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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a key, K1 combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, the test substance was administered daily to rats at dose levels up to 1000 mg/kg bodyweight/day (OECD 422; Martell A., 2013). The parental and embryofetal NOAEL were established as 1000 mg/kg body weight/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2011-07-20 to 2012-03-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD 422, Section 4: Health Effects, 1996
Deviations:
yes
Remarks:
No chemical analysis of the test solutions prepared was carried out
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3650.2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The study followed the SOP-M 285 and accomplished in agreement with the OECD 422, 1996, Section 4, and OPPTS 870.3650.2000.
Limit test:
no
Specific details on test material used for the study:
- Source and lot/batch No.of test material:
8684-90
- Storage of the test item: the samples were stored in a specific room, at room temperature, protected from humidity and light
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Currently, no acceptable non-animal alternative methodology is accepted by the relevant regulatory agencies. The rat was selected because this species has proven to be sensitive to chemicals; the rat is the preferred rodent species for prenatal developmental toxicity studies because of its small size, short gestation period, high fertility rate, large litter size and ease of maintenance. The Wistar Hannover rat was selected due to the large amount of background knowledge accumulated on this strain.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BIOAGRI Laboratorios
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11 - 12 weeks old
- Weight at study initiation: Males 352.3±20.7 - 359.5±19.9 grams; Females 216.2± 8.6 - 220.4 ± 9.6 grams
- Fasting period before study: no data
- Housing: Each animal was housed individually, except during cohabitation. After acclimation one male was placed into each female cage for pairing. After pairing, females that presented vaginal smears with the presence of sperm were considered mated and housed individually. The rats were housed in polypropylene cages (41x34x19 cm) with wire mesh tops and bedding material (wood shavings). Clean cages were provided twice weekly for all animals. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
During gestation period all females were housed individually in the cages. Females without copulation date were housed in individual cages at the end of the mating period. Females showing no evidence of copulation were euthanized 24-26 days after the last day of mating period.
- Diet (e.g. ad libitum): ad libitum, Nuvilab CR-1 diet for rats supplied by Nuvital Nutrientes Ltda.
- Water (e.g. ad libitum): ad libitum, supplied by CAESB (Compania de Saneamento Ambiental do Distrito Federal) in water bottles
- Acclimation period: 5 days prior to dosing


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 - 24.4° C
- Humidity (%): 40.8 - 70.0%
- Air changes (per hr): 10-20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- For each dosage group, the appropriate amount of JEFFSOL® AG 1700 was weighed into a precalibrated beaker. The vehicle (corn oil) was added in sufficient quantity to achieve the desired concentration. Each solution was homogenized and dispensed into individual containers properly identified. A sufficient quantity of the vehicle was similarly dispensed for administration to control animals.
- the prepared dolutions were stored at room temperature.
- Test solutions were prepared daily at the testing facility and were administered within 2 hours after preparation. The test solutions were stirred continuously during the administration to maintain homogeneity.

- VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Amount of vehicle (if gavage): 1 ml/250g body weight.
Details on mating procedure:
- M/F ratio per cage: 1 female: 1 male
- Length of cohabitation: until evidence of copulation is observed or 2 weeks had elapsed
- Proof of pregnancy: . Vaginal smears were collected daily during mating period and examined for the presence of sperm.
Day O of gestation was defined as the day a sperm was found in the vaginal smear.
Analytical verification of doses or concentrations:
no
Frequency of treatment:
daily administration, on a 7 day per week basis
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1, vehicle only
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Group 2, the expected dose which causes no signs of toxicity
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Group 3, the intermediate dose level
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4, the expected dose which causes signs of systemic toxicity, but not death or severe suffering
No. of animals per sex per dose:
12 animals/sex/group
5 animals/sex/satllite group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosage levels (in mg/kg body weight/day) were selected in agreement with the Sponsor, based upon the results of a dose range-finding study (P.T. 3984.329.003.11).
- Rationale for animal assignment (if not random): randomized
Positive control:
No
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
- All animals underwent a daily clinical observation for overt signs of ill health. These included, but were not limited to, changes in skin and fur, eye and mucous membranes, respiratory, circulatory, autonomic and central nervous system, motor activity and behavioral patterns.


BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed on the first day of dosing and weekly thereafter (including mating and post-mating periods). Females were weighed on first day of dosing and once a week during premating and mating periods, on days 0, 7, 14 and 20 of gestation, and during lactation on the same days as the weighing of litters (on days 0 and 4 postnatal).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption of females was determined on the same day of body weight determination during premating and lactation periods. During the gestation period, food consumption was determined on days 3, 6, 9, 12, 15, 18 and 20. After the mating period, food consumption of males was determined weekly. Food consumption was not determined during the mating period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes, animals were anesthetized by CO2 prior to blood collection (cardiac puncture).
- Animals fasted: Yes , overnight
- How many animals: 5 parental animals/sex/group, randomly selected
- Parameters examined: Red Blood Cell Count (RBC), Hemoglobin (HB), Hematocrit (HCT), Platelets (PLT), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Total White Blood Cell Count (WBC), Differential Leukocyte Count, Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy.
- Animals fasted: Yes, overnight
- How many animals: 5 parental animals/sex/group, randomly selected
- Parameters examined: Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), Alkaline Phosphatase (ALP), Total Protein (TP), Albumin (ALB), Glucose (GLU), Total Cholesterol (CHOL), Urea Nitrogen (BUN), Creatinine (CREA), Sodium (Na), Potassium (K), Calcium (Ca), Globulin (GLOB), Albumin/Globulin Ratio (A/G)

FUNCTIONAL OBSERVATIONS: yes
Sensory reactivity to stimuli and motor activity assessment were performed in 5 animals/sex/group. For males these evaluations were performed at the end of the dosing period before scheduled necropsy, and for females, these evaluations were performed during the lactation period (day 3). The following parameters were assessed:
A - Autonomic Functions: lacrimation, salivation, palpebral closure, prominence of the eye, piloerection and respiration.
B - Reactivity and Sensitivity: sensor motor responses to approach tactile and tail flick. C - Excitability: reactions to handling and behavior in an open field.
D - Gait and Sensor Motor Coordination: degree of mobility and gait pattern in an open field.
E - Abnormal Clinical Signs: including convulsions, tremors, unusual behavior and deposits around the eyes, nose or mouth.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
Live pups were counted, sexed and weighed on days 0 and 4 postnatal.
The day when delivery is completed was designated day O of lactation (postnatal day 0). On day O of lactation the number of alive and dead pups/sex were recorded. Dams with offspring were euthanized on day 4 postnatal. All pups were euthanized at day 4 postnatal.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
At termination, all parental animals were examined macroscopically for any structural abnormalities or pathological changes. The animals were euthanized in a carbon dioxide chamber. The numbers of implantation sites and corpora lutea were recorded. Animals found dead were necropsied, discarded in biological garbage and incinerated.

ORGAN WEIGHTS:
- At scheduled necropsy, testes and epididymides of all males were weighed.
- Organ weights were obtained for the following organs from 5 animals/sex/group: liver; kidneys; adrenals; thymus; spleen; brain; heart.

