogenicity (2-year) assays, commensurate with accepted standards at the time.
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Benzyl acetate was obtained in two lots (8743-84 and 845585) from GivaudanCorporation (Clifton, NJ). Identity and purity analyses were conducted by the analytical chemistry laboratory, Midwest Research Institute (Kansas City, MO), and confirmed by the study laboratory. Purity was determined to be > 98% for both lots.
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL: Stability studics performed at the analytical chemistry laboratory indicated that benzyl acetate was stable as a bulk chemical for 2 weeks at temperatures as high as 60" C. The stability of the bulk chemical was monitorcd periodically by the study laboratory using infrarcd and spectroscopy gas ultraviolet and chromatography; no change in purity was observed. - Species:
- other: both rat and mouse are reported by Morrissey et al.
- Strain:
- other: F344/N rats; B6C3F1 mice
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Frederick Cancer Research Facility (Frederick, MD)
- Age at study initiation: Average of 30 days old.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days prior to study initiation
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.6 +/- 3.0 degrees C
- Humidity (%): 46-65%
- Air changes (per hr): Minimum of 10 changes/hour.
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark - Route of administration:
- oral: feed
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
- Rate of preparation of diet (frequency): diet with test material prepared weekly.
- Storage temperature of food: Dose formulations stored at -20 degrees C in sealed, double plastic bags. - Details on mating procedure:
- Not Applicable: The study is a 13-week repeated dose study, and is included regarding the collection of data on effects on reproductive parameters, not including mating.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- "Periodic analyses of the dose formulations of benzyl acctate were conducted at the sludy laboratory and the analytical chemistry laboratory using gas chromatography. The stability of 330ppm dose formulations stored in the dark at -20 degrees C was established for at least 3 weeks. Dose formulations were analyzed four times for the 13-week studies and approximately every 6 to 8 weeks during the 2-year studies. All dose formulations for rats and mice were within 10% of the target concentrations throughout the studies." "The results of periodic referee analysis pcrformed by the analytical chemistry laboratory indicated agreement with the results obtained by the study laboratory."
- Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- Feed containing test material provided ad libitum.
- Details on study schedule:
- The appropriate feed was supplied every 1 to 2 days. Feed and water were available ad libitum. Feed consumption was recorded daily by cage. Rats were housed five per cage and mice were housed individually. Clinical findings were recorded once weekly. The animals were weighed at the beginning of the studies, weekly, and at the end of the studies.
- Dose / conc.:
- 0 ppm
- Remarks:
- Control for both rats and mice
- Dose / conc.:
- 3 130 ppm
- Remarks:
- Resulted in 235 mg/kg bw/day in rats (average of Male and Female), and 537.5 mg/kg bw/day in mice (average of Male and Female).
- Dose / conc.:
- 6 250 ppm
- Remarks:
- Resulted in 470 mg/kg bw/day in rats (average of Male and Female), and 1,140 mg/kg bw/day in mice (average of Male and Female).
- Dose / conc.:
- 12 500 ppm
- Remarks:
- Resulted in 915 mg/kg bw/day in rats (average of Male and Female), and 2,490 mg/kg bw/day in mice (average of Male and Female).
- Dose / conc.:
- 25 000 ppm
- Remarks:
- Resulted in 1,810 mg/kg bw/day in rats (average of Male and Female), and 4,000 mg/kg bw/day in mice (average of Male and Female).
- Dose / conc.:
- 50 000 ppm
- Remarks:
- Resulted in 4,200 mg/kg bw/day in rats (average of Male and Female), and 8,650 mg/kg bw/day in mice (average of Male and Female).
- No. of animals per sex per dose:
- 10 males/group
10 females/group - Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: Not described in NTP report.
- Positive control:
- None stated
- Parental animals: Observations and examinations:
- N/A - This study endpoint record is focused on reproductive parameters as reported by Morrissey et al., 1988.
- Oestrous cyclicity (parental animals):
- Estrous cyclicity evaluated in mice and rats.
- Sperm parameters (parental animals):
- Sperm quality parameters were evaluated at the end of the 13-week in-life phase, including:
- Sperm motility
- Sperm count
- Sperm head staining for morphology - Litter observations:
- Not examined in this study.
- Postmortem examinations (parental animals):
- Regarding male reproductive tissues, in the basic NTP study, the right testis and seminal vesicle was necropsied, and histopathology was examined on testis with epididymis, and seminal vesicle. For testis w/ epididymis, all doses were evaluated, not just the two high doses. Regarding female reproductive tissues, in the basic NTP study, the uterus was necropsied, and histopathology of the uterus was examined.
- Postmortem examinations (offspring):
- N/A - This study endpoint record is focused on reproductive parameters as reported by Morrissey et al., 1988.
- Statistics:
- For body, organ and relative organ weight, either William's or Dunnett's test was used. Because Dunnett's test makes no allowance for dose-response relationships, Jonckheere's test (a non-parametric test for dose-response relationship) was used to determine whether Dunnett's or Williams' would be used. Potential decreases in sperm motility and increases in percentage abnormal sperm were evaluated using Jonckheere's test. Sperm density was evaluated using the Kruskal-Wallis test.
- Reproductive indices:
- Not examined in this study.
- Offspring viability indices:
- Not examined in this study.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Overall toxicity findings were not discussed in Morrissey et al. (1988), but were discussed in the original NTP study. The discussion of clinical findings in rats was limited to the description of "tremors, ataxia and urine stains" in the high-dose animals. The discussion of clinical findings in mice was as follows:
"The significant clinical finding was tremors, which occurred only in females and was predominant in the 50,000 ppm group; however, one 12,500
ppm female and one 25,000 ppm female also exhibited tremors. The tremors occurred first on day 16 of the study in three females receiving 50,000
ppm, day 94 in one female receiving 25,000 ppm, and day 93 in one female receiving 12,500 ppm, and continued until the end of the study." - Mortality:
- mortality observed, treatment-related
- Description (incidence):
- In rats, nine male and nine female animals receiving the highest dose died or were killed moribund between weeks 2 and 8 of the study. One 12,500 ppm female died under anesthesia during blood collection at the end of the study.
In mice, one 50,000 ppm male mouse died, and one 50,000 ppm female mouse was sacrificed as moribund before the end of the study. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- In rats, the mean body weight gain and the final mean body weight of 25,000 ppm males were significantly lower than control. The final mean body weight of 25,000 ppm males was 10% lower than that of controls, whereas the final body weights of the surviving 50,000 ppm male and female animals was less than 50% of controls. Final mean body weights of males and females of other exposed groups were similar to or slightly lower than those of controls.
In mice, statistically significant dose-related decreases in final mean body weights occurred in all groups of exposed male and female mice. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- In rats, feed consumption was comparable to control in all but the 25,000 and 50,000 ppm groups, where reduced consumption was attributed to decreased palatability and/or toxicity.
In mice, the mean feed consumption was lower in all groups when compared to control. - Key result
- Dose descriptor:
- NOEL
- Effect level:
- > 50 000 ppm
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- > 50 000 ppm
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- > 50 000 ppm
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- > 50 000 ppm
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Reproductive effects observed:
- no
- Lowest effective dose / conc.:
- 50 000 ppm
- Treatment related:
- no
- Conclusions:
- These data supplement missing endpoints from the Kieckebusch and Lang (1960) Key Study, and fill the gap in that study design. In the National Toxicology Program (Morrissey et al., 1988) 13-week feeding benzyl acetate studies in rats and mice, both sperm parameters (epididymis, cauda epididymis, testis weights, and sperm motility, density and percent abnormal sperm) and estrus cyclicity measurements were collected. Dose levels of 2000 to 3000 mg/kg/day did not affect any endpoints of male reproduction. Estrus cycles were lengthened in female mice receiving >3000 mg/kg/day but this only occurred at a dose levels also causing significant decreases in growth (body weights and body weight gain).
- Executive summary:
Regulation (EC) No 1907/2006 (REACH), Annex XI 1.5., establishes, that substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group of substances. The similarity may also be based on the likelihood of common breakdown products via physical and biological processes. Given that the grouping of substances and read-across approach can be justified, data gaps may be fulfilled from data of reference substance(s) within the group. This avoids the need to test every substance for every endpoint, which is highly recommended in the context of animal welfare considerations (Directive 2010/63/EU, 2010).
For sodium benzoate, the fertility and developmental toxicity endpoints are filled by read across from a group of structurally similar substances.
Benzyl acetate, benzyl alcohol, benzaldehyde, and benzoic acid and its sodium and potassium salt were considered as a single category regarding human health by JECFA as they are all rapidly metabolized and excreted via a common pathway within 24 hrs (JECFA 1997).
The data presented below if for benzyl acetate using the read across justification described above and is used as read across for sodium benzoate as requested by ECHA in its letter ‘’Submission FP609849-12 – request for dossier update’’ received by the lead registrant on October 03, 2019 and attached in Section 13 of the IUCLID dossier.
These data supplement missing endpoints from the Kieckebusch and Lang (1960) Key Study, and fill the gap in that study design. In the National Toxicology Program (Morrissey et al., 1988) 13-week feeding benzyl acetate studies in rats and mice, both sperm parameters (epididymis, cauda epididymis, testis weights, and sperm motility, density and percent abnormal sperm) and estrus cyclicity measurements were collected. Dose levels of 2000 to 3000 mg/kg/day did not affect any endpoints of male reproduction. Estrus cycles were lengthened in female mice receiving >3000 mg/kg/day but this only occurred at a dose levels also causing significant decreases in growth (body weights and body weight gain).
- Endpoint:
- multi-generation reproductive toxicity
- Remarks:
- The effect of benzoic acid on reproduction was studied through four generations.
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Remarks:
- In a chronic feeding study, male and female rats were exposed to up to 1% benzoic acid in the diet through four generations.
- Adequacy of study:
- key study
- Study period:
- Benzoic acid was fed in the diet to male and female rats for 11-12 weeks before the first mating and then through four generations.
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Please see remarks for details.
- Remarks:
- While the design of this four-generation study for reproductive toxicity does not exactly match the endpoints included within the EOGRTS, it does include almost all of the endpoints associated with the OECD Guideline 416 – two generation reproductive toxicity that was previously considered adequate to satisfy the reproductive toxicity endpoint under the REACh regulation. The only missing reproductive endpoints within the Kiekebusch and Lang publication are collection of sperm parameters or estrous cyclicity data. However, the endpoints that are missing from the four-generation study with benzoic acid are available for benzyl acetate, a chemical that is metabolized completely to benzoic acid. In the National Toxicology Program (Morrissey et al., 1988) 13-week feeding benzyl acetate studies in rats and mice, both sperm parameters (epididymis, cauda epididymis, testis weights, and sperm motility, density and percent abnormal sperm) and estrus cyclicity measurements were collected. Dose levels of 2000 to 3000 mg/kg/day did not affect any endpoints of male reproduction. Estrus cycles were lengthened in female mice receiving >3000 mg/kg/day but this only occurred at a dose levels also causing significant decreases in growth (body weights and body weight gain). Therefore, it appears that all of the reproductive toxicity endpoints have been collected, albeit in two separate studies on benzoic acid and benzyl acetate.
- Justification for type of information:
- See Read Across Justification document in Section 13.2 of IUCLID
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
- Version / remarks:
- Study predates current published regulatory guidelines for GLP compliance and reproductive toxicity studies; study contains most of the same elements found in a present-day two generation reproductive toxicity test and continued exposure through four generations. The premating exposure intervals were at least 10 weeks and the dose levels used were comparable to the limit dose level of 1000 mg/kg/day required under the current regulatory guidelines. Endpoints examined included collection of body weight and feed consumption data, determination of effects on sexual maturity and fertility, collection of litter and pup data, necropsy data including organ weights and histopathology, and effect on onset of sexual senescence, and lifespan. The study did not collect sperm parameters and estrous cyclicity data but these endpoints are addressed in a separate 13-week NTP feeding study in rats with the analogue benzyl acetate.
