Benzyl benzoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Remarks:

investigation of chemotherapeutic activity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

published in 1965

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published literature from peer reviewed journal

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1965

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Various assays were completed with rats and mice with implanted tumours and the inhibitory effects or cytostatic effects of benzyl benzoate investigated.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzyl benzoate

EC Number:

204-402-9

EC Name:

Benzyl benzoate

Cas Number:

120-51-4

Molecular formula:

C14H12O2

IUPAC Name:

benzyl benzoate

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): benzyl benzoate
No other information

Test animals

Species:

rat

Strain:

other: Wistar WAG

Sex:

male

Details on test animals or test system and environmental conditions:

Male Wistar WAG rats were grouped in batches having approximately similar weights (circa 150 g) and approximately the same age (circa 2.5 months).

Administration / exposure

Route of administration:

oral: gavage

Details on oral exposure:

Use of benzyl benzoate in palliative care of terminal cancer patients resulted in several systematic investigations of the cytostatic effects of benzyl benzoate therapies.
In the first of these assays , two tumour types were investigated - either the solid form of the Erlich carcinoma or the Walker 256 carcinoma. In the Erlich carcinoma test, treatment was initiated 5 days after implantation of the tumour, in the form of intraperitoneal injections for 6 consecutive days. At a dose of 10 mg/kg/day, a 15% inhibition of tumour growth was noted in the treated animals.
In the rat study with the Walker 256 carcinoma, treatment was initiated 3 days after subcutaneous inoculation of the tumour, in the form of intraperitoneal injections for 6 consecutive days. The maximum dose used was 100 mg/kg/day and the maximum tumour inhibition observed was 20%.

The Ornellas publication describes a third and fourth assay in which the lymphotrophic and transplantable Guerin T8 tumour (rat uterus epithelioma) was grafted into rats (this virulent tumour has an almost 100% positive grafting success rate, shows an absence of tumour regression and a constancy of lymphatic metastases). In the first assay with this tumour, inoculation was achieved by implanting the graft subcutaneously on the dorsum. Oral cytostatic treatment commenced 5 days after grafting (Group I) or on the day of grafting (group II). The doses of benzyl benzoate administered were based on human exposure (6g/day) and equated to 166 mg/kg/day.

In the second assay 0.5 mL of pulverised tumour material, equating to circa 0.25 g of tumour was injected under the dorsal skin rather than grafting. Oral cytostatic treatment commenced on the day of injection. Benzyl benzoate was administered at 200 mg/kg /day (Group I) or 800 mg/kg /day (Group II).

The animals were retained until death occurred naturally and the tumours and metastases were excised and weighed separately.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

The cytostatic phase was completed and then all of the negative animals were dosed on a daily basis at 800 mg/kg /day such that the total exposure time was 7 months.

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
166 , 200 or 800 mg/kg /day
Basis:
no data

No. of animals per sex per dose:

20 males per group for the cytostatic phase

Control animals:

yes

Details on study design:

Use of benzyl benzoate in palliative care of terminal cancer patients resulted in several systematic investigations of the cytostatic effects of benzyl benzoate therapies.
In the first of these assays , two tumour types were investigated - either the solid form of the Erlich carcinoma or the Walker 256 carcinoma. In the Erlich carcinoma test, treatment was initiated 5 days after implantation of the tumour, in the form of intraperitoneal injections for 6 consecutive days. At a dose of 10 mg/kg/day, a 15% inhibition of tumour growth was noted in the treated animals.
In the rat study with the Walker 256 carcinoma, treatment was initiated 3 days after subcutaneous inoculation of the tumour, in the form of intraperitoneal injections for 6 consecutive days. The maximum dose used was 100 mg/kg/day and the maximum tumour inhibition observed was 20%.

The Ornellas publication describes a third and fourth assay in which the lymphotrophic and transplantable Guerin T8 tumour (rat uterus epithelioma)was grafted into rats (this virulent tumour has an almost 100% positive grafting success rate, shows an absence of tumour regression and a constancy of lymphatic metastases). In the first assay with this tumour, inoculation was achieved by implanting the graft subcutaneously on the dorsum. Oral cytostatic treatment commenced 5 days after grafting (Group I) or on the day of grafting (group II). THe doses of benzyl benzoate administered were based on human exposure (6g/day) and equated to 166 mg/kg/day.

