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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Remarks:
Studies on Effects of Sodium Benzoate on Fetuses and Offspring of Wistar Rats
Type of information:
other: read-across based on grouping of substances (category approach): Onodera et al. is provided as a supplement to the Morgareidge et al. (1972) studies to also address potential limitations regarding dose levels in Morgareidge.
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
Justification for type of information:
Test material used was Sodium Benzoate, which is rapidly metabolized to benzoic acid. Please see Toxicokinetic section for details.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1978

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
No data given.
Specific details on test material used for the study:
Source of test material and purity not specified, assumed to be > 99.0% pure.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals 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.

Administration / exposure

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.
Doses / concentrationsopen allclose all
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.

Examinations

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: NoPOST-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: YesExaminations 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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

open allclose all
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:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
early or late resorptions
dead fetuses
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:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
early or late resorptions
dead fetuses
Remarks on result:
other: Effect level given in %, which is not a choice in drop down.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

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.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified
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.

Effect levels (fetuses)

open allclose all
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.

Fetal abnormalities

open allclose all
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.

Overall developmental toxicity

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.