- Deviations:
- yes
- Remarks:
- Not specified
- Principles of method if other than guideline:
- A feeding study was performed, in which 4 generations of rats received the test substance (0.5% or 1%). The first generation was exposed for 8 weeks an then allowed to mate (1/1 for a period of 14 days). 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. Some reproduction parameters were assessed: percentage of infertility, delayed sexual maturation, litter size, total pups, surviving pups. These parameters were assessed for all generations (summed) and for thefirst two generations separately.
- GLP compliance:
- no
- Remarks:
- Study pre-dated GLPs.
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Not provided in publication.
- Expiration date of the lot/batch: Not provided in publication.
- Purity test date: Not provided in publicaton.
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not provided in publication.
- Stability under test conditions: Not provided in publication.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Not provided in publication. - Species:
- rat
- Strain:
- not specified
- Details on species / strain selection:
- TEST ANIMALS
- Source: Farbwerken Bayer (Elherfeld)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Not provided in publication.
- Weight at study initiation: 40-50g
- Fasting period before study: Not provided in publication.
- Housing: Rats were kept in double cages in a simplified Columbus-type feed apparatus.
- Diet (e.g. ad libitum): For the first 8 weeks, they were fed according to the “paired feed” method, and then ad libitum.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: Not provided in publication.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not provided in publication.
- Humidity (%): Not provided in publication.
- Air changes (per hr): Not provided in publication.
- Photoperiod (hrs dark / hrs light): Not provided in publication.
IN-LIFE DATES: From: To: Not provided in publication. - Sex:
- male/female
- Route of administration:
- oral: feed
- Details on exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): Not provided in publication.
- Mixing appropriate amounts with (Type of food): Feed mixtures were produced in a feed mixing machine made of stainless steel. Diet consisted of commercial standard rat feed made by Lutz (Euskirchen) with sufficient benzoic acid added to achieve feed concentrations of 0.5% and 1.0%
- Storage temperature of food: Not provided in publication. - Details on mating procedure:
- - M/F ratio per cage: 1M/1F
- Length of cohabitation: 14 days
- Proof of pregnancy: Not provided in publication.
- After 14 days of unsuccessful pairing ,the pairing was repeated 8 weeks later to investigate delays in sexual maturity or full sterility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Not provided in publication. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Not provided in publication.
- Duration of treatment / exposure:
- 11-12 weeks prior to mating and through 4 generations.
- Frequency of treatment:
- Feed containing test material provided ad libitum.
- Details on study schedule:
- Details on study schedule were not provided in the publication.
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 0.5 mg/kg bw/day (actual dose received)
- Remarks:
- Overall, the dose level from the 0.5% diet for the entire premating period was approximately 450 and 600 mg/kg/day for the male and female rats, respectively.
- Dose / conc.:
- 1 mg/kg bw/day (actual dose received)
- Remarks:
- Overall, the dose level from the 1% diet for the entire premating period was approximately 900 and 1176 mg/kg/day for the male and female rats, respectively.
- No. of animals per sex per dose:
- 20 males/group/generation
20 females/group/generation - Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: The dietary concentrations were selected based upon a previous probe study where rats consuming 5% benzoic acid in the diet died within 3 weeks due to lack of palatability of the diet and corrosive effects of the free acid on the digestive tract.
- Positive control:
- None stated
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: No data
BODY WEIGHT: Yes
- Time schedule for examinations: During the first 8 weeks of the experiment, weight checks were done weekly; after that, weight was checked in 4-week intervals.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined: Yes; feed consumption data were collected throughout the exposure period over the four generations; for reporting purposes in the publication, feed consumption data are presented as “Protein efficiency (weight increase per gram of dietary protein)”
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: calculated from body weight and feed consumption data include within the publication. - Oestrous cyclicity (parental animals):
- Not examined in this study.
- Sperm parameters (parental animals):
- Testis weight was examined in the 3rd generation.
- Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No; Litter size was recorded on the 1st and 2nd day as well as the number of surviving young on the 21st day.
PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: Since there was no demonstratable effect of benzoic acid on reproduction and since the data from all four generations were similar, the data were combined. Parameters examined included total number of pups born, pup survival, and litter size.
GROSS EXAMINATION OF DEAD PUPS: No information provided.
ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not examined.
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not examined. - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: The third generation of animals was necropsied after 16 weeks of exposure. Organ weights of the brain, heart, spleen, liver, kidneys and testis were collected. Organs were examined histopathologically.
- Maternal animals: The third generation of animals was necropsied after 16 weeks of exposure. Organ weights of the brain, heart, spleen, liver, and kidneys were collected. Organs were examined histopathologically.
HISTOPATHOLOGY / ORGAN WEIGHTS: The following organs from the third generation animals were prepared for microscopic examination and weighed, respectively.: brain, heart, spleen, liver, kidneys, testis. - Postmortem examinations (offspring):
- None stated.
- Statistics:
- Not provided in publication.
- Reproductive indices:
- Not provided in publication.
- Offspring viability indices:
- No differences were observed between generations so all data were combined.
- Clinical signs:
- not specified
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- There was no difference between the 1% group and the control group in terms of lifespan (similar number of short vs long-lived animals) but there was a statistically significant increase in the number of long-lived animals in the 0.5% group.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on body weight gain or body weights over the four generations of rats tested in this study.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on feed consumption (efficiency) over the four generations of rats tested in this study.
- Food efficiency:
- no effects observed
- Description (incidence and severity):
- Feed consumption data in this study are presented as “Protein efficiency (weight increase per gram of dietary protein)”. There was no effect on feed consumption (efficiency) in either test group.
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- Histopathology was only reported for 3rd generation animals.
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- no effects observed
- Reproductive function: oestrous cycle:
- not examined
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- no effects observed
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- > 1 other: %
- Based on:
- other:
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Remarks:
- Generational data were not reported separately. At concentrations up to 1% benzoic acid in the diet, there were no adverse effects on parents or offspring through four generations of test substance administration. Overall, the dose level from the 1% diet for the entire premating period was approximately 900 and 1176 mg/kg/day for the male and female rats, respectively.
- Key result
- Critical effects observed:
- no
- Clinical signs:
- not specified
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- There was no difference between the 1% group and the control group in terms of lifespan (similar number of short vs long-lived animals) but there was a statistically significant increase in the number of long-lived animals in the 0.5% group.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on body weight gain or body weights over the four generations of rats tested in this study.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on feed consumption (efficiency) over the four generations of rats tested in this study.
- Food efficiency:
- no effects observed
- Description (incidence and severity):
- Feed consumption data in this study are presented as “Protein efficiency (weight increase per gram of dietary protein)”. There was no effect on feed consumption (efficiency) in either test group.
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- Organ weights were only reported for the 3rd generation animals.
- Gross pathological findings:
- not specified
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- Histopathology was only reported for 3rd generation animals.
- Histopathological findings: neoplastic:
- not examined
- Reproductive function: oestrous cycle:
- not examined
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- no effects observed
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- > 1 other: %
- Based on:
- other:
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Remarks:
- Generational data were not reported separately. At concentrations up to 1% benzoic acid in the diet, there were no adverse effects on parents or offspring through four generations of test substance administration. Overall, the dose level from the 1% diet for the entire premating period was approximately 900 and 1176 mg/kg/day for the male and female rats, respectively.
- Key result
- Critical effects observed:
- no
- Clinical signs:
- not specified
- Mortality / viability:
- mortality observed, treatment-related
- Description (incidence and severity):
- There was no difference between the 1% group and the control group in terms of lifespan (similar number of short vs long-lived animals) but there was a statistically significant increase in the number of long-lived animals in the 0.5% group.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on body weight gain or body weights over the four generations of rats tested in this study.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on feed consumption (efficiency) over the four generations of rats tested in this study.
- Food efficiency:
- no effects observed
- Description (incidence and severity):
- Feed consumption data in this study are presented as “Protein efficiency (weight increase per gram of dietary protein)”. There was no effect on feed consumption (efficiency) in either test group.
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- Organ weights were only reported for the 3rd generation animals.
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- not examined
- Description (incidence and severity):
- Histopathology was only reported for the 3rd generation animals.
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOEL
- Generation:
- F1
- Effect level:
- > 1 other: %
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Remarks:
- There were no adverse effects on viability, sexual maturation, mortality, body weight and weight gain, food consumption and efficiency, body weights, select organ weight and pathology for any of the four generations in this study receiving up to 1% benzoic acid in the diet. Overall, the dose level from the 1% diet for the entire premating period was approximately 900 and 1176 mg/kg/day for the male and female rats, respectively.
- Key result
- Critical effects observed:
- no
- Clinical signs:
- not specified
- Mortality / viability:
- mortality observed, treatment-related
- Description (incidence and severity):
- There was no difference between the 1% group and the control group in terms of lifespan (similar number of short vs long-lived animals) but there was a statistically significant increase in the number of long-lived animals in the 0.5% group.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on body weight gain or body weights over the four generations of rats tested in this study.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on feed consumption (efficiency) over the four generations of rats tested in this study.
- Food efficiency:
- no effects observed
- Description (incidence and severity):
- Feed consumption data in this study are presented as “Protein efficiency (weight increase per gram of dietary protein)”. There was no effect on feed consumption (efficiency) in either test group.
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- Organ weights were only reported for the 3rd generation animals.
- Gross pathological findings:
- not specified
- Histopathological findings:
- no effects observed
- Description (incidence and severity):
- Histopathology was only reported for 3rd generation animals.
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOEL
- Generation:
- F2
- Effect level:
- > 1 other: %
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Remarks:
- There were no adverse effects on viability, sexual maturation, mortality, body weight and weight gain, food consumption and efficiency, body weights, select organ weight and pathology for any of the four generations in this study receiving up to 1% benzoic acid in the diet. Overall, the dose level from the 1% diet for the entire premating period was approximately 900 and 1176 mg/kg/day for the male and female rats, respectively.
- Key result
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- no
- Conclusions:
- In a chronic feeding study, there were no adverse effects on reproductive parameters including fertility measures, delayed sexual maturity, total number of pups born, pup survival, onset of reproductive senescence or litter size when male and female rats were fed up to 1% benzoic acid in the diet over four generations. Under conditions of this study, benzoic acid is not a reproductive toxicant.
- Executive summary:
Regulation (EC) No 1907/2006 (REACH), Annex XI 1.5., establishes, that substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group of substances. The similarity may also be based on the likelihood of common breakdown products via physical and biological processes. Given that the grouping of substances and read-across approach can be justified, data gaps may be fulfilled from data of reference substance(s) within the group. This avoids the need to test every substance for every endpoint, which is highly recommended in the context of animal welfare considerations (Directive 2010/63/EU, 2010).
For sodium benzoate, the fertility endpoint is filled by read across from a group of structurally similar substances.
Benzyl acetate, benzyl alcohol, benzaldehyde, and benzoic acid and its sodium and potassium salt were considered as a single category regarding human health by JECFA as they are all rapidly metabolized and excreted via a common pathway within 24 hrs (JECFA 1997). Sodium benzoate is the sodium salt of benzoic acid, and is completely metabolized to benzoic acid prior to excretion via the hippuric acid pathway.
The data presented below is for benzoic acid using the read across justification described above and is used as read across for sodium benzoate as requested by ECHA in its letter ‘’Submission FP609849-12 – request for dossier update’’ received by the lead registrant on October 03, 2019 and attached in Section 13 of the IUCLID dossier.
In a long-term feeding study, benzoic acid was added to standard feed to achieve concentration of 0, 0.5% or 1.0% in the diet. Diets were provided ad libitum to groups of 20 rats/sex through four generations. Body weights and feed consumption data were collected throughout the exposure period. Other endpoints examined included: onset of sexual maturity, evidence of permanent sterility, onset of menopause, litter sizes, number of pups born, surviving young, organ weights and histology, and effect on lifespan. Feed consumption, body weights, and weight gain were unaffected by exposure to benzoic acid at concentrations up to 1% in the diet. There was actually an unexplained statistically significant increase in the lifespan of rats in the 0.5% exposure group, i.e., a higher percentage of rats lived longer. There were no differences between the groups exposed to benzoic acid and the control group for fertility measures, delayed sexual maturity, total number of pups born, pup survival, onset of reproductive senescence or litter size. In addition, organ weights and histopathologic findings were similar for all groups. Under conditions of this study, there were no dose-related adverse effects on either reproductive or developmental parameters in both sexes of rats fed 1% benzoic acid in the diet over four generations.