HISTOPATHOLOGY: Yes
- For all animals necropsied, tissues were preserved in 10% neutral buffered formalin (except for the testes, which were preserved in Davidson's fixative)
- At scheduled necropsy, the following organs of all animals were preserved: testes, epididymides, ovaries, prostate, seminal vesicle and coagulating gland , bulbourethral gland ,organs showing alterations
- The following organs and tissues of 5 animals/sex/group were preserved: adrenals (right and left); bone marrow (femur); brain (cerebrum, cerebellum and pons); heart; intestine (duodenum, jejunum, ileum - including Peyer's patches, colon, rectum/anus); kidneys (right and left); liver (3 lobes); lungs; lymph nodes (mesenteric and submaxillary); peripheral nerve (sciatic); spinal cord (cervical, midthoracic and lumbar sections); spleen; stomach (glandular and non-glandular); trachea; thymus; thyroid; urinary bladder; uterus; all gross lesions.
- Full histopathology of the preserved organs and tissues listed above were performed in high dose and control animals.
Postmortem examinations (offspring):
All pups were grosly examined for abnormalities of the oral, thoracic and abdominal cavities.
Statistics:
Quantitative variables such as body weights, food consumption and organs weights were analyzed by One Way Analysis of Variance (ANOVA), followed by Dunnett's test if significance is detected, or by the non-parametric test of Kruskal-Wallis, according to the results of tests for normality and homogeneity of variance. For qualitative or non-parametric data such as clinical findings, macroscopic and microscopic findings and fetal findings, comparison between means were carried out using Fisher's Exact Test or the Chi-Square Test.
The level of significance was set at 5%.
Reproductive indices:
Mating index index (%): (number of female mated x 100/ number of females paired)
Fertility index (%): (number of pregnant animals/number of presumed pregnant animals) x 100
Preimplantation loss (%): (no. of corpora lutea - number of implantations/number of corpora lutea) x 100
Offspring viability indices:
Postimplantation loss (%): (number of implantations-number of live fetuses/ number of implantations) x 100
Gestation index (%): (number of female with live pups at birth x 100 /number of pregnant animals) x 100
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were observed.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No alteration on body weight in treated males was observed when compared to the control group. Lower body weight gain was observed in males exposed to 250 mg/kg/day between days 7 to 14 (-11.3%) and days 28 to 35 (-16%). Similar diminution on body weight gain was observed in males at 500 mg/kg/day between days 14 to 21 (-55.8%) and at 1000 mg/kg/day between days 7 to 14 and days 14 to 21 (-6.5% and -97.7%, respectively). In all cases, these changes were not considered to be test item related, because they were isolated, not statistically significant and did not affect overall body weight gain.
In treated satellite males, at the end of the first observation interval no alteration on body weight was observed, but some variations on body weight gain occurred, however, these variations did not affect overall body weight gain. In the recovery period, lower body weight gain occurred (- 29.7%), but was not statistically significant and did not affect the body weight. This finding was not considered to be test item related.
No alteration on body weight was observed in treated females compared to the control group. Differences on body weight gain were observed at several observation periods, affecting overall body weight gain in the gestation period (-15.7% low, -11.9% mid and -10.6% high dose) and lactation period (-60.9% low, -18.6% mid and -51.9% high dose). These differences were not dose related and not statistically significant, and therefore were not considered to be test item related.
In treated satellite females, at the end of the first observation period no alteration on body weight was observed, but some variations on body weight gain, statistically significant between days 0 to 21 were observed, but did not affect total body weight gain. In the recovery period, although the body weight gain was decreased (-57.5%), this change did not affect overall body weight, therefore this finding was considered incidental.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Differences in food consumption were observed in treated males when compared to the control group.
Similar effects occurred in treated satellite males. Although these differences were sometimes statistically significant, they did not affect overall food consumption in either treated males or treated satellite males.
In treated females, differences in food consumption were observed, with statistical significance at some observation periods. In the lactation period, these differences were observed in high dose females, statistically significantly affecting overall food consumption (-24.46%) compared to the control group. This finding was not considered to be test item related since the body weight was not affected.
Statistically significant differences in food consumption were observed at several observation periods in treated satellite females, affecting total food consumption (-5%) in the first observation period and (-8.8%) in the recovery period compared to the control group. These findings were small and did not affect overall body weight, and, therefore are not considered to be test item related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In treated males at 500 mg/kg/day, statistically significant higher lymphocytes counts were observed (+7.8%) but considered incidental because they occurred at the mid dose group. No other alterations on hematological and clotting parameters were observed in treated males.
Mean hemoglobin in treated satellite males was statistically significant lower (-6.7%) compared to the control group, but this finding was not considered to be test item related, because the difference was very small, and within Bioagri's historical range (14.1-17.9). No other hematological parameters were affected.