In the second assay 0.5 mL of pulverised tumour material, equating to circa 0.25g of tumour was injected under the dorsal skin rather than grafting. Oral cytostatic treatment commenced on the day of injection. Benzyl benzoate was administered at 200 mg/kg /day (Group I) or 800 mg/kg /day (Group II).

The animals were retained until death occurred naturally and the tumours and metastases were excised and weighed separately.

The negative animals, following completion ofte cytostatic phase were dosed daily for a period of seven months.

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

Cytostatic activity. Inhibition of tumour growth was assessed in rats that died naturally following tumour graft or subcutaneous injection.

Weight gains were monitored through the treatment phase.

Survival, prolongation of life, and tumour or metastases development were monitored.

Sacrifice and pathology:

No details provided. The rats were allowed to die naturally and tumours and metastases were excised and weighed at time of death.

Other examinations:

No data

Statistics:

No data

Results and discussion

Results of examinations

Clinical signs:

not examined

Mortality:

not examined

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

increased weight gain in treated animals

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 specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Details on results:

Cytostatic activity

Inhibition of tumour growth in group I (200 mg/kg benzyl benzoate) was about 16%, relative to the controls. In the assay with a tumour graft rather than pulverised tumour material , the level of inhibition was 33%, the differences were attributed to differences in the assay techniques employed. In addition, the development of tumours from pulverized material is less influenced by external conditions: operative shock, local infections and early ulcerations.
Group II (800 mg/kg /day) gave higher mean values than the control group because of two excessively high figures. The individual influence can only be removed by working with a much larger number of animals and in this case it appears the outlier results were retained in the data set..
Statistical analysis showed no significant results, even when the weight of the tumours and of the metastases of each group with the control are analysed together.

Bodyweight gains
Treated animals had larger weight gains than the controls. This was taken to indicate the animals' general condition was not disturbed by dosing with benzyl benzoate, and possibly that the treated animals ate more than the control animals.
The weight gain curve of the control animals was compared with the weight gain curve of normal animals. There was a very slight gap between the two curves, which can be attributed to the graft itself.

Survival
The survival curve for rats dosed at 200 or 800 mg/kg /day showed no significant benefit for treated animals in comparison with controls. Although there was a indication of prolongation of life for group II (800 mg/kg/day), the development of tumours and of metastases was not delayed. Overall statistical analysis was not significant, when the values for each group were compared with the controls.


Numerous compounds of the aromatic series have been investigated for cytostatic or mito-inhibitory effects. Details on the mito-inhibitory activity of most of the functions of the aromatic series on the root meristem of wheat were previiously investigated and the authors noted that absence of the carboxyl function or its esterification are the only practical conditions of the mito-inhibitory activity (which resembles that of colchicine). The particular cytostatic activity of benzyl benzoate had not been investigated prior to the assays detailed in this publication.
Benzoic acid is toxic and inactive as a cytostatic agent, showing a distinction between toxicity and specific activity. However, not all aromatic acids are devoid of mito-inhibitory capacity. Furthermore, the excitomitotic action at low dose levels seems to be associated with the presence of a carboxyl function. By comparing certain physical properties of the acid and of the ester of the benzene series, the authors concluded that the salts and esters are less polar than free benzoic acid and the lower water-solubility of the salts and esters must influence their mito-inhibitory capacity
Fat-solubility also has a role in mito-inhibition . The authors concluded that there is an inverse ratio between intensity of action and solubility in water. The mito-inhibitory action cannot be explained by a purely lipoid mechanism, but a more direct action must be assumed in the synthesis of proteins.
It should also be noted that most benzoic antiseptics, and generally the commonly used external antiseptics, exert a direct action on cellular respiration, specifically on the dehydrogenases. Cytotoxicity is exerted by different effects on the cytoplasm or on the nucleus.