Referenceopen allclose all
This citation is being presented to cover apparent gaps in the Kieckebusch and Lang (1960) study regarding effects on reproducitve tissues/organs. Table 1 of the Morrissey et al. paper provides the following information regarding benzyl acetate on male reproductive tissues, relative to control:
Species |
Terminal Body Weight |
Organ Weight |
Sperm Parameters |
|||||||
R. cauda |
R. epididymis |
R. Testis |
||||||||
Absolute |
Relative |
Absolute |
Relative |
Absolute |
Relative |
Motility (%) |
Density |
Abnormal (%) |
||
Mouse |
Decrease |
Decrease |
Increase |
Decrease |
Increase |
Decrease |
Increase |
No difference |
No difference |
No difference |
Rat |
Decrease |
No difference |
No difference |
No difference |
No difference |
No difference |
No difference |
No difference |
No difference |
No difference |
Regarding female reproductive tissue outcomes, benzyl acetate was noted to significantly increase the mean cycle length in the high-dose group of mice.
There were no differences in reproduction or development of the young in the 4 observed generations exposed to 1% benzoic acid in the diet. See Table 1 in "Any other information on results incl. tables".
There were no differences in reproduction or development of the young in the 4 observed generations exposed to 1% benzoic acid in the diet. See Table 1 in "Any other information on results incl. tables".
Table 1: Reproduction of rats given chronic feeding of benzoic acid over 4 generations
| % Benzoic Acid in Feed | |||
| 0 | 0.5 | 1.0 | |
| Total number of females used | 80 | 78 | 80 |
| Sterile females in % | 14% | 10% | 4% |
| Females with delayed sexual maturity | 7.5 |
17 |
9 |
Litter Size |
9.0 + 0.33 |
9.5 + 0.26 |
9.6 + 0.29 |
|
|
|
|
Total number of pups born |
625 |
688 |
741 |
Surviving young in % of number of pups born |
74% |
66% |
65% |
| Age Pairing (2nd Generation) | |||
| Number of females used | 37 | 38 | 38 |
| Litter size | 6.9 |
7.7 |
7.5 |
|
|
|
|
| Total number of pups born | 173 | 139 | 171 |
| Surviving young in % of number of pups born | 72% | 61% | 73% |
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 500 mg/kg bw/day
- Study duration:
- chronic
- Species:
- rat
- Quality of whole database:
- 2 (reliable with restrictions)
Additional information
No data on reproductive effects and effects on fertility of the test substance are available.
Regulation (EC) No 1907/2006 (REACH), Annex XI 1.5., establishes, that substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group of substances. The similarity may also be based on the likelihood of common breakdown products via physical and biological processes. Given that the grouping of substances and read-across approach can be justified, data gaps may be fulfilled from data of reference substance(s) within the group. This avoids the need to test every substance for every endpoint, which is highly recommended in the context of animal welfare considerations (Directive 2010/63/EU, 2010).
For sodium benzoate, the fertility endpoint is filled by read across from a group of structurally similar substances.
Benzyl acetate, benzyl alcohol, benzaldehyde, and benzoic acid and its sodium and potassium salt were considered as a single category regarding human health by JECFA as they are all rapidly metabolized and excreted via a common pathway within 24 hrs (JECFA 1997). Sodium benzoate is the sodium salt of benzoic acid, and is completely metabolized to benzoic acid prior to excretion via the hippuric acid pathway.
The data presented below is for benzoic acid and benzyl acetate using the read across justification described above and is used as read across for sodium benzoate as requested by ECHA in its letter ‘’Submission FP609849-12 – request for dossier update’’ received by the lead registrant on October 03, 2019 and attached in Section 13 of the IUCLID dossier.
From the studies that are available on repeated dose and carcinogenicity no report on effects on the reproductive organs/tissues in both male and female laboratory animals are reported.
In a 4-generation study with the structural analogue benzoic acid (Kieckebusch 1970), no effects on reproductive performance and off-spring were reported at 1% bezoic acid in feed (500 mg/kg bw).
Kiekebusch and Lang (1960) describes an experiment where 0, 0.5 and 1% benzoic acid was mixed into the feed and fed to rats (groups of 20 males and females/group) for a period of 11-12 weeks prior to breeding. The dietary concentrations were selected based upon a previous probe study where rats consuming 5% benzoic acid in the diet died within three weeks due to lack of palatability of the diet and corrosive effects of the free acid on the digestive tract. The weight range of the animals at the start of the four generation study was 40-50 grams. Based on the mean value of the feed consumed, the authors reported that the rats in the 1% dietary group received “150 mg of benzoic acid / day’’. Based upon the starting body weights and the mean body weight increases reported in Table 1 of the paper, the starting dose level in the males and female animals was in the range of 3750 mg/kg/day for the 1% dose level. After four weeks of feeding, the dose level decreased to 1304 and 1485 mg/kg/day in the male and female rats, respectively. At eight weeks, the dose levels decreased further to 802 and 867 mg/kg/day in the male and female rats, respectively (as the rats grew and consumed roughly the same amount of feed, the effective dose level decreased with the increase in body weight). At 12 weeks of age, the dose level in the male animals was 542 mg/kg/day. The dose level for the female animals was not calculated as they were pregnant by 12 weeks and not included in the body weight gain table. Overall, the dose level for the entire premating period was approximately 900 and 1176 mg/kg/day for male and female rats, respectively. This pattern of feed consumption, body weight gain and corresponding dose levels repeated itself over the four generations of rats consuming these diets within this study. These premating exposure intervals and dose levels are generally comparable to the ten week premating exposure period and limit dose level of 1000 mg/kg/day required under the current EOGRTS test guideline.
Body weights and feed consumption data were collected throughout the exposure periods over the four generations. The body weight data within this publication is presented as body weight “increase” (Table 1) and the feed consumption data is presented as “Protein efficiency (weight increase per gram of dietary protein)” in Table 2. There was no effect of feeding 0.5 or 1% benzoic acid in the diet on feed consumption (efficiency), body weight gain or body weights over the four generations of rats tested in this study.
Effects of benzoic acid on sexual maturity and fertility were determined by pairing male and female animals for 14 days in the 11th and 12th weeks of exposure. Unsuccessful matings were repeated eight weeks later to determine if sexual maturity was delayed or if the mating pairs were infertile. Finally, during the 48thweek of exposure, mating trials were again conducted to evaluate the onset of reproductive senescence in the older animals. Litter data was collected on postnatal days 1, 2 and 21 in each of the four generations. Since there was no demonstrable effect of benzoic acid on reproduction and since the data from all four generations was similar, the data from all four generations was combined into a single table (Table 3). There was no difference between the groups with benzoic acid exposure and the control group for fertility measures, delayed sexual maturity, total number of pups born, pup survival, onset of reproductive senescence or litter size.
The Kiekebusch and Lang paper also evaluated the effect of feeding 0.5 or 1.0% benzoic acid on the lifespan of rats. There was no difference between the 1.0% group and the control animals in terms of lifespan, while the incorporation of 0.5% benzoic acid in the diet appeared to lengthen the lifespan of the rats consuming this dietary concentration. The third generation of animals was necropsied after 16 weeks of exposure and organ weights of the brain, heart, liver, spleen kidneys and testes were collected. Tissues were saved and histopathological examination of the tissues did not reveal any difference between the control and two treated groups for either the organ weights or the histopathology findings.
While the design of this four-generation study for reproductive toxicity does not exactly match the endpoints included within the EOGRTS, it does include almost all of the endpoints associated with the OECD Guideline 416 – two generation reproductive toxicity that was previously considered adequate to satisfy the reproductive toxicity endpoint under the REACH regulation. The only missing reproductive endpoints within the Kiekebusch and Lang publication are collection of sperm parameters or estrous cyclicity data. However, the endpoints that are missing from the four-generation study with benzoic acid are available for benzyl acetate, a chemical that is metabolized completely to benzoic acid. In the National Toxicology Program (Morrissey et al., 1988) 13-week feeding benzyl acetate studies in rats and mice, both sperm parameters (epididymis, cauda epididymis, testis weights, and sperm motility, density and percent abnormal sperm) and estrus cyclicity measurements were collected. Dose levels of 2000 to 3000 mg/kg/day did not affect any endpoints of male reproduction. Estrus cycles were lengthened in female mice receiving >3000 mg/kg/day but this only occurred at a dose levels also causing significant decreases in growth (body weights and body weight gain). Therefore, it appears that all of the reproductive toxicity endpoints have been collected, albeit in two separate studies on benzoic acid and benzyl acetate. Based upon the recent information that has become available following receipt of the English translation of the Kiekebusch and Lang publication, the reproductive toxicity endpoint for benzoic acid should be satisfied based upon the available study data. Shortcomings of the Kiekebusch and Lang 1960 study can be addressed using the data from the 13-week feeding study with benzyl acetate (Morrissey et al., 1988). For these reasons, ECHA should consider the reproductive toxicity endpoint for benzoic acid to be satisfied with the available study data. Overall, taking into consideration both the Kieckebusch and Lang (1960), and Morrissey et al. (1988) studies, no effects on reproductive performance and off-spring were reported at 1% the test substance in feed (500 mg/kg bw). Therefore, the NOAEL for toxicity to reproduction is set at 500 mg/kg bw.
From the toxicokinetic assessment, it was concluded that uptake of the test substance after oral dosing would be in the form of benzoic acid. Therefore it is considered acceptable to use the data from the 4-generation study, especially as from the studies that are available on repeated dose and carcinogenicity no report on effects on the reproductive organs/tissues in both male and female laboratory animals are reported. The NOAEL for toxicity to reproduction is set at 500 mg/kg bw.
Effects on developmental toxicity
Description of key information
In the available studies on developmental toxicity (Morgareidge, 1972), no effects on the off-spring were seen in four species ( rats, mice, hamsters and rabbits) at the highest dose levels tested (175-300 mg/kg bw). In these studies no signs of maternal toxicity were reported. An additional study (Onodera, 1978 ) showed no effects at 2% in diet (1000 mg/Kg bw), but provided limited information on maternal toxicity.
Link to relevant study records
- Endpoint:
- developmental toxicity
- Remarks:
- Studies on Effects of Sodium Benzoate on Fetuses and Offspring of Wistar Rats
- Type of information:
- experimental study
- Remarks:
- The study was designed to show the effects of sodium benzoate on pregnant rats, the teratogenic effect on the fetuses, and the influence on the growth of the offspring.
- Adequacy of study:
- key study
- Study period:
- Sodium benzoate in diet was administered to groups of pregnant Wistar rats during the entire gestation period (GD 0 until termination on GD 20); a subgroup of rats was selected to continue exposure through delivery and lactation, an additional 3 weeks.
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- Study predates current published regulatory guidelines for GLP compliance and developmental toxicity studies; the study design closely follows the OECD 414 test guideline with additional postnatal investigations for developmental endpoints. Some differences from the OECD 414 guideline include: use of top two dose levels which greatly exceeded the MTD with severe effects due to marked decreases in feed consumption; a next highest dose level which exceeded the 1000 mg/kg/day limit dose for OECD 414 studies; an extended exposure period and group sizes which exceeded those required in an OECD 414 guideline study; and assignment of three-quarters of viable fetuses for skeletal examination and one-quarter for visceral examination on GD 20 rather than the 50/50 ratio currently used.
- Principles of method if other than guideline:
- Groups of 27-30 pregnant Wistar rats were fed CE-2 diets containing 0, 1, 2, 4 or 8% sodium benzoate in the diet from gestation day (GD) 0 through 20.
- GLP compliance:
- not specified
- Limit test:
- no
- Specific details on test material used for the study:
- Source of test material and purity not specified, assumed to be > 99.0% pure.
- Species:
- rat
- Strain:
- Wistar
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Japan Rat Ltd.
- Age at study initiation: 8 - 9 week old males, and 7 - 10 week old females.