Statistically significant higher hematocrit (+6.6%) was observed in females exposed to 250 mg/kg/day, but it was an isolated finding at low dose, and therefore not considered to be test item related.
Statistically significant higher mean corpuscular hemoglobin concentration was noted in treated satellite females (+2.8%), but the difference was very small and considered incidental, as a normal biological variation.
In treated satellite males, white blood cell counts (-30.1%) and total lymphocyte counts (-34%) were lower than control group. Total lymphocyte count is within Bioagri's historical range and total white blood cells count is slightly outside (-3.9%). Considering this difference very small in magnitude and no other white cells were affected, this finding was not considered to be test item related. No other changes in white blood cells were observed.
Changes in clotting parameters were observed in both prothrombin time in females at 1000 mg/kg/day (+11.8%) and in treated satellite females (+7.2%), statistically significant in both cases. These
values were small and are within Bioagri's baseline (13.5-23.1) and are not considered to be test item related.
For more detailed information, see section 'Any other information on results'
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In treated males, statistically significantly lower creatinine (-20%) at 500 mg/kg/day and albumin (-7.7% low and -15.4% mid dose) were observed compared to the control group, but not considered to be dose related.
No alteration in the clinical chemistry parameters were noted in treated female groups.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No alteration in the Functional Observation Battery was found in male or female treated groups compared to the control.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Slight reduction of germ cells (bilateral) was the most common lesion noted. This finding was observed in three males exposed to 1000 mg/kg/day. This change should not be considered to be test item related, since this focal isolated reduction of germ cells unilateral in epididymis was also observed in two treated satellite males and two control satellites.
Other microscopic lesions were observed in two animals; one male, at 1000 mg/kg/day presented erosion in the stomach mucosa, and in one female presented focal chronic proliferative nephropathy in the left kidney. These lesions were considered normal background findings and were not considered to be test item related.
For more detailed information, see section 'Any other information on results'
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Similar gestation index were observed when treated groups were compared to the controls and no differences were observed. In dams exposed to the high dose, the number of total pups and the number of live pups at day 0 were lower compared to the control group. These findings, while decreased compared to controls, were not statistically significant and are not likely to be test item related.
For further detailed information, see section 'Any other information on results'
The test substance did not cause any mortality or clinical signs of toxicity during the study. No effects on body weight, body weight gain or food consumption were observed. No effects were observed in the functional observational battery, hematology and clotting parameters, clinical chemistry parameters or organ weights. No macroscopic findings related to the test item were observed.
In this study, the alterations occurred at high dose levels in male and female. These changes were observed on testes (slight reduction of germ cells) and reproductive parameters. Slight reduction of germ cells was observed in three males exposed to the highest dose level. This result was not considered test item related because this lesion appeared in equal proportions in both control and high dose satellites and the frequency of this lesion was not statistically significant. Despite that these males produced offspring from their respective females.
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
No specific findings in all animals.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
While the absolute number of dead pups in the high dose group was statistically significantly increased when compared to controls on day 0, the mean number of live and dead pups per litter was not significantly increased when compared to controls on day 4. No differences were observed when delivery day 1 was compared to day 4 postnatal.
For further information, see section 'Any other information on results'
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No difference in pup body weight was observed between the treated and control groups at day 0. The body weight of pups at day 4 postnatal were lower compared to the control group. This finding, while decreased compared to controls, was not statistically significant and is not likely to be test item related.
For further information, see section 'Any other information on results'
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic finding was observed in treated or control groups.
Histopathological findings:
no effects observed
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
In dams exposed to the high dose, the number of total pups, live pups at day 0, and body weight of pups at day 4 postnatal were lower compared to the control group. These findings, while decreased compared to controls, were not statistically significant and are not likely to be test item related. While the absolute number of dead pups in the high dose group was statistically significantly increased when compared to controls on day 0, the mean number of live and dead pups per litter was not significantly increased when compared to controls on day 4.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Hematology: data is presented as mean+/-SD (No. animals)

