Calcium dibenzoate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not specified

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Abstract in English language only. Study summary available only.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Benzoic acid and sodium benzoate

Author:

WHO

Year:

2000

Bibliographic source:

Concise International Chemical Assessment. Document No. 26. WHO, Geneva

Reference 2

Reference Type:

publication

Title:

Provisional peer reviewed toxicity values for benzoic acid (CASRN 65-85-0)

Author:

U.S. EPA

Year:

2005

Bibliographic source:

EPA/690/R-05/008F

Reference 3

Reference Type:

publication

Title:

SIDS Initial Assessment Report for benzoates Cas N°: 65-85-0, 532-25-2, 100-51-6

Author:

OECD SIDS

Year:

2001

Bibliographic source:

OECD SIDS, UNEP Publications

Reference 4

Reference Type:

publication

Title:

Scientific opinion on the re-evaluation of benzoic acid (E 210), sodium benzoate (E 211), potassium benzoate (E 212) and calcium benzoate (E 213) as food additives

Author:

EFSA

Year:

2016

Bibliographic source:

EFSA Journal 14(3) : 4433.

Reference 5

Reference Type:

publication

Title:

Studies on effects of sodium benzoate on fetuses and offspring of Witar rats

Author:

Onodera, H. et al.

Year:

1978

Bibliographic source:

Eisei Shikensho Hokoku. 96: 47 - 55.

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

In the current study, sodium benzoate was administered via diet to groups of pregnant female Wistar rats (n = 27 – 30) at concentrations of 1, 2, 4 and 8 % (equivalent to 700, 1310, 1875 and 965 mg/kg bw/day based on total food intake). The rats were dosed daily from gestation day 1 up to gestation day 20. A control group receiving plain diet was run concurrently. Dams were sacrificed on gestation day 20 (n = 22- 25/rats/group) and foetuses were examined. Some dams (5 rats/group) were allowed to littered and the pups were investigating up to 8 weeks of age.

GLP compliance:

not specified

Remarks:

not specified in the publication

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium benzoate

EC Number:

208-534-8

EC Name:

Sodium benzoate

Cas Number:

532-32-1

Molecular formula:

C7H6O2.Na

IUPAC Name:

sodium benzoate

Test material form:

not specified

Details on test material:

not specified

Specific details on test material used for the study:

not specified

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CE-2 (manufactured by Nippon Kure)
- Age (at purchase): males: 8 -9 weeks old; females: 7 - 10 weeks old
- Weight (on gestation day 0): females: approx. 265 g
- Diet: CE-2
- Water: tap water
- Acclimation period: approx. 4 weeks

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
The treatment groups were fed diet containing the test item for the entire maternal period.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused all night
- M/F ratio per cage: 1 male / 1 female
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

gestation days 1 to 20

Frequency of treatment:

daily

Duration of test:

20 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 other: %

Remarks:

equivalent to 700 mg/kg bw/day (based on total food intake)

Doses / concentrations 2

Dose / conc.:

2 other: %

Remarks:

equivalent to 1310 mg/kg bw/day (based on total food intake)

Doses / concentrations 3

Dose / conc.:

4 other: %

Remarks:

equivalent to 1875 mg/kg bw/day (based on total food intake)

Doses / concentrations 4

Dose / conc.:

8 other: %

Remarks:

equivalent to 965 mg/kg bw/day (based on total food intake)

No. of animals per sex per dose:

27 - 30 pregnant rats

Control animals:

yes, plain diet

Details on study design:

not specified

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked: mortality and clinical signs

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: feed intake was measured daily

WATER CONSUMPTION : No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
22- 25 pregnant females were sacrifice using anaesthesia and the abdomen was open immediately. Abnormalities of maternal organs were recorded.

- Sacrifice 3 weeks after birth
The remaining 5 pregnant rats were allowed to litter and they were subjected to necropsy three weeks after birth.