- Weight at study initiation: Not stated, but based on Figure 1, females were between 260 - 270 g at GD 0.
- Fasting period before study: Not stated.
- Housing: Not stated.
- Diet (e.g. ad libitum): ad libitum access to CE-2 generic feed or CE-2 feed containing test material.
- Water (e.g. ad libitum): Ad libitum access to tap water.
- Acclimation period: Not stated, assumed to be 4 weeks based on statement that treatment began when dams were 15 - 17 weeks old.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not stated.
- Humidity (%): Not stated.
- Air changes (per hr): Not stated.
- Photoperiod (hrs dark / hrs light): Not stated. - Route of administration:
- oral: feed
- Details on exposure:
- The delivered dose calculations were 0, 699, 1306, 1874 and 965 mg benzoic acid/kg body weight/day for the 0, 1, 2, 4 and 8% diet groups, respectively.
PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
- Rate of preparation of diet (frequency): Not provided in publication.
- Mixing appropriate amounts with (Type of food): sodium benzoate was added to generic feed to achieve concentrations of 1%, 2%, 4% and 8%.
- Storage temperature of food: Not provided in publication.
- Analytical verification of doses or concentrations:
- not specified
- Details on mating procedure:
- - Impregnation procedure: Animals were co-housed.
- M/F ratio per cage: 2 males/5 females per cage.
- Length of cohabitation: Overnight.
- Proof of pregnancy: Both vaginal plug and sperm in vaginal smear were used to confirm pregnancy, and was considered Day 0 of pregnancy. - Duration of treatment / exposure:
- Throughout the entire period of gestation (GD 0 – GD 20); a subgroup continued exposure throughout delivery and lactation.
- Frequency of treatment:
- Test material in feed was available ad libitum.
- Duration of test:
- 22-25 dams were provided test material in feed ad libitum from GD 0 until termination on GD 20; 5 rats continued exposure to test diets from GD 0 through delivery and lactation.
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Calculated dose based on 0% in feed, corrected for actual feed consumption.
- Dose / conc.:
- 699 mg/kg bw/day (actual dose received)
- Remarks:
- Calculated dose based on 1% in feed, corrected for actual feed consumption.
- Dose / conc.:
- 1 306 mg/kg bw/day (actual dose received)
- Remarks:
- Calculated dose based on 2% in feed, corrected for actual feed consumption.
- Dose / conc.:
- 1 874 mg/kg bw/day (actual dose received)
- Remarks:
- Calculated dose based on 4% in feed, corrected for actual feed consumption.
- Dose / conc.:
- 965 mg/kg bw/day (actual dose received)
- Remarks:
- Calculated dose based on 8% in feed, corrected for actual feed consumption.
- No. of animals per sex per dose:
- 27-30/females/dose group on GD 0; on GD 20, 22-25 rats were sacrificed; 5 dams/group continued exposure and were sacrificed following natural delivery and weaning. At the GD 20 sacrifice, the number of pregnant rats examined in each exposure group were: 0% (15); 1% (15); 2% (16); 4% (18) and 8% (12).
- Control animals:
- yes, plain diet
- Details on study design:
- Not provided in publication.
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Not provided in publication.
BODY WEIGHT: Yes
- Time schedule for examinations: Every 5 days.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20; additional 5 dams/group were sacrificed following natural delivery and weaning.
- Organs examined: Dams were examined for internal organ abnormalities; ovaries and uterus plus other unspecified organs. - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
- Other: Total number of dead fetuses and resorptions were counted; resorptions were not identified as early/late; also number of retained placentas and placental scars were recorded; placenta and ovary weights were recorded; fetal weights were recorded. - Fetal examinations:
- - External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 25% per litter
- Skeletal examinations: Yes: 75% per litter
- Head examinations: Yes: 25% per litter - Statistics:
- Means and standard deviations were used for most endpoints; Wilcoxon Rank Sum test was used to evaluate fetal visceral and skeletal data.
- Historical control data:
- No information provided in publication.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Signs of tonic spasms and restricted movement were observed in animals that died.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- In the 4% group, one rat died on GD 10 and one on GD 20; in the 8% group, one rat died on GD 17 and two rats died on GD 20. Cause of death was unknown.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Steady increase in BW with no significant difference from control observed in the 1% and 2% groups; body weight losses occurred in the 4% and 8% groups during the entire gestation period; 4% group body weight values were able to recover to the original starting body weight by GD 20 but 8% group body weights were 25% lower on GD 20 compared to GD 0.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Feed consumption values in the 4% and 8% dose groups were decreased 58 and 87%, respectively, when compared to the control group. Feed consumption values in the 1% and 2% dose groups were similar to the control value.
- Food efficiency:
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not specified
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- not examined
- Number of abortions:
- no effects observed
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- No data were provided on the number of pre- and post-implantation losses. However, there were no significant differences in the average number of implantations among the groups when compared to the control.
- Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- effects observed, treatment-related
- Description (incidence and severity):
- Total number of resorptions per group were not reported separately or specified as early or late; data for dead fetuses and resorbed embryos were grouped together; in both the 4% group and the 8% group, there was an increase in the number of dead fetuses/resorbed embryos. Effects in the 1% and 2% groups were similar to the control.
- Dead fetuses:
- effects observed, treatment-related
- Description (incidence and severity):
- Total number of dead fetuses per group were not reported; data for dead fetuses and resorbed embryos were grouped; in both the 4% group and the 8% group, there was an increase in the number of dead fetuses/resorbed embryos. Effects in the 1% and 2% groups were similar to the control.
- Changes in pregnancy duration:
- no effects observed
- Changes in number of pregnant:
- no effects observed
- Other effects:
- no effects observed
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 2 other: %
- Based on:
- test mat.
- Basis for effect level:
- clinical signs
- Remarks on result:
- other: Effect level given in %, which is not a choice in drop down.
- Key result
- Dose descriptor:
- conc. level:
- Effect level:
- 4 other: %
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- clinical signs
- dead fetuses
- early or late resorptions
- food consumption and compound intake
- mortality
- Remarks on result:
- other: Effect level given in %, which is not a choice in drop down.
- Key result
- Dose descriptor:
- conc. level:
- Effect level:
- 8 other: %
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- clinical signs
- dead fetuses
- early or late resorptions
- food consumption and compound intake
- mortality
- Remarks on result:
- other: Effect level given in %, which is not a choice in drop down.
- Key result
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- In the 1% and 2% dose groups, there was no significant difference from the control for average body weight of viable fetuses. There was a 29-40% decrease in average fetal body weights in the 4 and 8% groups.
- Reduction in number of live offspring:
- effects observed, treatment-related
- Description (incidence and severity):
- In the 1% and 2% dose groups, there was no significant difference from the control group for combined number of dead fetuses and resorbed embryos. There was a 3 to 5-fold increase in combined number of dead fetuses and resorbed embryos in the 4 and 8% groups.
- Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- not specified
- Changes in postnatal survival:
- effects observed, treatment-related
- Description (incidence and severity):
- The rate of perinatal death was 100% in the 4 and 8% groups and 0% in the 1 and 2% groups.
- External malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- Slight edema was observed in 17/151 fetuses in the 4% group and 1/117 in the 8% group. No external abnormalities were observed in the 1% and 2% groups.
- Skeletal malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- In the 4% group, 97% of the fetuses had skeletal abnormalities. In the 8% group, the percentage was 100%. For the 1 % and 2% groups, there were no significant skeletal differences from the control group.
- Visceral malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- In the 4% group, 12/36 (33%) examined had abnormalities including unilateral microphthalmia (5), bilateral microphthalmia (1), unilateral anophthalmia (2), hydrocephalus (3), bilateral pyelectasis (2), and unilateral renal hypoplasia (1). In the 8% group, 11/26 (42%) examined had abnormalities including unilateral microphthalmia (6), unilateral anophthalmia (1), hydrocephalus (3), cerebral hypoplasia (1), and bilateral pyelectasis (2). For the 1 % and 2% groups, there were no significant visceral differences from the control group.
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 2 other: %
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: At a concentration of 2% in the diet, there were no significant effects on any fetal endpoints in rats when compared to the control.
- Remarks on result:
- other: Effect level given in %, which is not a choice in drop down.
- Key result
- Dose descriptor:
- conc. level:
- Effect level:
- 4 other: %
- Based on:
- test mat.
- Sex:
- not specified
- Basis for effect level:
- reduction in number of live offspring
- fetal/pup body weight changes
- changes in postnatal survival
- external malformations
- skeletal malformations
- visceral malformations
- Remarks on result:
- other: Effect level given in %, which is not a choice in drop down.
- Key result
- Dose descriptor:
- conc. level:
- Effect level:
- 8 other: %
- Based on:
- test mat.
- Sex:
- not specified
- Basis for effect level:
- reduction in number of live offspring
- fetal/pup body weight changes
- changes in postnatal survival
- external malformations
- skeletal malformations
- visceral malformations
- Remarks on result:
- other: Effect level given in %, which is not a choice in drop down.
- Key result
- Abnormalities:
- effects observed, non-treatment-related
- Localisation:
- external: eye
- skeletal: sternum
- skeletal: rib
- visceral/soft tissue: urinary
- Description (incidence and severity):
- There were no external or visceral abnormalities in the control group. The only external or visceral findings in the 1 and 2% groups were bilateral anophthalmia in a single fetus in the 1% group and unilateral pyelectasis (dilatation of renal pelvis) in a single fetus in the 2% group. Incidences of skeletal abnormalities in the 1 and 2% groups were similar to the control group. The most frequent findings were the presence of lumbar ribs and varied sternebrae. The NOEL for fetal abnormalities was considered to be 2% sodium benzoate in the diet.
- Key result
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- external: eye
- skeletal: sternum
- skeletal: rib
- visceral/soft tissue: central nervous system
- Description (incidence and severity):
- A mild level of systemic edema was observed in the 4% and 8% groups. In the 4% group, 33% had visceral abnormalities. Out of 36 fetuses examined, there were 5 cases of unilateral microphthalmia (right or left), 1 case of bilateral microphthalmia, 2 cases of unilateral anophthalmia (right or left), 3 cases of ventricular dilation, 2 cases of pyelectasis, and 1 case of hypoplasia of the left kidney. In the 8% group, 42% had visceral abnormalities. Out of the 26 fetuses examined in the 8% group, there were 6 cases of unilateral microphthalmia (right or left), 1 case of anophthalmia on the left, 3 cases of ventricular dilation, 1 case of cerebral hypoplasia, and 2 cases of pyelectasis. In the 4% group, 97% had skeletal abnormalities while in the 8% group, 100% had skeletal abnormalities. Similar incidences of abnormalities were found in the control, 1% and 2% dose groups. Skeletal abnormalities in the 4% and 8% groups primarily involved lumber ribs, cervical ribs, and varied sternebrae.
- Key result
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 4 other: %
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects as a secondary non-specific consequence of maternal toxicity effects
- Dose response relationship:
- yes
- Relevant for humans:
- yes
- Conclusions:
- The administration of up to 2% sodium benzoate in the diet to pregnant rats throughout the entire gestation period, delivery and weaning had no adverse effects on any maternal or fetal parameters examined in this study and did not cause developmental delays. The number of abnormalities seen in either soft or skeletal tissues of fetuses and weanlings in the 1 and 2% test groups did not significantly differ from the number occurring spontaneously in offspring of the control group. The NOEL for maternal and developmental toxicity was considered to be 2% in the diet.