Parameter

unit

group

0 mg/kg/day (Control)

250 mg/kg/day

500 mg/kg/day

1000 mg/kg/day

WBC

/mm3

Males

5360+/-2304

(5)

6640+/-1569 (5)

5500+/-1158 (5)

5040+/-2556 (5)

WBC

/mm3

Satellite males

5180+/-832 (5)

 

 

3620+/-1246* (5)

WBC

/mm3

Females

3260+/-1316 (5)

3040+/-1234 (5)

3500+/-1030 (5)

4680+/-2989 (5)

WBC

/mm3

Satellite females

3180+/-853 (5)

 

 

3580+/-1743 (5)

Lymphocytes

/mm3

Males

4333+/-2148 (5)

5027+/-1550 (5)

4744+/-981 (5)

3917+/-1886 (5)

Lymphocytes

WBC%

Males

78.6+/-10.1 (5)

76.0+/-13.2 (5)

86.4+/-4.2* (5)

79.0+/-6.5 (5)

Lymphocytes

/mm3

Satellite males

4249+/-705 (5)

 

 

2801+/-876* (5)

Lymphocytes

WBC%

Satellite males

82.0+/-2.0 (5)

 

 

78.2+/-6.4 (5) (5)

Lymphocytes

/mm3

Females

2546+/-1140 (5)

2420+/-1028 (5)

2611+/-890 (5)

3867+/-2563 (5)

Lymphocytes

WBC%

Females

77.2+/-4.6 (5)

79.2+/-5.6 (5)

74.0+/-9.0 (5)

81.4+/-3.4 (5)

Lymphocytes

/mm3

Satellite females

3455+/-741 (5)

 

 

2735+/-1348 (5)

Lymphocytes

WBC%

Satellite females

76.8+/-7.4 (5)

 

 

76.6+/-5.0 (5)

PT

sec

Males

14.4+/-1.2 (5)

14.1 +/-1.0 (5)

14.8+/-0.8 (5)

14.2+/-1.3 (5)

PT

sec

Satellite males

19.9+/-0.6 (5)

 

 

19.5+/-0.9 (5)

PT

sec

Females

13.6+/-0.7 (5)

14.6+/-0.8 (5)

13.6+/-1.0 (5)

15.2+/-0.8* (4)

PT

sec

Satellite females

19.5+/-0.7 (5)

 

 

20.9+/-0.8* (4)

hemoglobin

g/dL

Males

15.66+/-3.11 (5)

16.36+/-0.61 (5)

15.96+/-1.00 (5)

16.32+/-0.76 (5)

hemoglobin

g/dL

Satellite males

16.18+/-0.44 (5)

 

 

15.10+/-0.75* (5)

hemoglobin

g/dL

Females

14.74+/-0.46 (5)

15.36+/-0.51 (5)

15.30+/-0.64 (5)

13.16+/- 3.97 (5)

hemoglobin

g/dL

Satellite females

15.18+/-0.87 (5)

 

 

15.38+/-0.41 (5)

Hematocrit

%

Males

48.64+/-10.08 (5)

50.32+/-2.09 (5)

49.52+/-3.38 (5)

50.56+/-2.71 (5)

Hematocrit

%

Satellite males

47.66+/-0.74 (5)

 

 

45.06+/-2.81 (5)

Hematocrit

%

Females

45.04+/-1.60 (5)

48.02+/-1.35* (5)

47.46+/-3.64 (5)

41.28+/-12.65 (5)

Hematocrit

%

Satellite females

45.54+/-2.49 (5)

 

 

44.92+/-1.47

MCHC

g/dL

Males

32.22+/-0.48 (5)

