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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Remarks:
Teratologic Evaluation of Sodium Benzoate in Rats
Type of information:
other: read-across based on grouping of substances (category approach)
Remarks:
Sodium benzoate is rapidly metabolized to benzoic acid, therefore the primary exposure in this study was to the substance of record.
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1972

Materials and methods

Test guideline
Qualifier:
no guideline available
Version / remarks:
Predates published regulatory guidelines; used internal guidelines of test facility.
Principles of method if other than guideline:
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

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
- 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.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals 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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): All pregnancies were carried to planned termination on GD 20.
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

Effect levels (maternal animals)

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

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

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

Effect levels (fetuses)

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

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
175 mg/kg bw/day
Treatment related:
no
Relation to maternal toxicity:
not specified
Dose response relationship:
no
Relevant for humans:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.