- Executive summary:
In a developmental toxicity study using a method similar to the OECD 414 test guideline, pregnant Wistar rats were fed diets containing 0, 1%, 2%, 4% or 8% sodium benzoate from GD 0 through termination on GD 20. The delivered dose calculations were 0, 699, 1306, 1874 and 965 mg/kg/day for the 0, 1, 2, 4 and 8% diet groups, respectively. A subgroup of dams continued on the diet through natural delivery and lactation. Maternal feed consumption was measured daily and body weights were recorded every five days. On GD20, 22-25 rats per group were euthanized and the number of living/dead fetuses, resorbed fetuses, retained placentas, and placental scars were recorded, along with fetus weight, and placenta and ovary weight. Dams were examined for internal organ abnormalities; fetuses were sexed and examined for external, visceral and skeletal abnormalities. Five dams per group were allowed to deliver naturally; their offspring were examined externally, weighed, and survival rate recorded. Juvenile rats were weaned after 3 weeks and approximately half were euthanized and examined for visceral and skeletal abnormalities. The remaining juveniles were raised to eight weeks, euthanized and examined for abnormalities. Under conditions of this study, there were no concentration-related adverse effects observed in the dams or fetuses in the 0, 1% or 2% groups sacrificed on GD 20. There were also no adverse effects on delivery rate, perinatal death rate, lactation rate, or survival rate for offspring at 8 weeks of age in the 0, 1% or 2% groups.
By comparison, maternal feed consumption values in the 4% and 8% groups were decreased 58% and 87%, respectively, when compared to the control group and resulted in body weight loses in both treatment groups during the entire gestation period. The study authors considered these to reflect a palatability issue with the test diet rather than a consequence of the toxicity of sodium benzoate. Adverse clinical signs of toxicity (including lethality and tonic spasms) were observed in dams in both the 4 and 8% groups. Dose dependent adverse effects in fetuses of dams in the 4 and 8% groups sacrificed on GD 20 included: an increase in number of dead or resorbed fetuses, a decrease in average fetal body weight, and an increase in the number of visceral and skeletal abnormalities. For dams that were allowed to deliver naturally, there was a significant adverse effect on delivery rate and perinatal death rate in the 4% and 8% groups. Delivery rates were 50% in the 4% group and 8.2% in the 8% group compared to 75%, 80.3% and 81.8% in the 0, 1% and 2% groups, respectively. While there were no perinatal deaths in the 0, 1% or 2% groups, the death rate in the 4 and 8% groups was 100%.
In the absence of maternal toxicity, there were no developmental effects or delays in the offspring of dams receiving up to 2% sodium benzoate in the diet throughout the entire gestation period and lactation. The NOEL in this study was considered to be 2% in the diet.
- Endpoint:
- developmental toxicity
- Remarks:
- Teratologic Evaluation of Sodium Benzoate in Rats
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 10 consecutive days from Gestation Day (GD) 6 through GD 15; animals were sacrificed on GD 20
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Remarks:
- There were 3 significant differences between the Morgareidge study and the current OECD 414 guideline, which were 1) selection of dose levels, 2) exposure period during gestation, and 3) group size requirements. The inclusion of Onodera et al. (1978) addresses the dose level issue. The availabilty of pre-guideline studies in 4 different species (rat, mouse, hamster and rabbit), all showing no developmental effects, suggests that there is a low probability that a contemporary OECD 414 would change that outcome.
- Justification for type of information:
- The study has been used in support of various risk assessments for the use of Sodium Benzoate as a food additive (EFSA, JECFA). In these reviews, the study is deemed acceptable for use in the risk assessment, and allowed for the establishment of an Acceptable Daily Intake (ADI) for Sodium Benzoate as a direct food additive.
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- Predates published regulatory guidelines; used internal guidelines of test facility.
Study predates GLP compliance requirements and published regulatory guidelines for teratology evaluation; test conducted according to internal test facility guidelines. Virgin adult female albino rats (Wistar derived stock) were individually housed in mesh bottom cages in temperature and humidity-controlled quarters with free access to food and fresh tap water. Groups of 24 females were bred to young adult males to achieve test group sizes of approximately 24 pregnant females. Presence of a vaginal sperm plug was considered Day 0 of gestation. Beginning on GD 6 and continuing daily, pregnant dams were dosed by oral gavage with 0, 1.75, 8.0, 38.0 or 175.0 mg/kg sodium benzoate on GD 6-15. Positive control dams were similarly treated with 250 mg/kg of aspirin. Negative controls were sham-treated. Dams were observed daily for appearance and behavior; maternal body weights were recorded on GD 0, 6, 11, 15 and 20. On GD 20, all surviving dams were subjected to Caesarean section under anesthesia, and numbers of corpora lutea, implantation sites, resorptions sites, live/dead fetuses, and body weights of live pups were recorded. The urogenital tract of each dam was examined in detail. Fetuses were sexed, and examined for external congenital abnormalities. One-third of the fetuses of each litter underwent detailed visceral examinations while the remaining two-thirds were examined for skeletal defects. - GLP compliance:
- no
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Not provided in report.
- Expiration date of the lot/batch: Not provided in report.
- Purity test date: Not provided in report.
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not provided in report.
- Stability under test conditions: Not provided in report.
- Solubility and stability of the test substance in the solvent/vehicle: Not provided in report.
FORM AS APPLIED IN THE TEST (if different from that of starting material): Administered as a water solution at 1.0 mL/kg body weight. - Species:
- rat
- Strain:
- Wistar
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Not provided in report
- Age at study initiation: Adult; specific age not provided in report
- Weight at study initiation: The average maternal body weights on GD 0 were 228, 233, 229, 227, and 224 g for the 0, 1.75, 8.0, 38.0 and 175.0 mg/kg sodium benzoate dose groups, respectively and 226 g for the positive control.
- Fasting period before study: Not provided in report
- Housing: Females housed individually in mesh bottom cages
- Diet (e.g. ad libitum): ad libitum; specific diet not provided in report
- Water (e.g. ad libitum): ad libitum to fresh tap water
- Acclimation period: Not provided in report
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Controlled; specific number not provided in report
- Humidity (%): Controlled; specific number not provided in report
- Air changes (per hr): Not provided in report
- Photoperiod (hrs dark / hrs light): Not provided in report
IN-LIFE DATES: From: To: Not provided in report - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: Test material was administered as a water solution , 1.0 mL/kg of body weight
- Analytical verification of doses or concentrations:
- not specified
- Details on mating procedure:
- - Impregnation procedure: co-housed
- If cohoused:
- M/F ratio per cage: Not specified but presumed 1/1
- Length of cohabitation: Not specified in report.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: Not specified in report.
- Further matings after two unsuccessful attempts: Not specified in report.
- Verification of same strain and source of both sexes: Not specified in report.
- Proof of pregnancy: presence of a vaginal plug considered as day 0 of pregnancy.
- Any other deviations from standard protocol: Not specified in report. - Duration of treatment / exposure:
- 10 days; Females were administered the indicated dosages beginning on GD 6 and continuing daily through GD 15.
- Frequency of treatment:
- Once per day.
- Duration of test:
- Animals were sacrificed on GD 20.
- Dose / conc.:
- 0 mg/kg bw/day
- Dose / conc.:
- 1.75 mg/kg bw/day
- Dose / conc.:
- 8 mg/kg bw/day
- Dose / conc.:
- 38 mg/kg bw/day
- Dose / conc.:
- 175 mg/kg bw/day
- No. of animals per sex per dose:
- 24/mated females/dose
23-24/pregnant females/dose - Control animals:
- yes, sham-exposed
- Details on study design:
- - Dose selection rationale: Not provided in report.
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
DETAILED CLINICAL OBSERVATIONS: No data
BODY WEIGHT: Yes
- Time schedule for examinations: On Day 0 and GD 6, 11, 15, and 20
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Urogenital tract of each dam was examined in detail for abnormalities - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
- Other: Total resorption sites were provided but no differentiation was made between early and late resorptions - Fetal examinations:
- - External examinations: Yes: all per litter.
- Soft tissue examinations: Yes: 1/3 per litter.
- Skeletal examinations: Yes: 2/3 per litter.
- Head examinations: Yes: 2/3 per litter. - Statistics:
- Not provided in report.
- Historical control data:
- Not provided in report.
- Clinical signs:
- not specified
- Mortality:
- no mortality observed
- Description (incidence):
- No dams in the sham, sodium benzoate-treated, or aspirin-treated groups died prior to study termination.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There was no adverse effect on average maternal body weight in sodium benzoate-treated dams when measured at sacrifice on GD 20. The average body weights were 347 (23), 359 (24), 345 (23), 345 (23) and 342 (23) g for animals in the 0, 1.75, 8.0, 38.0 and 175.0 mg/kg sodium benzoate-treated groups, respectively. The number in ( ) indicates the number of surviving dams. The average weight for dams in the positive control group was lower at 308 (24) g. Each test and control group contained 23-24 dams at sacrifice.
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- not specified
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not specified
- Histopathological findings: neoplastic:
- not examined
- Number of abortions:
- no effects observed
- Description (incidence and severity):
- There were no abortions in the sodium benzoate-treated groups or in the negative or positive control groups.
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- No data were provided on the number of pre- and post-implantation losses. However, there was no significant dose-related difference from the sham group in the ratio of total live fetuses/total implant sites at dose levels up to 175.0 mg/kg of sodium benzoate. By comparison, there was a significant decrease in the number of live fetuses in the positive control when compared to total implant sites.
0 mg/kg = 243/249
1.75 mg/kg = 294/300
8.0 mg/kg = 228/238
38.0 mg/kg = 263/268
175.0 mg/kg = 232/252
250.0 mg/kg aspirin (positive control) = 160/263
The average numbers of implant sites per dam were 10.8, 12.5, 10.3, 11.7, and 11.0 for the 0, 1.75, 8.0 38.0 and 175.0 mg/kg sodium benzoate-treated groups, respectively and 11.0 for the positive control. By comparison, the average numbers of live fetuses per dam were 10.6, 12.3, 9.91, 11.4, and 10.1 in the 0, 1.75, 8.0, 38.0 and 175.0 mg/kg sodium benzoate-treated groups compared to 6.67 for the positive control. - Total litter losses by resorption:
- effects observed, non-treatment-related
- Description (incidence and severity):
- For dams examined at term, there were a total of 6 resorptions in the sham (0 mg/kg) group compared to 6, 10, 5 and 20 in the 1.75, 8.0, 38.0 and 175.0 mg/kg sodium benzoate-treated groups, respectively. By comparison, there were 102 resorptions in the positive control group. A single dam in the 38.0 mg/kg sodium benzoate-treated group had all sites resorbed compared with 6 dams in the positive control group.
- Early or late resorptions:
- not specified
- Description (incidence and severity):
- Resorptions were not specified as early or late. There was no significant dose-related difference from the sham group in the total number of resorptions at dose levels up to 175.0 mg/kg of sodium benzoate. For dams examined at term, there were a total of 6 resorptions in the sham (0 mg/kg) group compared to 6, 10, 5 and 20 in the 1.75, 8.0, 38.0 and 175.0 mg/kg sodium benzoate-treated groups, respectively. By comparison, there were 102 resorptions in the positive control group.
- Dead fetuses:
- no effects observed
- Description (incidence and severity):
- There were no dead fetuses in the sham or sodium benzoate-treated groups. A single fetus died in the positive control group.
- Changes in pregnancy duration:
- no effects observed
- Description (incidence and severity):
- No dams in the sodium benzoate-treated groups died or aborted before GD 20. All litters were carried to term.
- Changes in number of pregnant:
- no effects observed
- Description (incidence and severity):
- The total number of pregnancies was similar across all sodium benzoate-treated groups and the positive control.
0 mg/kg = 23
1.75 mg/kg = 24
8.0 mg/kg = 23
38.0 mg/kg = 23
175.0 mg/kg = 23
250.0 mg/kg aspirin (positive control) = 24 - Key result
- Dose descriptor:
- NOEL
- Effect level:
- > 175 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- other: At dose levels up to 175.0 mg/kg sodium benzoate, there were no adverse effects on maternal toxicity or pregnancy in rats.
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- no effects observed
- Description (incidence and severity):
- There was no significant difference in average fetal weight in the sodium benzoate-treated groups when compared to the sham-treated group. Average fetal weight was 3.67, 3.70, 3.70, 3.89, and 3.81 g in the 0, 1.75, 8.0, 38.0 and 175.0 mg/kg groups, respectively. The average fetal weight in the positive control group was significantly lower at 2.15 g.