32.48+/-0.26 (5)

32.24+/-0.34 (5)

32.28 +/- 0.72 (5)

MCHC

g/dL

Satellite males

33.96+/-0.59 (5)

 

 

33.58+/-0.74 (5)

MCHC

g/dL

Females

32.74+/-0.99 (5)

31.94+/-0.76 (5)

32.34+/-1.22 (5)

31.98+/-0.45 (5)

MCHC

g/dL

Satellite females

33.34+/-0.56 (5)

 

 

34.26+/-0.43* (5)

* statistically significant p<0.05 difference between the means of the group treated and control group

Organ weight: data is presented as mean+/-SD (N° of animals)

Organ

 

group

0 mg/kg/d (control)

250 mg/kg/day

500 mg/kg/day

1000 mg/kg/day

Right testis

Absolute weight

Males

2.027+/-0.153 (12)

2.038+/-0.180 (12)

1.978+/-0.158 (12)

1.879+/-0.186 (12)

Right testis

Relative to body weight

Males

0.527+/-0.043 (12)

0.509+/-0.048 (12)

0.492+/-0.048 (12)

0.476+/-0.044* (12)

Right testis

Relative to brain weight

Males

99.36+/-7.73

96.44+/-8.55

101.96+/-6.94

95.13+/-7.23

Left testis

Absolute weight

Males

2.061+/-0.209 (12)

2.048+/-0.198 (12)

2.030+/-0.162 (12)

1.937+/-0.194 (12)

Left testis

Relative to body weight

Males

0.535+/-0.047 (12)

0.511+/-0.054 (12)

0.506+/-0.053 (12)

0.491+/-0.051 (12)

Left testis

Relative to brain weight

Males

98.71+/-9.58 (12)

94.97+/-7.02 (12)

103.45+/-8.34 (12)

96.97+/-7.94 (12)

Right testis

Absolute weight

Satellite males

2.120+/-0.188 (5)

 

 

1.866+/-0.212 (5)

Right testis

Relative to body weight

Satellite males

0.505+/-0.058 (5)

 

 

0.450+/-0.034 (5)

Right testis

Relative to brain weight

Satellite males

102.18+/-11.32 (5)

 

 

95.19+/-10.67 (5)

Left testis

Absolute weight

Satellite males

2.033+/-0.201 (5)

 

 

1.990+/-0.185 (5)

Left testis

Relative to body weight

Satellite males

0.483+/-0.052 (5)

 

 

0.481+/-0.040 (5)

Left testis

Relative to brain weight

Satellite males

97.99+/-11.82 (5)

 

 

101.45+/-8.77 (5)

* statistically significant (p<0.05)

Histopathology: individual data (presented as x out of y animals)

Organ

observation

Group

0 mg/kg/day

250 mg/kg/day

500 mg/kg/day

1000 mg/kg/day

Testis (bilateral)

Slight reduction of germ cells

Males

0/12

-

-

3/12

Epididymis (right)

Focal isolated reduction of germ cells

Satellite males

2/5

-

-

2/5

 

Reproduction data of dams on day 0

Test group

Animal/litter number

 

Total pups

N° of live pups

N° of dead pups

0 mg/kg/day (control)

13

Not pregnant

-

-

-

 

14

 

11

11

0

 

15

 

11

11

0

 

16

No viable pups

0

0

0

 

17

 

11

11

0

 

18

 

6

6

0

 

19

Not pregnant

-

-

-

 

20

Not pregnant

-

-

-

 

21

 

8

8

0

 

22

 

13

13

0

 

23

No viable pups

0

0

0

 

24

 

11

11

0

 

Total

 

71

71

0

 

Mean+/-SD (N° animals)

 

7.9+/-4.9 (9)

7.9+/-4.9 (9)

0+/-0 (9)

250 mg/kg/day

47

Not pregnant

-

-

-

 

48

 

3

3

0

 

49

 

10

10

0

 

50

 