Effect levels

Dose descriptor:

other: cytostatic activity

Effect level:

800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

While the overall results from these investigations were generally consistent they cannot be regarded as biologically significant.
Benzyl benzoate shows definite cytostatic activity, which is limited by rapid hydrolysis in the tissues or in the duodenum.

The action by direct contact when the product is administered per os must definitely be important.
It seems to us to be logical to think that tumours localized in the upper part of the gastrointestinal tract would be the most sensitive.
In fact, clinical experience confirms this hypothesis.
However, this confirmation does not allow generalization.
Cancer is not a localized disease, and with benzyl benzoate it is not possible to achieve control of the development of malignancy.
Only clinical experience can justify or not its use as a palliative in view of the almost complete absence of toxicity.

Executive summary:

Use of benzyl benzoate in palliative care of terminal cancer patients resulted in several systematic investigations of the cytostatic effects of benzyl benzoate therapies. In the first of these assays , two tumour types were investigated - either the solid form of the Erlich carcinoma or the Walker 256 carcinoma. In the Erlich carcinoma test, treatment was initiated 5 days after implantation of the tumour, in the form of intraperitoneal injections for 6 consecutive days. At a dose of 10 mg/kg/day, a 15% inhibition of tumour growth was noted in the treated animals. In the rat study with the Walker 256 carcinoma, treatment was initiated 3 days after subcutaneous inoculation of the tumour, in the form of intraperitoneal injections for 6 consecutive days. The maximum dose used was 100 mg/kg/day and the maximum tumour inhibition observed was 20%. The Ornellas publication describes a third and fourth assay in which the lymphotrophic and transplantable Guerin T8 tumour (rat uterus epithelioma)was grafted into rats (this virulent tumour has an almost 100% positive grafting success rate, shows an absence of tumour regression and a constancy of lymphatic metastases). In the first assay with this tumour, inoculation was achieved by implanting the graft subcutaneously on the dorsum. Oral cytostatic treatment commenced 5 days after grafting (Group I) or on the day of grafting (group II). THe doses of benzyl benzoate administered were based on human exposure (6g/day) and equated to 166 mg/kg/day. In the second assay 0.5 mL of pulverised tumour material, equating to circa 0.25g of tumour was injected under the dorsal skin rather than grafting. Oral cytostatic treatment commenced on the day of injection. Benzyl benzoate was administered at 200 mg/kg /day (Group I) or 800 mg/kg /day (Group II). The animals were retained until death occurred naturally and the tumours and metastases were excised and weighed separately. The negative animals, following completion ofte cytostatic phase were dosed daily for a period of seven months.

Cytostatic activity Inhibition of tumour growth in group I (200 mg/kg benzyl benzoate) was about 16%, relative to the controls. In the assay with a tumour graft rather than pulverised tumour material , the level of inhibition was 33%, the differences were attributed to differences in the assay techniques employed. In addition, the development of tumours from pulverized material is less influenced by external conditions: operative shock, local infections and early ulcerations. Group II (800 mg/kg /day) gave higher mean values than the control group because of two excessively high figures. The individual influence can only be removed by working with a much larger number of animals and in this case it appears the outlier results were retained in the data set.. Statistical analysis showed no significant results, even when the weight of the tumours and of the metastases of each group with the control are analysed together. Bodyweight gains Treated animals had larger weight gains than the controls. This was taken to indicate the animals' general condition was not disturbed by dosing with benzyl benzoate, and possibly that the treated animals ate more than the control animals. The weight gain curve of the control animals was compared with the weight gain curve of normal animals. There was a very slight gap between the two curves, which can be attributed to the graft itself. Survival The survival curve for rats dosed at 200 or 800 mg/kg /day showed no significant benefit for treated animals in comparison with controls. Although there was a indication of prolongation of life for group II (800 mg/kg/day), the development of tumours and of metastases was not delayed. Overall statistical analysis was not significant, when the values for each group were compared with the controls.

he cytostatic activity and general systemic toxicity of benzyl benzoate was investigated following administration of 200 or 800 mg/kg bw/day to rats over a seven month period. The primary endpoint for the investigation was potential for tumour inhibition or life prolongation, no endpoints were determined for systemic toxicity other than tolerance of repeated administration of a dose of 800 mg/kg bw.