- Reduction in number of live offspring:
- no effects observed
- Description (incidence and severity):
- There was no significant difference in number of live fetuses in the sodium benzoate-treated groups when compared to the sham-treated group. The total number of live fetuses was 243, 294, 228, 263, and 232 in the 0, 1.75, 8.0, 38.0 and 175.0 mg/kg groups, respectively. The average number of live fetuses per dam was also similar at 10.6, 12.3, 9.91, 11.4, and 10.1 in the 0, 1.75, 8.0, 38.0 and 175.0 mg/kg groups, respectively. There was a significant decrease in the number of live fetuses and average number of fetuses per dam in the positive control group. The total number of live fetuses was 160 and the average number of live fetuses per dam was 6.67 for the positive control.
- Changes in sex ratio:
- effects observed, non-treatment-related
- Description (incidence and severity):
- When compared to the sham-treated control, there was no dose-dependent effect on M/F sex ratio in the sodium benzoate-treated groups or in the aspirin positive control. The ratios (M/F) were 1.15, 1.10, 0.81, 0.80, and 1.0 for the 0, 1.75, 8.0, 38.0 and 175.0 mg/kg sodium benzoate-treated groups, respectively and 1.03 for the positive control.
- Changes in litter size and weights:
- not specified
- Description (incidence and severity):
- There was no dose-dependent difference in number of fetuses (live + dead) in the sodium benzoate-treated groups when compared to the sham-treated group. There was a significant decrease in the number of fetuses observed with the positive control. Litter weights were not reported but the average fetus weight was similar across all sodium benzoate-treated groups but significantly depressed in the aspirin-treated positive control.
- Changes in postnatal survival:
- not examined
- External malformations:
- no effects observed
- Skeletal malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There was no dose-dependent increase in the number of skeletal abnormalities in sodium benzoate-treated groups when compared to the number occurring spontaneously in the sham-treated controls. Most of the abnormalities observed in the sham control and test groups were incomplete ossification of sternebrae and vertebrae, and incomplete closure of the skull. Missing sternebrae, wavy ribs, and missing or reduced hyoid were also observed at similar rates across the sham and all sodium benzoate-treated groups. By comparison, rates and types of skeletal defects of the sternebrae, ribs, vertebrae, skull, extremities, and hyoid were significantly increased in the aspirin-treated control.
- Visceral malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Except for subcutaneous edema observed in one pup in the 38.0 mg/kg test group, no other soft tissue abnormalities were reported in the sham or test groups receiving up to 175 mg/kg sodium benzoate. By comparison, four of the litters from dams treated with 250 mg/kg aspirin had pups with exencephaly (17), spina bifida (16), and enterohepatocele (10).
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- > 175 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: At dose levels up to 175.0 mg/kg sodium benzoate, there were no adverse effects on fetal parameters in rats examined in this study.
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Abnormalities:
- no effects observed
- Key result
- Developmental effects observed:
- no
- Lowest effective dose / conc.:
- 175 mg/kg bw/day
- Treatment related:
- no
- Conclusions:
- The administration of up to 175.0 mg/kg (body weight) of sodium benzoate to pregnant rats for 10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in offspring in the sham-treated controls.
- Executive summary:
In a developmental toxicity study, pregnant Wistar rats were administered 0, 1.75, 8.0, 38.0, or 175.0 mg/kg sodium benzoate by oral gavage once daily on gestation days (GD) 6 through 15 while positive control animals received 250 mg/kg aspirin. Maternal body weights were recorded on GD 0, 6, 11, 15 and 20. All animals were observed daily for appearance and behavior with particular attention to food consumption and weight. On GD 20, all surviving dams were subjected to Caesarean section under anesthesia, and the numbers of corpora lutea, implantation sites, resorptions sites, and live and dead fetuses were recorded. Body weights of live fetuses were also recorded. The urogenital tract of each dam was examined in detail. All fetuses were examined for gross congenital abnormalities. In each litter, one-third of the fetuses underwent detailed visceral examination while the remaining two-thirds were treated and examined for skeletal defects. Under conditions of this study, no dose-related adverse effects were observed in the dams or fetuses in any of the groups receiving up to 175.0 mg/kg sodium benzoate during gestation days 6 through 15. By comparison, the positive control aspirin administered at a dose level of 250 mg/kg on GD 6-15 had a significant effect on maternal pregnancy and fetal development and survival.
- Endpoint:
- developmental toxicity
- Remarks:
- Teratologic Evaluation of Sodium Benzoate in Rabbits
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13 consecutive days from Gestation Day (GD) 6 through GD 18; animals were sacrificed on GD 29
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Remarks:
- There were 3 significant differences between the Morgareidge study and the current OECD 414 guideline, which were 1) selection of dose levels, 2) exposure period during gestation, and 3) group size requirements. The inclusion of Onodera et al. (1978) addresses the dose level issue. The availabilty of pre-guideline studies in 4 different species (rat, mouse, hamster and rabbit), all showing no developmental effects, suggests that there is a low probability that a contemporary OECD 414 would change that outcome.
- Justification for type of information:
- The study has been used in support of various risk assessments for the use of Sodium Benzoate as a food additive (EFSA, JECFA). In these reviews, the study is deemed acceptable for use in the risk assessment, and allowed for the establishment of an Acceptable Daily Intake (ADI) for Sodium Benzoate as a direct food additive.
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- Predates published regulatory guidelines; used internal guidelines of test facility.
Study predates GLP compliance requirements and published regulatory guidelines for teratology evaluation; test conducted according to internal test facility guidelines. Virgin, adult, Dutch-belted female rabbits were individually housed in mesh bottom cages in temperature and humidity-controlled quarters with free access to food and fresh tap water. On Day 0, each doe was given an injection of 0.4 ml of human chorionic gonadotropin (400 IU) via the marginal ear vein. Three hours later, each doe was artificially inseminated with 0.3 ml of diluted semen from a proven donor buck using approximately 20 x 106 motile sperm. Beginning on Day 6 and continuing daily, pregnant does were dosed by oral gavage with 0, 2.5, 12.0, 54.0 or 250.0 mg/kg of sodium benzoate on GD 6-18. Positive control does received a single 2.5 mg/kg dose of 6-aminonicotinamide on GD 9. Negative controls were sham-treated. Does were observed daily for appearance and behavior; body weights were recorded on GD 0, 6, 12, 18 and 29. On GD 29, all surviving does were subjected to Caesarean section under anesthesia, and numbers of corpora lutea, implantation sites, resorption sites, and live/dead fetuses were recorded. Body weights of the live pups were also recorded. The urogenital tract of each dam was examined in detail. All fetuses underwent a detailed gross examination for the presence of external congenital abnormalities. The live fetuses of each litter were placed in an incubator for 24 hours for evaluation of neonatal survival. All surviving pups were then sacrificed and examined for visceral abnormalities. All fetuses were then cleared in potassium hydroxide, stained, and examined for skeletal defects. - GLP compliance:
- no
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Not provided in report
- Expiration date of the lot/batch: Not provided in report
- Purity test date: Not provided in report
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not provided in report
- Stability under test conditions: Not provided in report
- Solubility and stability of the test substance in the solvent/vehicle: Not provided in report
FORM AS APPLIED IN THE TEST (if different from that of starting material) Administered as a water solution at 1.0 mL/kg body weight. - Species:
- rabbit
- Strain:
- Dutch
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Not provided in report
- Age at study initiation: Adult; specific age not provided in report.
- Weight at study initiation: The average maternal body weights on GD 0 were 2.69, 2.25, 2.30, 2.53 and 2.62 for the 0, 2.5, 12.0, 54.0 and 250 mg/kg sodium benzoate dose groups, respectively and 2.78 for the 6-AN positive control.
- Fasting period before study: Not provided in report.
- Housing: Females housed individually in mesh bottom cages.
- Diet (e.g. ad libitum): ad libitum; specific diet not provided in report.
- Water (e.g. ad libitum): ad libitum to fresh tap water.
- Acclimation period: Not provided in report
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Controlled; specific number not provided in report.
- Humidity (%): Controlled; specific number not provided in report.
- Air changes (per hr): Not provided in report.
- Photoperiod (hrs dark / hrs light): Not provided in report.
IN-LIFE DATES: From: To: Not provided in report - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: Test material was administered as a water solution , 1.0 mL/kg of body weight.
- Analytical verification of doses or concentrations:
- not specified
- Details on mating procedure:
- - Impregnation procedure: artificial insemination
- Any other deviations from standard protocol: Not specified in report - Duration of treatment / exposure:
- 13 days; Females were administered the indicated dosages beginning on GD 6 and continuing daily through GD 18.
- Frequency of treatment:
- Once per day.
- Duration of test:
- Animals were sacrificed on GD 29.
- Dose / conc.:
- 0 mg/kg bw/day
- Dose / conc.:
- 2.5 mg/kg bw/day
- Dose / conc.:
- 12 mg/kg bw/day
- Dose / conc.:
- 54 mg/kg bw/day
- Dose / conc.:
- 250 mg/kg bw/day
- No. of animals per sex per dose:
- 14-32/inseminated females/dose
10-12/pregnant females/dose - Control animals:
- yes, sham-exposed
- Details on study design:
- - Dose selection rationale: Not provided in report
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
DETAILED CLINICAL OBSERVATIONS: No data
BODY WEIGHT: Yes
- Time schedule for examinations: On Day 0 and GD 6, 12, 18, and 29.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29.
- Organs examined: Urogenital tract of each dam was examined in detail for abnormalities. - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
- Other: Total resorption sites were provided but no differentiation was made between early and late resorptions. - Fetal examinations:
- - External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter - Statistics:
- Not provided in report.
- Historical control data:
- Not provided in report.
- Clinical signs:
- not specified
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- A single doe in the 54.0 mg/kg dose group died on GD 18; a single doe in the 250.0 mg/kg group died on GD 17. No deaths occurred in any other sodium benzoate-treated group or in the negative or positive control groups.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There was no adverse effect on average maternal body weight of surviving does when measured at sacrifice on GD 29. The average body weights were 2.90 (10), 2.47 (10), 2.38 (10), 2.64 (11), and 2.86 (9) kg for animals in the 0, 2.5, 12.0, 54.0 and 250.0 mg/kg sodium benzoate-treated groups, respectively. The number in ( ) indicates the number of surviving does. The average weight for dams in the positive control group was 2.95 (10) kg. Each test and control group contained 9-11 does at sacrifice.
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- not specified
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not specified
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Number of abortions:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no dose-related effects on number of abortions. Two of 12 pregnant does in the 2.5 mg/kg sodium benzoate-treated group aborted on GD 23. There were no other abortions in any of the other sodium benzoate-treated groups or in the negative or positive control groups.
- Pre- and post-implantation loss:
- not specified
- Description (incidence and severity):
- No data were provided on the number of pre- and post-implantation losses. However, in does examined at term, there was no significant dose-related difference from the sham group in the average number of implant sites per doe at dose levels up to 250.0 mg/kg of sodium benzoate or for the positive control group receiving 6-AN.
Dose Average implant sites/doe
0 mg/kg 5.90
2.5 mg/kg 5.40
12.0 mg/kg 5.50
54.0 mg/kg 4.64
250 mg/kg 7.22
6-AN (positive control) 7.10 - Total litter losses by resorption:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There was no dose-related effect on the number of does with all implant sites resorbed for animals dosed with sodium benzoate at dose levels up to 250 mg/kg. Results for dams examined at term are as follows:
Dose group Does with all sites resorbed
0 mg/kg 1
2.5 mg/kg 0
12.0 mg/kg 1
54.0 mg/kg 6
250 mg/kg 1
6-AN (positive control) 3 - Early or late resorptions:
- not specified
- Description (incidence and severity):
- Resorptions were not specified as early or late. There was no significant dose-related difference in the total number of resorptions at dose levels up to 250.0 mg/kg of sodium benzoate. In does examined at term, there were a total of 6 resorptions in the sham (0 mg/kg) group compared to 14, 13, 36, and 12 in the 2.5, 12.0, 54.0 and 250.0 mg/kg groups, respectively. Although there was an unusually high number of total resorptions in the 54.0 mg/kg group, this effect was not observed at 250 mg/kg, a dose five times greater, and is unlikely to be related to test substance administration. There were a total of 39 resorptions in the positive control group.