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: Yes
- Number of late resorptions: Yes
- Placental and ovarian weights

Blood sampling:

not specified

Fetal examinations:

NOTE: besides investigating foetuses at sacrifice on gestation day 20, the authors also let some dams delivered naturally and investigated the pups until 8 weeks of age.

1) Foetuses
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, about 25 % of the foetuses from treated animals were fixed with Bouin's solution and examined for visceral anomalies by Wilson's method.
- Skeletal examinations: Yes, about 75 % of the foetuses from treated animals were staine with alizarin red S for skeletal examination.
- Number of viable foetuses and dead foetuses were recorded
- Foetal weight was recorded.
- Sex ratio was determined

2) Pups
Five pregnant rats were allowed to litter and the following parameters were recorded at birth:
- number of pups
- survival rate and body weight of the pups
- presence and absence of external abnormalities

Three weeks after birth, all surviving pups were weaned and examined for gross abnormalities, and one-half of the pups were necropsied. The remaining pups were necropsied at 8 weeks of age, body weight and food intake being measured weekly until necropsy. In addition, the survival and lactation rate were determined. Lastly, the organ weights were recorded at 8 weeks of age.

Statistics:

mean and standard deviations

Indices:

not specified

Historical control data:

not specified

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

- 4 and 8 %: two dams at 4 % and three dams at 8 % died after convulsions and depressed motor activity.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 4 and 8 %: animals at the 4 % level did not gain weight and those at 8 % lost weight (statistical comparisons not presented)

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

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- 4 and 8 %: feed intake was markedly reduced in the dams.

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

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

BODY WEIGHT AND WEIGHT GAIN
- 1 and 2 %: maternal body weight and body weight gain were comparable in the controls and in animals receiving 1 and 2 % sodium benzoate in the diet.

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

FOOD CONSUMPTION
- 1 and 2 %: feed intake was comparable in the controls and animals at 1% and 2% dietary levels.

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

Maternal developmental toxicity

Number of abortions:

not specified

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

not specified

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

- 4 and 8 %: number dead or resorbed foetuses was significantly increased compared to the controls.

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

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

no effects observed

Details on maternal toxic effects:

DEAD FOETESUS
- 1 and 2 %: number of dead/resorbed or viable foetuses was not different compared to the control.

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

IMPLANTATION
- 1 %, 2 %, 4 % and 8 %: dams receiving sodium benzoate showed no differences compared wit controls in the average number of implants per female.

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

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

1 310 mg/kg bw/day

Based on:

test mat.

Remarks on result:

other: see "Remarks"

Remarks:

Mortality was observed in maternal animals at the 4 and 8 % concentration level. Two dams at 4 % and three dams at 8 % concentration level died after convulsions and depressed motor activity. Furthermore, maternal animals at the 4 % level did not gain weight and those at 8 % lost weight (statistical comparisons not presented) and feed intake was markedly reduced in the dams at both concentration levels. Furthermore, the number of dead or resorbed foetuses was significantly increased compared to the controls at the 4 and 8 % concentration levels.

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 4 and 8 %: average body weight of viable foetuses was significantly lower than that of controls.

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

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

- 4 and 8 %: among the pups that were delivered naturally, the groups at 4 % and 8 % were reported to have delivery rates reduced by 50 % and 8.2 %, respectively.

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

Changes in sex ratio:

not specified

Anogenital distance of all rodent fetuses:

not specified

Changes in postnatal survival:

effects observed, treatment-related

Description (incidence and severity):

- 4 and 8 %: among the pups that were delivered naturally, a complete loss of litters was observed after parturition.

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

External malformations:

effects observed, treatment-related

Description (incidence and severity):

- 4 and 8 %: significant abnormalities finding (mild systemic oedema) were observed in the foetuses.

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

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

- 4 and 8 %: delayed ossification, lumbar or cervical ribs, and varied sternebrae were reported in foetuses in both control and treated groups. The percentage of animals with these findings was not comparable to that in controls among animals at 4 % and 8 %. The percentage of animals was increased in the groups at 4 % (96.5 %) and 8 % (100 %). Additional anomalies seen in the treated groups included a higher incidence of wavy ribs and abnormal vertebrae in the rats at 4 %, but not at 8 %.

