Methyl benzoate

Toxicity to reproduction

Administrative data

Endpoint:

multi-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: very concise study summary is available

Data source

Reference

Reference Type:

publication

Title:

Die Verträglichkeit der Benzoesäure in chronischen Fütterungsversuch

Author:

Kieckebusch W, Lang K

Year:

1960

Bibliographic source:

Arzneimittel Forschung 10, 1001-1003

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

4-generation study

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzoic acid

EC Number:

200-618-2

EC Name:

Benzoic acid

Cas Number:

65-85-0

Molecular formula:

C7H6O2

IUPAC Name:

Benzoic acid

Details on test material:

- Name of test material (as cited in study report): Benzoesäure

Test animals

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Farbwerke Bayer (Elberfeld)
- Age at study initiation: no data
- Weight at study initiation: 40-50 g
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): first 8 weeks "paired feed" technique was applied and after ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: no data
- Further matings after two unsuccessful attempts: yes
- After successful mating each pregnant female was caged (how): no data
- Any other deviations from standard protocol: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

every 6 weeks

Duration of treatment / exposure:

Generation 1 and 2: lifelong; generation 3: 16 weeks; generation 4: until breeding

Frequency of treatment:

in 8 first weeks a "paired feed" technique was applied and after ad libitum

Details on study schedule:

no data

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 0.5, 1 %
Basis:
nominal in diet

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): no data

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: in the first 8 weeks body weight was measured weekly and later in four week interval

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

no data

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: total number of offspring, % of survived offspring in litter

GROSS EXAMINATION OF DEAD PUPS:
no data

Postmortem examinations (parental animals):

no data

Postmortem examinations (offspring):

no data

Statistics:

no data

Reproductive indices:

no data

Offspring viability indices:

no data

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
no effects

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
no effects

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
no effects

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
no data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
no data

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
no data

ORGAN WEIGHTS (PARENTAL ANIMALS)
no effects: except in 3rd generation females liver weight increase caused by weight increase during lactation and variation in liter size

GROSS PATHOLOGY (PARENTAL ANIMALS)
no effects

HISTOPATHOLOGY (PARENTAL ANIMALS)
no effects

Effect levels (P0)

1

Dose descriptor:

NOEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 1% of benzoic acid in food, assuming 150 mg (1%) of benzoic acid per day per rat and 300 g bw of rat

2

Dose descriptor:

NOEL

Effect level:

557 mg/kg bw/day (nominal)

Based on:

other: methyl benzoate

Sex:

male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
no effects

CLINICAL SIGNS (OFFSPRING)
no effects

BODY WEIGHT (OFFSPRING)
no effects

SEXUAL MATURATION (OFFSPRING)
no data

ORGAN WEIGHTS (OFFSPRING)
no effects

GROSS PATHOLOGY (OFFSPRING)
no effects

HISTOPATHOLOGY (OFFSPRING)
no effects

Effect levels (F1)

1

Dose descriptor:

NOEL

Generation:

F1

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

2

Dose descriptor:

NOEL

Generation:

F1

Effect level:

557 mg/kg bw/day (nominal)

Based on:

other: methyl benzoate

Sex:

male/female

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1. Body weight gain in g (mean ± deviation)