12

12

0

 

51

 

10

10

0

 

52

 

4

4

0

 

53

 

6

6

0

 

54

Not pregnant

-

-

-

 

55

Not pregnant

-

-

-

 

56

 

7

7

0

 

57

 

8

8

0

 

58

 

10

10

0

 

Total

 

70

70

0

 

Mean+/-SD (N° animals)

 

7.8+/-3.0 (9)

7.8+/-3.0 (9)

0+/-0 (9)

500 mg/kg/day

71

 

5

4

1

 

72

Not pregnant

-

-

-

 

73

 

1

1

0

 

74

 

7

7

0

 

75

 

9

9

0

 

76

 

2

2

0

 

77

 

11

11

0

 

78

 

12

11

1

 

79

 

11

11

0

 

80

 

9

9

0

 

81

 

10

10

0

 

82

Not pregnant

-

-

-

 

Total

 

77

75

2

 

Mean+/-SD (N° animals)

 

7.7+/-3.9 (10)

7.5+/-3.8 (10)

0.2+/-0.4 (10)

1000 mg/kg/day

95

No viable pups

0

0

0

 

96

 

7

5

2

 

97

 

7

0

7

 

98

 

3

0

3

 

99

 

12

12

0

 

100

 

10

10

0

 

101

 

10

10

0

 

102

 

4

4

0

 

103

 

5

5

0

 

104

 

3

3

0

 

105

Not pregnant

-

-

-

 

106

Not pregnant

-

-

-

 

Total

 

61

49

12

 

Mean+/-SD (N° animals)

 

6.1+/-3.8 (10)

4.9+/-4.5 (10)

1.2+/-2.3 (10)

  Individual body weights (g) of pups

Test group

Litter number

Day 0 postnatal

mean pup weight

Day 4 postnatal

mean pup weight

0 mg/kg/day (control)

14

6.4

9.9

 

15

6.6

11.2

 

17

6.7

10.6

 

18

5.3

8.1

 

21

6.7

13.0

 

22

6.4

9.7

 

24

5.7

9.3

 

Mean+/-SD (N° of animals)

6.3+/-0.5 (7)

10.3+/-1.6 (7)

250 mg/kg/day

48

7.6

12.3

 

49

6.7

9.7

 

50

5.7

8.8

 

51

6.0

9.4

 

52

7.3

12.2

 

53

7.0

11.4

 

56

6.5

9.1

 

57

6.1

9.1

 

58

6.2

10.1

 

Mean+/-SD (N° of animals)

8.6+/-0.6 (9)

10.2+/-1.4 (9)

500 mg/kg/day

71

8.5

10.2

 

73

4.6

-

 

74

6.5

11.6

 

75

6.7

11.8

 

76

7.1

9.1

 

77

5.6

10.1

 

78

5.9

10.7

 

79

7.3

12.7

 

80

6.4

10.3

 

81

6.1

10.5

 

Mean+/-SD (N° of animals)

6.3+/-0.8 (10)

10.8+/-1.1 (9)

1000 mg/kg/day

96

5.4

3.6

 

99

5.9

8.0

 

100

6.3

11.2

 

101

5.3

7.8

 

102

7.4

13.0

 

103

6.6

8.7

 

104

6.9

11.8

 

Mean+/-SD (N° of animals)

6.3+/-0.8 (7)

9.2+/-3.2 (7)