- Dead fetuses:
- effects observed, non-treatment-related
- Description (incidence and severity):
- All six fetuses in a single doe in the 54.0 mg/kg sodium benzoate dose group died. Based on the small total number of dead fetuses in all other dose groups, the absence of dead fetuses in two higher dose groups, and the limited number of does with 1 or more dead fetuses, the results are unlikely to be related to test substance administration.
Total dead fetuses/ does with 1 or more dead:
0 mg/kg = 1/1
2.5 mg/kg = 1/1
12.0 mg/kg = 6/1
54.0 mg/kg= 0/0
250.0 mg/kg = 0/0
6-AN (positive control) = 2/2 - Changes in pregnancy duration:
- no effects observed
- Description (incidence and severity):
- Except for two does that died and two does that aborted prior to study termination, all litters were carried to term.
- Changes in number of pregnant:
- no effects observed
- Description (incidence and severity):
- The total number of pregnancies was similar across all sodium benzoate-treated groups and the positive control.
0 mg/kg = 10
2.5 mg/kg = 12
12.0 mg/kg = 10
54.0 mg/kg = 12
250.0 mg/kg = 10
6-AN (positive control) = 10 - Other effects:
- not specified
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- > 250 mg/kg bw/day
- Based on:
- test mat.
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Remarks:
- At dose levels up to 250.0 mg/kg sodium benzoate, there were no adverse effects on maternal toxicity or pregnancy in rabbits.
- Key result
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- no effects observed
- Description (incidence and severity):
- There was no significant difference in average fetal weight in the sodium benzoate-treated groups when compared to the sham-treated group. Average fetal weights were 38.0, 35.4, 36.3, 41.9 and 38.6 g in the 0, 2.5, 12.0, 54.0 and 250.0 mg/kg dose groups, respectively. The average fetal weight in the positive control group was slightly lower at 32.4 g.
- Reduction in number of live offspring:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There was no dose-dependent difference in number of live fetuses in the sodium benzoate-treated groups when compared to the sham-treated group. Values for the highest sodium benzoate dose group tested were comparable to those of the sham control. The total number of live fetuses was 52, 39, 36, 15, and 53 in the 0, 2.5, 12.0, 54.0 and 250.0 mg/kg groups, respectively while the average number of live fetuses per doe for does examined at term was 5.20, 3.90, 3.60, 1.36, and 5.89. The total number of live fetuses was 30 and the average number of live fetuses per doe was 3.0 for the positive control group.
- Changes in sex ratio:
- effects observed, non-treatment-related
- Description (incidence and severity):
- When compared to the sham-treated control, there was no dose-dependent effect on M/F sex ratio in the sodium benzoate-treated groups or in the 6-AN positive control. The ratios (M/F) were 0.73, 1.44, 0.89, 0.50 and 0.77 for the 0, 2.5, 12.0, 54.0 and 250.0 mg/kg sodium benzoate-treated groups, respectively and 0.85 for the positive control.
- Changes in litter size and weights:
- not specified
- Description (incidence and severity):
- There was no dose-dependent difference in number of fetuses (live + dead) in the sodium benzoate-treated groups when compared to the sham-treated group. Similar values were observed with the positive control. Litter weights were not reported but the average fetus weight was similar across all sodium benzoate-treated groups.
- Changes in postnatal survival:
- effects observed, non-treatment-related
- Description (incidence and severity):
- When compared to the sham-treated control, there was no dose-dependent effect on postnatal survival in the sodium benzoate-treated groups. The numbers of neonatal deaths were 3, 1, 1, 1, and 3 in the 0, 2.5, 12.0, 54.0, and 250 mg/kg sodium benzoate-treated groups. There was a significant effect on neonatal survival in the 6-AN positive control group. Twenty-one (21) of 30 positive control fetuses died.
- External malformations:
- no effects observed
- Description (incidence and severity):
- No external malformations were observed in the pups of groups treated with up to 250.0 mg/kg sodium benzoate. Four (4) does in the 6-AN treated group had one or more pups with external malformations including anopia, short tail, and club foot.
- Skeletal malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There was no dose-dependent increase in the number of skeletal abnormalities in sodium benzoate-treated groups at dose levels up to 250.0 mg/kg when compared to the number occurring spontaneously in the sham-treated controls. The only skeletal finding in the sham-treated group was extra sternebrae in a total of 2 fetuses in 2 litters. By comparison, there were a total of 3 fetuses similarly affected in 2 litters in the highest sodium benzoate-treated group tested. Findings at intermediate dose levels included incomplete ossification of sternebrae, bipartite sternebrae, or fused or extra sternebrae and were limited to 1 or 2 fetuses in 1 or 2 litters. A single fetus in the 12.0 mg/kg dose group had craniostosis. By comparison, there were a significant number of skeletal abnormalities in the seven 6-AN litters examined. These included tail defects in all 26 fetuses examined as well as sternebrae, rib, and vertebrae findings in 20-40 % of the fetuses and 2 instances of craniostosis in one litter.
- Visceral malformations:
- no effects observed
- Description (incidence and severity):
- No visceral malformations were observed in the pups of groups treated with up to 250.0 mg/kg sodium benzoate. Four (4) does in the 6-AN treated group had one or more pups with visceral malformations including cleft palate.
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- > 250 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Remarks:
- At dose levels up to 250.0 mg/kg sodium benzoate, there were no adverse effects on fetal parameters in rabbits examined in this study.
- Key result
- Abnormalities:
- no effects observed
- Description (incidence and severity):
- There were no dose-dependent external, skeletal or visceral abnormalities observed in offspring of does receiving up to 250 mg/kg sodium benzoate on GD 6-18.
- Key result
- Developmental effects observed:
- no
- Lowest effective dose / conc.:
- 250 mg/kg bw/day
- Treatment related:
- no
- Conclusions:
- The administration of up to 250.0 mg/kg (body weight) of sodium benzoate to pregnant rabbits for 13 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in offspring in the sham-treated controls.
- Executive summary:
In a developmental toxicity study, pregnant Dutch-belted female rabbits were administered 0, 2.5, 12.0, 54.0, or 250.0 mg/kg sodium benzoate by oral gavage once daily on gestation days (GD) 6 through 18 while positive control animals received a single 2.5 mg/kg dose of 6-aminonicotinamide on GD 9. Maternal body weights were recorded on GD 0, 6, 12, 18 and 29. All animals were observed daily for appearance and behavior with particular attention to food consumption and weight. On GD 29, all surviving does were subjected to Caesarean section under anesthesia, and the numbers of corpora lutea, implantation sites, resorptions sites, and live and dead fetuses were recorded. Body weights of live pups were also recorded. The urogenital tract of each doe was examined in detail. All fetuses underwent a detailed gross examination for the presence of external congenital abnormalities. To evaluate neonatal survival, the live fetuses of each litter were placed in an incubator for 24 hours. All surviving pups were then sacrificed and examined for visceral and skeletal abnormalities. Under conditions of this study, no dose-related adverse effects were observed in the does or fetuses in any of the sodium benzoate-treated groups. A decrease in neonatal survival as well as an increase in the numbers of skeletal defects was observed in litters of does exposed to 2.5 mg/kg 6-aminonicotinamide on GD 9.
Referenceopen allclose all
Of the remaining dams that were allowed to litter, there were 5, 5, 4, 4, and 5 dams in the control, 1, 2, 4, and 8% groups, respectively. The rate of perinatal death was 100% in the 4 and 8% groups while there were no deaths in the other dose groups. The delivery rate, lactation rate and survival at 3 and 8 weeks were unaffected in the 1 and 2% groups. Body weights of the offspring at birth, three weeks and eight weeks of age in the 1 and 2% groups were similar to control group values. There were no significant differences in average organ weights of 8-week-old male or female offspring rats in control, 1% and 2% groups. The incidence of pathological findings (primarily cervical and lumbar ribs) in the litters at three and eight weeks (combined) were similar in the control, 1% and 2% groups.
Table 2 of Study Report, "Reproduction Data":
|
Sham |
+ Control |
1.75 mg/kg bw |
8.0 mg/kg bw |
38.0 mg/kg bw |
175.0 mg/kg bw |
|
Endpoint |
|
|
|
|
|
|
|
Pregnancies |
|
|
|
|
|
|
|
|
Total No. |
23 |
24 |
24 |
23 |
23 |
23 |
|
Died or Aborted (before Day 20) |
0 |
0 |
0 |
0 |
0 |
0 |
|
To Term (on Day 20) |
23 |
24 |
25 |
23 |
23 |
23 |
Corpora Lutea |
|
|
|
|
|
|
|
|
Total No. |
276 |
271 |
304 |
285 |
288 |
269 |
|
Average/dam mated |
11.5 |
11.3 |
12.7 |
11.9 |
12.0 |
11.2 |
Live Litters |
|
|
|
|
|
|
|
|
Total No. |
23 |
18 |
24 |
23 |
22 |
23 |
Implant Sites |
|
|
|
|
|
|
|
|
Total No. |
249 |
263 |
300 |
238 |
268 |
252 |
|
Average/dam |
10.8 |
11.0 |
12.5 |
10.3 |
11.7 |
11.0 |
Resorptions |
|
|
|
|
|
|
|
|
Total No. |
6 |
102 |
6 |
10 |
5 |
20 |
|
Dams with 1 or more sites resorbed |
4 |
19 |
4 |
8 |
5 |
7 |
|
Dams with all sites resorbed |
-- |
6 |
-- |
-- |
1 |
-- |
|
Per cent partial resorptions |
17.4 |
79.2 |
16.7 |
34.8 |
21.7 |
30.4 |
|
Per cent complete resorptions |
-- |
25.0 |
-- |
-- |
4.35 |
-- |
Live fetuses |
|
|
|
|
|
|
|
|
Total No. |
243 |
160 |
294 |
228 |
263 |
232 |
|
Average/dam |
10.6 |
6.67 |
12.3 |
9.91 |
11.4 |
10.1 |
|
Sex ratio (M/F) |
1.15 |
1.03 |
1.10 |
0.81 |
0.80 |
1.00 |
Dead Fetuses |
|
|
|
|
|
|
|
|
Total No. |
-- |
1 |
-- |
-- |
-- |
-- |
|
Dams with 1 or more dead |
-- |
1 |
-- |
-- |
-- |
-- |
|
Dams with all dead |
-- |
-- |
-- |
-- |
-- |
-- |
|
Per cent partial dead |
-- |
4.16 |
-- |
-- |
-- |
-- |
|
Per cent all dead |
-- |
-- |
-- |
-- |
-- |
-- |
Average Fetus Weight (g) |
|
3.67 |
2.15 |
3.70 |
3.70 |
3.89 |
3.81 |
Table 2 of Study Report, "Reproduction Data":
|
Sham |
+ Control |
2.5 mg/kg bw |
12.0 mg/kg bw |
54.0 mg/kg bw |
250.0 mg/kg bw |
|
Endpoint |
|
|
|
|
|
|
|
Pregnancies |
|
|
|
|
|
|
|
|
Total No. |
10 |
10 |
12 |
10 |
12 |
10 |
|
Died or Aborted (before Day 29) |
1 |
0 |
4 |
0 |
2 |
2 |
|
To Term (on Day 29) |
10 |
10 |
10 |
10 |
11 |
9 |
Corpora Lutea |
|
|
|
|
|
|
|
|
Total No. |
170 |
185 |
302 |
204 |
217 |
207 |
|
Average/dam mated |
10.0 |
12.3 |
9.44 |
9.71 |
9.86 |
14.8 |
Live Litters |
|
|
|
|
|
|
|
|
Total No. |
9 |
7 |
10 |
8 |
5 |
8 |
Implant Sites |
|
|
|
|
|
|
|
|
Total No. |
59 |
71 |
54 |
55 |
51 |
65 |
|
Average/dam |
5.90 |
7.10 |
5.40 |
5.50 |
4.64 |
7.22 |
Resorptions |
|
|
|
|
|
|
|
|
Total No. |
6 |
39 |
14 |
13 |
36 |
12 |
|
Dams with 1 or more sites resorbed |
3 |
9 |
6 |
4 |
11 |
5 |
|
Dams with all sites resorbed |
1 |
3 |
-- |
1 |
6 |
1 |
|
Per cent partial resorptions |
30.0 |
90.0 |
60.0 |
40.0 |
100.0 |
55.6 |
|
Per cent complete resorptions |
10.0 |
30.0 |
-- |
10.0 |
54.6 |
11.1 |
Live fetuses |
|
|
|
|
|
|
|
|
Total No. |
52 |
30 |
39 |
36 |
15 |
53 |
|
Average/dam |
5.20 |
3.00 |
3.90 |
3.60 |
1.36 |
5.89 |
|
Sex ratio (M/F) |
0.73 |
0.85 |
1.44 |
0.89 |
0.50 |
0.77 |
Dead Fetuses |
|
|
|
|
|
|
|
|
Total No. |
1 |
2 |
1 |
6 |
-- |
-- |
|
Dams with 1 or more dead |
1 |
2 |
1 |
1 |
-- |
-- |
|
Dams with all dead |
-- |
-- |
-- |
-- |
-- |
-- |
|
Per cent partial dead |
10.0 |
20.0 |
10.0 |
10.0 |
-- |
-- |
|
Per cent all dead |
-- |
-- |
-- |
10.0 |
-- |
-- |
Average Fetus Weight (g) |
|
38.0 |
32.4 |
35.4 |
36.3 |
41.9 |
38.6 |
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 175 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- 2 (reliable with restrictions)
Additional information
The Onodera, et al., (1978) publication describes a developmental toxicity study in rats conducted by the National Institute of Hygiene and Science in Japan. The study design closely follows the OECD 414 test guideline with additional postnatal investigations for developmental endpoints. Groups of 27-30 pregnant Wistar rats were fed CE-2 diets containing 0, 1, 2, 4 or 8% sodium benzoate in the diet from gestation day (GD) 0 through 20. Feed consumption measures were collected daily and body weight measurements were collected every five days. On GD 20, five dams from each group were selected to continue exposure throughout delivery and lactation while the remaining dams were necropsied for evaluation of maternal and fetal toxicity endpoints. Full necropsies were conducted on all dams included within this study. The five dams selected for the postnatal portion of the study were necropsied on postnatal day 21 along with one-half of the surviving pups. The remaining pups were necropsied at eight weeks of age. On GD 20, three-fourths of the fetuses were processed for skeletal examination using alizarin Red S staining and one-quarter of the fetuses were processed for fetal examination according to Wilson’s method. The weanling pups and the eight-week old pups were necropsied for gross pathology (including weights of eight organs) and examined for external, visceral and skeletal malformations and variations.