	Control		0.5% Benzoic acid		1% benzoic acid
	Male	Female	Male	Female	Male	Female
I Generation
Week 4	70 ± 5.0	67 ± 4.1	69 ± 2.7	64 ± 2.4	75 ± 1.4	61 ± 2.2
Week 8	159 ± 6.6	124 ± 5.2	160 ± 5.0	135 ± 5.4	147 ± 2.8	133 ± 2.4
Week 12	219 ± 6.3	-	221 ± 5.6	-	237 ± 6.0	-
II Generation
Week 4	69 ± 2.3	57 ± 3.3	71 ± 2.2	60 ± 6.7	73 ± 3.3	70 ± 2.4
Week 8	167 ± 2.0	122 ± 3.6	161 ± 2.6	131 ± 3.5	158 ± 4.6	123 ± 5.3
Week 12	243 ± 5.5	-	238 ± 3.3	-	231 ± 5.6	-
III Generation
Week 4	73 ± 3.0	60 ± 3.5	68 ± 3.8	71 ± 2.4	72 ± 3.5	67 ± 3.7
Week 8	157 ± 4.5	134 ± 4.1	158 ± 5.3	137 ± 3.9	168 ± 2.0	136 ± 4.5
Week 12	240 ± 4.6	-	226 ± 5.8	-	233 ± 3.8	-
IV Generation
Week 4	93 ± 1.7	76 ± 4.8	84 ± 2.9	70 ± 3.1	73 ± 5.3	72 ± 3.5
Week 8	189 ± 5.3	137 ± 4.3	168 ± 3.7	143 ± 2.4	159 ± 6.2	134 ± 4.5
Week 12	255 ± 4.6	-	245 ± 4.4	-	240 ± 4.4	-

Table 2. The organ weights of animals of the 3rd generation (% of bodyweight)

	Control		0.5% Benzoic acid		1% Benzoic acid
	Male	Female	Male	Female	Male	Female
Bodyweight, g	326 ± 5	262 ± 5	329 ± 5	276 ± 5	316 ± 3	248 ± 6
Brain	0.57 ± 0.01	0.69 ± 0.02	0.58 ± 0.01	0.68 ± 0.01	0.61 ± 0.00	0.75 ± 0.02
Heart	0.34 ± 0.02	0.35 ± 0.01	0.32 ± 0.01	0.39 ± 0.01	0.33 ± 0.01	0.35 ± 0.01
Liver	3.49 ± 0.1	5.32 ± 0.3	3.34 ± 0.1	5.19 ± 0.2	3.49 ± 0.1	5.40 ± 0.2
Spleen	0.21 ± 0.00	0.25 ± 0.01	0.21 ± 0.04	0.23 ± 0.01	0.22 ± 0.01	0.25 ± 0.01
Rein	0.72 ± 0.02	0.8 ± 0.03	0.70 ± 0.04	0.79 ± 0.01	0.74 ± 0.02	0.78 ± 0.02
Testes	0.85 ± 0.03	-	0.83 ± 0.04	-	0.92 ± 0.01	-

Table 3. Reproduction in rats in chronic benzoic acid feeding throughout 4 generations

	Benzoic acid in food, %
	0	0.5	1.0
Total number of pregnant females	80	78	80
Sterile females, %	14	10	4
Females with delayed sexual maturity	7.5	17	9
Litter per generation	9.0 ± 0.33	9.5 ± 0.26	9.6 ± 0.29
Total number of offspring	625	688	741
% of survived offspring in litter	74	66	65
Old pairing (2nd generation) the number of pregnant females	37	38	38
Litter per generation	6.8	7.7	7.5
Total number of offspring	173	139	171
% of survived offspring in litter	72	61	73

Applicant's summary and conclusion

Conclusions:

No effects were noted after chronic dietary exposure of rats (parents and offspring) to 1% benzoic acid (equivalent to 500 mg/kg bw/day). The NOEL for benzoic acid can be converted to a NOEL for methyl benzoate of 557 mg/kg bw/day.

Executive summary:

In a life long chronic multi-generation study 2 experimental groups of 20 male and 20 female rats were treated with 0.5 and 1% of benzoic acid in food. The control group received untreated food and consisted also of 20 male and 20 female rats. The first and the second generations were treated lifelong, the 3rd generation for 16 weeks and the 4th generation until breeding. No effects on body weight and food intake were detected in all 4 generations. Liver weight increase in females of the 3rd generation was considered as not treatment related effect but may be caused by weight increase during lactation and due to variation in litter size. Otherwise no effects after histopathological examinations were detected in the treated animals of the 3rd generation. No effects were detected on reproduction and offspring raise in both dosing groups. Causes of death in this chronic study were not investigated. Based on the data of this study, the NOEL is 1% (500 mg/kg bw/day of benzoic acid and 557 mg/kg bw/day of methyl benzoate).