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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
multi-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1960
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Die verträglichkeit der benzoesäure im chronischen fütterungsvers-uch (The tolerability of benzoic acid in chronic feeding experiments)
Author:
Kieckebusch, W. and Lang, K.
Bibliographic source:
Arzeneimittel-Forschung volume 10: 1001-1003
Report Date:
1960

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Principles of method if other than guideline:
A feeding study was performed, in which 4 generations of rats received the test substance (0.5% or 1%). The first generation was exposed for 8 weeks and then allowed to mate (1/1 for a period of 14 days). Mating was repeated in week 48 to raise a second litter. Survival of the first and second generation was measured. The third generation was terminated after 16 weeks and examined histopathologically. In this generation weights of brain, heart, liver, spleen, kidneys and testes were determined. The fourth generation was terminated after weaning of the pups. Body weights were determined in week 4, 8 and 12 weeks of each generation (week 12 males only). Feeding efficiency was measured in all generations after 2,4, 6 and 8 weeks. Some reproduction parameters were assessed: percentage of infertility, sexual maturation, litter size, total pups and surviving pups. These parameters were assessed for all generations (summed) and for the first two generations separately.
GLP compliance:
no
Remarks:
Study conducted prior to GLP
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Benzoic acid

Test animals

Species:
rat
Strain:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
Source: Farbwerken Bayer
Weight at study initiation: 40-50g
Housing: Double cages
Diet: paired feeding for first 8 weeks, then ad libitum
Water: ad libitum

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: Mixed with diet
Details on exposure:
Feed mixtures were produced in a feed mixing machine made of stainless steel. Diet consisted of commercial standard rat feed made by Lutz (Euskirchen) with sufficient benzoic acid added to achieve feed concentrations of 0.5% and 1.0%.
Details on mating procedure:
1 male and 1 female cohabited for 14 days. If unsuccessful mating, this was repeated 8 weeks later to investigate delays in sexual maturity or sterility.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No futher details available
Duration of treatment / exposure:
11-12 weeks prior to mating and through 4 generations.
Frequency of treatment:
Feed available ad libitum
Details on study schedule:
No futher details available
Doses / concentrationsopen allclose all
Dose / conc.:
0 other: % in diet
Remarks:
Untreated control
Dose / conc.:
0.5 other: % in diet
Remarks:
Equivalent to 450 and 600 mg/kg bw/day for males and females, respectively.
Dose / conc.:
1 other: % in diet
Remarks:
Equivalent to 900 and 1116 mg/kg bw/day for males and females, respectively.
No. of animals per sex per dose:
20/sex/group/generation
Control animals:
yes, plain diet
Details on study design:
The dietary concentrations were selected based upon a previous range-finding study where rats consuming 5% benzoic acid in the diet died within 3 weeks due to lack of palatability of the diet and corrosive effects of the free acid on the digestive tract.
Positive control:
None stated

Examinations

Parental animals: Observations and examinations:
Body weight recorded weekly for the first 8 weeks, then every 4 weeks.
Food consumption for each animal recorded. Consumption data presented as 'protein efficiency (weight increase per gram of dietary protein)'. Compound intake calculated as time-weighted averages from the consumption and body weight gain.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Testes weight examined in the third generation
Litter observations:
Litter size recorded on first and second days. Surviving young determined on twenty-first day.
As there were no differences between generations, data were combined from the four generations.
Assessments included total number of pups born, pup survival and litter size.
Postmortem examinations (parental animals):
The third generation was subject to a necropsy after 16 weeks of exposure. Organ weights (brain, heart, spleen, liver, kidneys and testes) were taken. Organs were examined histopathologically.
Postmortem examinations (offspring):
None stated
Statistics:
Not provided
Reproductive indices:
Not provided
Offspring viability indices:
No differences noted between generations; all data were combined.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
There was no difference between the 1% group and the control group in terms of lifespan (similar number of short vs long-lived animals) but there was a statistically significant increase in the number of long-lived animals in the 0.5% group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on body weight gain or body weights over the four generations of rats tested in this study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on feed consumption over the four generations of rats tested in this study.
Food efficiency:
no effects observed
Description (incidence and severity):
Feed consumption data in this study are presented as “Protein efficiency (weight increase per gram of dietary protein)”. There was no effect on feed consumption (efficiency) in either test group.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathology was only reported for 3rd generation animals.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

For all four generations, there were no adverse effects on food consumption or efficiency, body weights, life span, organ weights, or organ pathology in male and female rats receiving 1% benzoic acid in the diet when compared to the control group. There were no differences in reproduction or development of the young in the 4 observed generations exposed to 1% benzoic acid in the diet.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 other: %
Based on:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Generational data were not reported separately. At concentrations up to 1% benzoic acid in the diet, there were no adverse effects on parents or offspring through four generations of test substance administration. Overall, the dose level from the 1% diet for the entire premating period was approximately 900 and 1176 mg/kg/day for the male and female rats, respectively.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
There was no difference between the 1% group and the control group in terms of lifespan (similar number of short vs long-lived animals) but there was a statistically significant increase in the number of long-lived animals in the 0.5% group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on body weight gain or body weights over the four generations of rats tested in this study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on feed consumption (efficiency) over the four generations of rats tested in this study.
Food efficiency:
no effects observed
Description (incidence and severity):
Feed consumption data in this study are presented as “Protein efficiency (weight increase per gram of dietary protein)”. There was no effect on feed consumption (efficiency) in either test group.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights were only reported for the 3rd generation animals.
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathology was only reported for 3rd generation animals.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P1)

For all four generations, there were no adverse effects on food consumption or efficiency, body weights, life span, organ weights, or organ pathology in male and female rats receiving 1% benzoic acid in the diet when compared to the control group.There were no differences in reproduction or development of the young in the 4 observed generations exposed to 1% benzoic acid in the diet.