Conclusions:
The No Observed Adverse Effect Level (NOAEL) of the test item in Wistar rats was 1000 mg/kg/day for males and females and 1000 mg/kg/day for embryo-fetal toxicity. The substance is not to be classified as reproductive toxicant.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

combined repeated dose toxicity study with reproductive/developmental screening

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was performed (OECD 422; Martell A., 2013. The test item was administered daily to Wistar rats (12 animals/sex/group and 5 animals/sex/satellite groups) by gavage, on a 7 day per week basis (from 14 days before mating period to 4 days post-delivery). The test item was administered at doses of 0, 250, 500 and 1,000 mg/kg/day. The dosage levels (in mg/kg/day) were selected in agreement with the Sponsor, based upon the results of a dose range-finding study. The control group received the vehicle alone (corn oil). Animals were checked daily for mortality or morbidity. Body weight of males was recorded on the first day of dosing and weekly thereafter (including mating and post-mating periods). Females were weighed on first day of dosing and once a week during premating and mating periods, on day 0, 7, 14 and 20 of gestation and during lactation on the same days as the weighing of the litters (on day 0 and 4 postnatal). Food consumption was determined on the same day of body weight determination during premating and lactation periods. During the gestation period, food consumption was determined on days 3, 6, 9, 12, 15, 18 and 20. After the mating period, food consumption of males was determined weekly. Animals were subjected to a careful clinical examination prior to the initiation of the treatment and once a week. At the end of the treatment period, the animals were submitted for overnight fasting. On the day of necropsy, the animals were weighed and anesthetized by CO2 inhalation. Blood samples were collected by cardiac puncture (from 5 animals/sex/group) for hemogram and clotting analyses, leukogram and clinical chemistry. During necropsy, the animals were subjected to a complete gross examination of organs and tissues. The number of implantations sites and corpora lutea were counted. The appropriate organs and tissues (from 5 animals/sex/group) were removed, weighed and submitted for histopathological fixation. Full histopathology of the preserved organs and tissues was performed in the high dose and control animals. The test substance did not cause any mortality or clinical signs of toxicity during the study. No effects on body weight, body weight gain or food consumption were observed. No effects were observed in the functional observational battery, hematology and clotting parameters, clinical chemistry parameters or organ weights. No macroscopic findings related to the test item were observed. During the microscopic tissue evaluations, three males exposed to 1000 mg/kg/day presented light reduction of germ cells. Despite the presence of this finding, their reproductive ability was not affected, since these males produced offspring from their respective females. Similar epididymides lesions were observed in treated and control satellites at the end of the recovery period, both cases were unilateral and occurred in two animals per group. Therefore, this finding was considered not test item related. In the high dose dams, the total number of pups, live pups at day 0 and body weight of pups at day 4 postnatal were lower compared to the control group. These findings, while decreased compared to controls, were not statistically significant and are not likely to be test item related. While the absolute number of dead pups in the high dose group was statistically significantly increased when compared to controls on day 0, the mean number of live and dead pups per litter was not significantly increased when compared to controls on day 4. Accordingly, in the experimental conditions of this study, the NOAEL of the test item in Wistar rats was 1000 mg/kg/day for males and females and 1000 mg/kg/day for embryo-fetal toxicity.


Effects on developmental toxicity

Description of key information

The administration of up to 2% (equivalent to 1306 mg/kg-bw/day) of the source chemical, 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. Therefore, it can be concluded that a similar NOEL would be observed for the target chemical, butyl benzoate.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
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
Endocrine 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:
1 306 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No observed effect level
Key result
Dose descriptor:
conc. level: causing effects
Effect level:
1 874 mg/kg bw/day
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
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:
no effects observed
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:
1 306 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No observed effect
Key result
Dose descriptor:
conc. level: causing abnormalities
Effect level:
1 874 mg/kg bw/day
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
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: urinary
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.:
1 874 mg/kg bw/day
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

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.

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 306 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results in the combined repeated dose toxicity study with reproductive/developmental screening (according to OECD guideline 422), combined with the absence of developmental effects in the absence of significant maternal toxicity in a prenatal developmental toxicity study (according to OECD guideline 414) on the analog substance sodium benzoate, the test substance does not warrant classification as reproductive toxicant. This finding is supported by additional data and review by the 2016 European Food Safety Authority (EFSA) 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 Panel on Food Additives and Nutrient Sources. EFSA Journal 14(3):4433, 110 p. doi:10.2903/j.efsa.2016.4433.

Additional information