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Toxicity to reproduction

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Administrative data

Endpoint:
multi-generation reproductive toxicity
Remarks:
The effect of benzoic acid on reproduction was studied through four generations.
Type of information:
experimental study
Remarks:
In a chronic feeding study, male and female rats were exposed to up to 1% benzoic acid in the diet through four generations.
Adequacy of study:
key study
Study period:
Benzoic acid was fed in the diet to male and female rats for 11-12 weeks before the first mating and then through four generations.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Please see remarks for details.
Remarks:
While the design of this four-generation study for reproductive toxicity does not exactly match the endpoints included within the EOGRTS, it does include almost all of the endpoints associated with the OECD Guideline 416 – two generation reproductive toxicity that was previously considered adequate to satisfy the reproductive toxicity endpoint under the REACh regulation. The only missing reproductive endpoints within the Kiekebusch and Lang publication are collection of sperm parameters or estrous cyclicity data. However, the endpoints that are missing from the four-generation study with benzoic acid are available for benzyl acetate, a chemical that is metabolized completely to benzoic acid. In the National Toxicology Program (Morrissey et al., 1988) 13-week feeding benzyl acetate studies in rats and mice, both sperm parameters (epididymis, cauda epididymis, testis weights, and sperm motility, density and percent abnormal sperm) and estrus cyclicity measurements were collected. Dose levels of 2000 to 3000 mg/kg/day did not affect any endpoints of male reproduction. Estrus cycles were lengthened in female mice receiving >3000 mg/kg/day but this only occurred at a dose levels also causing significant decreases in growth (body weights and body weight gain). Therefore, it appears that all of the reproductive toxicity endpoints have been collected, albeit in two separate studies on benzoic acid and benzyl acetate.

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.
Year:
1960
Bibliographic source:
Arzeneimittel-Forschung volume 10: 1001-1003

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
Study predates current published regulatory guidelines for GLP compliance and reproductive toxicity studies; study contains most of the same elements found in a present-day two generation reproductive toxicity test and continued exposure through four generations. The premating exposure intervals were at least 10 weeks and the dose levels used were comparable to the limit dose level of 1000 mg/kg/day required under the current regulatory guidelines. Endpoints examined included collection of body weight and feed consumption data, determination of effects on sexual maturity and fertility, collection of litter and pup data, necropsy data including organ weights and histopathology, and effect on onset of sexual senescence, and lifespan. The study did not collect sperm parameters and estrous cyclicity data but these endpoints are addressed in a separate 13-week NTP feeding study in rats with the analogue benzyl acetate.
Deviations:
yes
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 an 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, delayed sexual maturation, litter size, total pups, surviving pups. These parameters were assessed for all generations (summed) and for thefirst two generations separately.
GLP compliance:
no
Remarks:
Study pre-dated GLPs.
Limit test:
no

Test material

Reference
Name:
Unnamed
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 publication.- Expiration date of the lot/batch: Not provided in publication.- Purity test date: Not provided in publicaton.STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Not provided in publication.- Stability under test conditions: Not provided in publication.TREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: Not provided in publication.

Test animals

Species:
rat
Strain:
not specified
Details on species / strain selection:
TEST ANIMALS- Source: Farbwerken Bayer (Elherfeld)- Females (if applicable) nulliparous and non-pregnant: yes- Age at study initiation: Not provided in publication.- Weight at study initiation: 40-50g- Fasting period before study: Not provided in publication.- Housing: Rats were kept in double cages in a simplified Columbus-type feed apparatus.- Diet (e.g. ad libitum): For the first 8 weeks, they were fed according to the “paired feed” method, and then ad libitum.- Water (e.g. ad libitum): ad libitum.- Acclimation period: Not provided in publication.ENVIRONMENTAL CONDITIONS- Temperature (°C): Not provided in publication.- Humidity (%): Not provided in publication.- Air changes (per hr): Not provided in publication.- Photoperiod (hrs dark / hrs light): Not provided in publication.IN-LIFE DATES: From: To: Not provided in publication.
Sex:
male/female