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

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

- 4 and 8 %: significant abnormalities and pathological findings (anophthalmia, microphthalmia, hydrocephalus, pyelectasis, renal hydroplasia and cerebral hypoplasia) were observed in the foetuses.

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

Details on embryotoxic / teratogenic effects:

NOTE: besides investigating foetuses at sacrifice on gestation day 20, the authors also let some dams delivered naturally and investigated the pups until 8 weeks of age.

1) FOETAL EXAMINATION

FOETAL BODY WEIGHT CHANGES
- 1 and 2 %: average weight of viable foetuses showed no differences compared with controls.

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

EXTERNAL MALFORMATIONS
- 1 and 2 %: no significant abnormalities and pathological findings were seen in the foetuses.

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

- 1 and 2 %: delayed ossification, lumbar or cervical ribs, and varied sternebrae were reported in foetuses in both control and treated groups. The percentage of animals with these findings was comparable to that in controls among animals at 1 % and 2 % (each about 37 %).

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

2) PUP EXAMINATION

REDUCTION IN LIVE OFFSPRING
- 1 and 2 %: among the pups that were delivered naturally, no difference in the delivery rate, number of perinatal deaths, and lactation rate up to week 8 were reported at the 1 % and 2 % dietary levels.

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

CHANGES IN POSTNATAL SURIVIVAL
- 1 and 2 %: among the pups that were delivered naturally, no difference in the survival up to week 8 were reported at the 1 % and 2 % dietary levels.

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

CHANGES IN LITTER SIZE AND WEIGHTS
- 1 and 2 %: the surviving pups in the control group and at 1 % and 2 % showed no significant differences in birth weight and weight at week 3 or week 8.

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

GROSS PATHOLOGY
- 1 and 2 %: the surviving pups in the control group and at 1 % and 2 % showed no significant differences in incidence of abnormalities at week 3 or 8.

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

ORGAN WEIGHTS
- 1 and 2 %: the surviving pups in the control group and at 1 % and 2 % showed no significant differences in organ weights at week 8.

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

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

1 310 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: see "Remarks"

Remarks:

The average body weight of viable foetuses was significantly lower than that of controls and significant abnormalities finding (mild systemic oedema) were observed in the foetuses at the 4 and 8 % concentration levels. Also, delayed ossification, lumbar or cervical ribs, and varied sternebrae were reported in foetuses in both control and treated groups (4 and 8 %). The percentage of animals with these findings was not comparable to that in controls among animals at 4 % and 8 %. The percentage of animals was increased in the groups at 4 % (96.5 %) and 8 % (100 %). Additional anomalies seen in the treated groups included a higher incidence of wavy ribs and abnormal vertebrae in the rats at 4 %, but not at 8 %. Significant abnormalities and pathological findings (anophthalmia, microphthalmia, hydrocephalus, pyelectasis, renal hydroplasia and cerebral hypoplasia) were observed in the foetuses at the two highest concentration levels. Furthermore, among pups that were delivered naturally, groups at 4 % and 8 % were reported to have delivery rates reduced by 50% and 8.2%, respectively, with complete loss of litters after parturition.

Applicant's summary and conclusion

Conclusions:

In the current study, sodium benzoate was administered via diet to groups of pregnant female Wistar rats (n = 27 – 30) at concentrations of 1, 2, 4 and 8 % (equivalent to 700, 1310, 1875 and 965 mg/kg bw/day based on total food intake). The rats were dosed daily from gestation day 1 up to gestation day 20. A control group receiving plain diet was run concurrently. Dams were sacrificed on gestation day 20 (n = 22- 25/rats/group) and foetuses were examined. Some dams (5 rats/group) were allowed to littered and the pups were investigating up to 8 weeks of age.

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

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

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

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

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

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