Effect levels (P1)

Key result
Dose descriptor:
NOEL
Effect level:
> 1 other: %
Based on:
other:
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Generational data were not reported separately. At concentrations up to 1% benzoic acid in the diet, there were no adverse effects on parents or offspring through four generations of test substance administration. Overall, the dose level from the 1% diet for the entire premating period was approximately 900 and 1176 mg/kg/day for the male and female rats, respectively.

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
There was no difference between the 1% group and the control group in terms of lifespan (similar number of short vs long-lived animals) but there was a statistically significant increase in the number of long-lived animals in the 0.5% group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on body weight gain or body weights over the four generations of rats tested in this study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on feed consumption (efficiency) over the four generations of rats tested in this study.
Food efficiency:
no effects observed
Description (incidence and severity):
Feed consumption data in this study are presented as “Protein efficiency (weight increase per gram of dietary protein)”. There was no effect on feed consumption (efficiency) in either test group.
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:
no effects observed
Description (incidence and severity):
Organ weights were only reported for the 3rd generation animals
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Description (incidence and severity):
Histopathology was only reported for the 3rd generation animals.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

For all four generations, there were no adverse effects on food consumption or efficiency, body weights, life span, organ weights, or organ pathology in male and female rats receiving 1% benzoic acid in the diet when compared to the control group.

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
> 1 other: %
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
There were no adverse effects on viability, sexual maturation, mortality, body weight and weight gain, food consumption and efficiency, body weights, select organ weight and pathology for any of the four generations in this study receiving up to 1% benzoic acid in the diet. Overall, the dose level from the 1% diet for the entire premating period was approximately 900 and 1176 mg/kg/day for the male and female rats, respectively.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
not specified
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
There was no difference between the 1% group and the control group in terms of lifespan (similar number of short vs long-lived animals) but there was a statistically significant increase in the number of long-lived animals in the 0.5% group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on body weight gain or body weights over the four generations of rats tested in this study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on feed consumption (efficiency) over the four generations of rats tested in this study.
Food efficiency:
no effects observed
Description (incidence and severity):
Feed consumption data in this study are presented as “Protein efficiency (weight increase per gram of dietary protein)”. There was no effect on feed consumption (efficiency) in either test group.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights were only reported for the 3rd generation animals.
Gross pathological findings:
not specified
Histopathological findings:
no effects observed
Description (incidence and severity):
Histopathology was only reported for 3rd generation animals.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Details on results (F2)

For all four generations, there were no adverse effects on food consumption or efficiency, body weights, life span, organ weights, or organ pathology in male and female rats receiving 1% benzoic acid in the diet when compared to the control group.

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
1 other: %
Based on:
other:
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
There were no adverse effects on viability, sexual maturation, mortality, body weight and weight gain, food consumption and efficiency, body weights, select organ weight and pathology for any of the four generations in this study receiving up to 1% benzoic acid in the diet. Overall, the dose level from the 1% diet for the entire premating period was approximately 900 and 1176 mg/kg/day for the male and female rats, respectively.

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
There were no adverse effects on reproductive parameters including fertility measures, delayed sexual maturity, total number of pups born, pup survival, onset of reproductive senescence or litter size when male and female rats were fed up to 1% benzoic acid in the diet over four generations. Under conditions of this study, benzoic acid is not a reproductive toxicant.
Executive summary:

In a long-term feeding study, benzoic acid was added to standard feed to achieve concentration of 0.5% or 1.0% in the diet. Untreated controls were also included. Diets were provided ad libitum to groups of 20 rats/sex through four generations. Body weights and feed consumption data were collected throughout the exposure period. Other endpoints examined included: onset of sexual maturity, evidence of permanent sterility, onset of menopause, litter sizes, number of pups born, surviving young, organ weights and histology, and effect on lifespan. Feed consumption, body weights, and weight gain were unaffected by exposure to benzoic acid at concentrations up to 1% in the diet. There was actually an unexplained statistically significant increase in the lifespan of rats in the 0.5% exposure group, i.e., a higher percentage of rats lived longer. There were no differences between the groups exposed to benzoic acid and the control group for fertility measures, delayed sexual maturity, total number of pups born, pup survival, onset of reproductive senescence or litter size. In addition, organ weights and histopathologic findings were similar for all groups. Under conditions of this study, there were no dose-related adverse effects on either reproductive or developmental parameters in both sexes of rats fed 1% benzoic acid in the diet over four generations.


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