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION- Rate of preparation of diet (frequency): Not provided in publication.- Mixing appropriate amounts with (Type of food): 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%- Storage temperature of food: Not provided in publication.
Details on mating procedure:
- M/F ratio per cage: 1M/1F- Length of cohabitation: 14 days- Proof of pregnancy: Not provided in publication.- After 14 days of unsuccessful pairing ,the pairing was repeated 8 weeks later to investigate delays in sexual maturity or full sterility.- Further matings after two unsuccessful attempts: no - After successful mating each pregnant female was caged (how): Not provided in publication.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Not provided in publication.
Duration of treatment / exposure:
11-12 weeks prior to mating and through 4 generations.
Frequency of treatment:
Feed containing test material provided ad libitum.
Details on study schedule:
Details on study schedule were not provided in the publication.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
0.5 mg/kg bw/day (actual dose received)
Remarks:
Overall, the dose level from the 0.5% diet for the entire premating period was approximately 450 and 600 mg/kg/day for the male and female rats, respectively.
Dose / conc.:
1 mg/kg bw/day (actual dose received)
Remarks:
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.
No. of animals per sex per dose:
20 males/group/generation20 females/group/generation
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dietary concentrations were selected based upon a previous probe 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:
CAGE SIDE OBSERVATIONS: No data DETAILED CLINICAL OBSERVATIONS: No data BODY WEIGHT: Yes - Time schedule for examinations: During the first 8 weeks of the experiment, weight checks were done weekly; after that, weight was checked in 4-week intervals. FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes - Food consumption for each animal determined: Yes; feed consumption data were collected throughout the exposure period over the four generations; for reporting purposes in the publication, feed consumption data are 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 data: calculated from body weight and feed consumption data include within the publication.
Oestrous cyclicity (parental animals):
Not examined in this study.
Sperm parameters (parental animals):
Testis weight was examined in the 3rd generation.
Litter observations:
STANDARDISATION OF LITTERS- Performed on day 4 postpartum: No; Litter size was recorded on the 1st and 2nd day as well as the number of surviving young on the 21st day. PARAMETERS EXAMINEDThe following parameters were examined in [F1 / F2 / F3] offspring: Since there was no demonstratable effect of benzoic acid on reproduction and since the data from all four generations were similar, the data were combined. Parameters examined included total number of pups born, pup survival, and litter size. GROSS EXAMINATION OF DEAD PUPS: No information provided.ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not examined.ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not examined.
Postmortem examinations (parental animals):
SACRIFICE- Male animals: The third generation of animals was necropsied after 16 weeks of exposure. Organ weights of the brain, heart, spleen, liver, kidneys and testis were collected. Organs were examined histopathologically.- Maternal animals: The third generation of animals was necropsied after 16 weeks of exposure. Organ weights of the brain, heart, spleen, liver, and kidneys were collected. Organs were examined histopathologically.HISTOPATHOLOGY / ORGAN WEIGHTS: The following organs from the third generation animals were prepared for microscopic examination and weighed, respectively.: brain, heart, spleen, liver, kidneys, testis.
Postmortem examinations (offspring):
None stated.
Statistics:
Not provided in publication.
Reproductive indices:
Not provided in publication.
Offspring viability indices:
No differences were observed between generations so all data were combined.

Results and discussion

Results: P0 (first parental animals)

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 (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
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. See Table 1 in "Any other information on results incl. tables".

Effect levels (P0)

Key result
Dose descriptor:
NOEL
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. See Table 1 in "Any other information on results incl. tables".

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
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):
effects observed, treatment-related
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:
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:
NOEL
Generation:
F2
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 (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 1: Reproduction of rats given chronic feeding of benzoic acid over 4 generations

      % Benzoic Acid in Feed   
   0 0.5 1.0 
 Total number of females used 80  78  80 
 Sterile females in % 14% 10% 4%
       
 Females with delayed sexual maturity

 7.5

17 

9

 Litter Size

 9.0 + 0.33

9.5 + 0.26 

9.6 + 0.29 

 

 

 

 

 Total number of pups born

625 

688 

741 

 Surviving young in % of number of pups born

74%

66%

65%

       
 Age Pairing (2nd Generation)      
 Number of females used 37 38 38
 Litter size

6.9

7.7

7.5

 

 

 

 

 Total number of pups born  173 139  171 
 Surviving young in % of number of pups born  72% 61%  73% 

 

Applicant's summary and conclusion

Conclusions:
In a chronic feeding study, 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, 0.5% or 1.0% in the diet. 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.