Feed consumption values in the 4 and 8% dose groups were decreased 58 and 87% when compared to the control group and resulted in body weight losses in both of these treatment groups during the entire gestation period. The 4% dose group body weight values were able to recover to the original starting body weights by GD 20 but the 8% group body weights were 25% lower on GD 20 when compared to the GD 0 body weight values. Feed consumption values basically mirrored the body weight measures and both of these changes were considered by the study authors to reflect a palatability issue with the test diets rather than a consequence of the toxicity of sodium benzoate. This conclusion is reinforced by the delivered dose calculations with dose levels of 0, 699, 1306, 1874 and 965 mg/kg/day for the 0, 1, 2, 4 and 8% diet groups, respectively. The most significant effects were observed in the 8% group, despite having a delivered dose level approximately one-half that of the 4% dose group.
Adverse clinical signs of toxicity (including lethality) were observed in the 4 and 8% groups. Two dams died during gestation in the 4% group and three dams died during this period in the 8% group. In addition, tonic spasms were observed in these groups but a cause of death could not be determined. Of the dams that died, only one was pregnant (from the 4% group). There were no significant differences in body weights and feed consumption between the control group and the dams in the 1 and 2% groups. No adverse clinical signs of toxicity were noted in the control, 1% or 2% groups. The number of pregnant rats at necropsy on GD 20 was 15, 15, 16, 18 or 12 dams in the 0, 1, 2, 4 and 8% dietary groups, respectively. The number of pregnant dams that delivered in the 0, 1, 2, 4 and 8% dietary groups was 5, 5, 4, 4 and 5 dams, respectively. The remaining dams in each treatment group were found non-pregnant at necropsy and data were not included within the calculations.
In terms of fetal endpoints, an increase in number of dead or resorbed fetuses (3 to 5-fold) and a decrease in fetal body weights (29-43%) were observed in the 4 and 8% groups. Total number of implantations and sex ratios were unaffected. No differences in number of implantations, sex ratios, dead or resorbed fetuses and fetal body weights were observed in the 1 and 2% groups. Gross fetal pathology was limited to mild systemic edema in a few pups in the 4 and 8% groups. For fetal visceral and skeletal exams, no significant differences from the control group were noted for the 1 and 2% groups when evaluated using the Wilcoxon Rank Sum test. In the 4% group, the percentage of fetuses with visceral and skeletal abnormalities was 33% and 97%, respectively. In the 8% group, the percentage of fetuses with visceral and skeletal abnormalities was 42% and 100%, respectively. The study authors attributed the increase in malformation and variations in the 4 and 8% groups to the severe reduction in feed consumption and resulting body weight losses in these groups.
Of the remaining dams that were allowed to litter, there were 5, 5, 4, 4, and 5 dams in the control, 1, 2, 4 and 8% groups, respectively. The
rate of perinatal death was 100% in the 4 and 8% groups while the control, 1 and 2% groups had 0% perinatal deaths. The delivery rate, lactation rate and survival at eight weeks were unaffected in the 1 and 2% groups. Body weights of the offspring at birth, three weeks and eight weeks of age in the 1 and 2% groups were similar to control group values. The incidence of pathological findings (primarily cervical and lumbar ribs) in the litters at three and eight weeks (combined) were equal in the control, 1% and 2% groups.
Comparison of the study design used by Onodera, et al., (1978) with the OECD 414 study guideline yields some interesting differences. The
dose levels selected in the top two dose levels greatly exceeded the maximally tolerated dose level with severe consequences due to marked decreases in feed consumption, presumably due to palatability. The 2% dietary level did not affect feed consumption or body weight gain and yielded a dose level of 1306 mg/kg/day, exceeding the limit dose of 1000 mg/kg/day for OECD 414 studies. The inclusion of one dose level below the 2% dietary level should not be problematic as the 2% dietary level provides a NOEL for maternal and developmental toxicity in this study. The exposure period used in this study (throughout the entire period of gestation and into the postnatal and post-weaning period) exceeds the exposure period required in the OECD 414 study guideline. Use of group sizes of 27-30 dams/group also exceeds the OECD Guideline requirement of twenty dams/group, although the presence of nonpregnant animals on GD 20 resulted in group sizes of 15, 15 and 16 dams in the control, 1% and 2% groups, respectively, at necropsy. However, this study also included a postnatal component with collection of visceral and skeletal endpoints in the offspring at weaning. The OECD Guideline requirement that twenty litters/group be evaluated for developmental endpoints is accomplished by addition of all of the dams (GD 20 and postweaning necropsies). In addition, the inclusion of the postnatal component of this study provides useful information on the ability of the pups to survive and grow after birth until weaning on postnatal day 21 or eight weeks of age. Since external, visceral and skeletal exams that were similar to those conducted on GD 20 fetuses were included in the postnatal day 21 and eight week pup necropsies, the developmental toxicity data from all twenty litters/group are available for evaluation and analysis.
A variance from the current OECD 414 Guideline was the assignment of three-quarters of the viable fetuses for skeletal examination and
one-quarter for visceral examination on GD 20. While this was a common feature of developmental toxicity study designs at the time, the current OECD 414 study design assigns at least one-half of the fetuses to the visceral exam and the remaining one-half to skeletal examination. The concern for the presence of this variance from the Guideline is partially alleviated by the collection of external, visceral and skeletal endpoints for all of the pups that were necropsied on postnatal day 21 and at eight weeks of age (approximately 44-45 pups/group). Since the majority of the variations and malformations observed in the fetuses and pups from the control, 1% and 2% groups were skeletal findings (the only visceral findings were either visible on external exams (bilateral anopthalmia in one fetus) or represent a very common finding in rats (unilateral pyelectasis – dilatation of the renal pelvis)). Furthermore, the skeletal findings observed in the three and eight week pups were similar to those found in the GD 20 fetuses (albeit at a lower incidence as many of the skeletal findings resolve with postnatal skeletal remodeling). The primary findings (the presence of cervical or lumbar ribs) were considered variations and were present in the control group fetuses and pups in a similar incidence as those in the 1% and 2% groups. All of the above facts regarding the study design of the Onodera, et al., 1985 publication allows for the conclusion that the OECD Guideline 414 rat study requirement has been satisfied for sodium benzoate.
The lack of developmental toxicity finding in rats is supported by the results from developmental toxicity studies with sodium benzoate in rats,
hamsters and mice (FDA, 1972). This series of experiments were conducted in support of sodium benzoate as a food preservative and therefore have a different study design that the current OECD 414 Guideline. The three significant differences between the FDA study design and the current OECD 414 study design are 1) selection of dose levels, 2) exposure period during gestation and 3) assignment of fetuses to visceral and skeletal exams. Since the study was designed for evaluation of sodium benzoate for use as a food additive, it is clear that the dose level selection was based upon the concept of “margin-of-exposure” (multiples of known human exposure from consumption of food containing the additive) rather than based on toxicity endpoints. Therefore, the top dose level (and study NOAEL’s) were 175, 175 or 300 mg/kg/day for rats, mouse and hamsters, respectively. As was common for developmental toxicity studies of this era, the period of exposure during gestation was limited to the embryonic period rather than the embryonic and fetal period as currently mandated in the OECD 414 Guideline. The assignment of fetuses for skeletal versus visceral examinations were two-thirds for skeletal exam and one-third for visceral exam in the rat, mouse and hamsters studies. Group sizes were 22-24 pregnant animals/group for the rat, mouse and hamster studies.
Evaluation of the four rodent developmental toxicity studies described above resulted in NOAEL’s for maternal and developmental toxicity in
the rat of 1306 mg/kg/day, of 175 mg/kg/day for mice and 300 mg/kg/day for hamsters. Based on the results of these studies, sodium benzoate is not considered a developmental toxicant in rodents.
COMPARISON OF THE FDA RABBIT STUDY WITH THE CURRENT OECD 414 GUIDELINE:
The lack of a formal OECD 414 Guideline developmental toxicity study in rabbits is understandable, given the past testing in support of the historical use of sodium benzoate as a food preservative. The three significant differences between the rabbit FDA study design and the current OECD 414 study design are 1) selection of dose levels, 2) exposure period during gestation and 3) group size requirements. Since the study was designed for evaluation of sodium benzoate for use as a food additive, it is clear that the dose level selection was based upon the concept of “margin-of-exposure” (multiples of known human exposure from consumption of food containing the additive) rather than based on toxicity endpoints. Therefore, the top dose level (and study NOAEL’s) was 250 mg/kg/day for rabbits. As was common for developmental toxicity studies of this era, the period of exposure during gestation was limited to the embryonic period rather than the embryonic and fetal period as currently mandated in the OECD 414 Guideline. All of the rabbit fetuses were examined for visceral and skeletal examinations. Group sizes were 10-12 dams/group in this study, as was common for rabbit developmental toxicity studies of this time period.
When evaluating the need for the conduct of another rabbit developmental toxicity study to satisfy the REACH requirements for sodium benzoate, it is important to understand that sodium benzoate has been demonstrated to not be a developmental toxicant in four different species, including rabbits. Furthermore, the probability that a rabbit developmental toxicity study conducted according to the current OECD 414 Guidelines would change the current status of sodium benzoate as a developmental toxicant appears to be rather remote based upon the totality of the current evidence for this endpoint. That is, in spite of none of the studies being conducted strictly to the current OECD 414 guideline, the weight of evidence from a number of studies, in multiple species, there is no indication of developmental toxicity and/or teratogenicity.
Justification for classification or non-classification
Based on the outcome of the studies the substance does not need to be classified for reproductive and/or developmental effects.
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