Benzyl benzoate

Endpoint summary

Description of key information

The oral toxicity of benzyl benzoate was determined in a repeated exposure study primarily investigating cytostatic activity but there were no indications of adverse systemic effects up to the cytostatically effective dose level of 800 mg/kg bw/d.
In a subacute dermal toxicity study the lowest effective dose level was 1250 mg/kg bw/d. Mortalities occurred at 2000 mg/kg bw/d but no adverse systemic toxicity was noted at 1250 mg/kg bw/d or lower dose levels. No local dermal effects were identified.

Benzyl benzoate was shown to be hydrolysed relatively rapidly in human plasma in vitro; a half-life of 19 minutes was shown. Benzyl benzoate hydrolyses to benzoic acid (65-85-0) and the corresponding alcohol (benzyl alcohol, CAS 100-51-6). This hydrolysis rate indicates rapid metabolism in vivo and suggests that bioaccumulation of benzyl benzoate is unlikely.
(Nielsen NM & Bundgaard H, Prodrugs as delivery systems, 68. Chemical and plasma-catalyzed hydrolysis of various esters of benzoic acid: a reference system for designing prodrug esters of carboxylic acid agents, International Journal of Pharmaceutics 39: 75-85 (1987).
http://www.inchem.org/documents/jecfa/jecmono/v48je14.htm
Based on the fast hydrolysis to benzoic acid and benzyl alcohol the systemic effects of these two substances are considered after absorption of benzyl benzoate.

 

In a 13 week study male and female rats received once daily on 5 days/week 0, 50, 100, 200, 400, 800 mg/kg bw benzyl alcohol via gavage. Based on clinical signs and reduced body weight development in males and females and histopathological changes in the brain at 800 mg/kg bw the NOAEL was considered to be 400 mg/kg bw/day (NTP TR 343, 1989).

 

Benzoic acid (BA) was administered in the diet to male and female Sprague Dawley Crl:CD(SD) rats in an OECD Test Guideline 443 Extended One-Generation Reproductive Toxicity (EOGRT) study to test for effects that may occur as a result of pre- and postnatal exposure. The study included cohorts of F1 offspring to evaluate potential effects of benzoic acid on reproduction, the developing immune system, and the developing neurological system with the inclusion of learning and memory assessments. Benzoic acid was incorporated in the diet at concentrations of 0, 7,500, 11,500, and 15,000 mg/kg diet (ppm). These concentrations were selected based on the results of preliminary studies, and, based on average food consumption, were intended to supply BA doses of approximately 0, 500, 750, and 1000 mg/kg bw/day. To avoid exceeding these target dose levels, the dietary
The highest dietary concentration (15,000 ppm), providing a dosage of approximately 1000 mg/kg bw/day, was the NOAEL for benzoic acid in this EOGRT study.

 

The NOAEL of 400 mg/kg bw/day from the 13 week study with benzyl alcohol is used for the derivation of the systemic DNELs.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

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

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).

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

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

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

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.

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'

Critical effects observed:

not specified

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

400 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Non-standard assessment of toxicity

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 to 30 April 1980

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

no

Principles of method if other than guideline:

In principle the methods used are the same as those described in standard regulatory guidelines for subacute determination of dermal toxicity.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: CD SD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, USA
- Age at study initiation: no data
- Weight at study initiation: group mean bodyweights at study initiation was , males : 297-344g and females: 198-225g
- Fasting period before study: not applicable
- Housing: individually housed in stainless steel mesh cages
- Diet (e.g. ad libitum):Purina Rat Chow, supplied by Stover Feed Company, ad libitum
- Water (e.g. ad libitum): ad libitum from automatic watering system
- Acclimation period: 4 weeks

IN-LIFE DATES: From: 1 April 1980 To: 30 Apil 1980

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: not specified - seven shaved areas identified on the dorsum. Treatment of the prepared sites was rotated over each 7 day period
- % coverage: no information
- Type of wrap if used: none used
- Time intervals for shavings or clipplings: dorsum shaved before dosing commenced, no details for interim subsequent shaving occasions

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no details


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): six dose levels - 0.188; 0.301; 0.488; 0.781; 1.25 and 2.0 g/kg bw


USE OF RESTRAINERS FOR PREVENTING INGESTION: no details

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No information

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0.188; 0.301; 0.488; 0.781; 1.25 and 2.0 g/kg bw
Basis:
nominal per unit body weight

No. of animals per sex per dose:

three males and three females per group

Control animals:

no

Details on study design:

- Dose selection rationale: doses selected to provide range-finding information for a 90-day study
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: not applicable

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily assessment

BODY WEIGHT: Yes
- Time schedule for examinations: weekly recording

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to termination, obtained by cardiac puncture
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: all - 3 males and 3 females per group
- Parameters checked : haematocrit; haemoglobin; erythrocytes; total and differential leucocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:immediately prior to termination, obtained by cardiac puncture
- Animals fasted: Yes
- How many animals:all - 3 males and 3 females per group
- Parameters checked : alkline phosphatase AP; lactic dehydrogenase; serum glutamic-oxalacetic transaminase GOT; serum glutamic-pyruvic transaminase GPT; glucose, cholesterol, calcium, phosphorus; uric acid; blood urea nitrogen, total protein, total bilirubin and albumin

URINALYSIS: Yes
- Time schedule for collection of urine: samples collected directly from under housing cages on day prior to or day of terminal sacrifice
- Metabolism cages used for collection of urine: No
- Animals fasted: No
- Parameters checked : appearance, pH, ketones, occult blood, bilirubin, protein, glucose, microscopic examination of sediment .

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
On day 31, all surviving rats were sacrificed following an overdose of ether . All animals including any decedents were examined for gross pathology. The following tissues were processed for histoptholoical examination:

treated and untreated skin
adrenals
heart
lung and bronchi
mesenteric lymph nodes
thyroid
pituitary
brain (3 sections)
sternal bone marrow
spinal cord
testes with epididymus
ovaries
spleen
urinary bladder
nerve with muscle
kidney
gross lesions

Other examinations:

no data

Statistics:

no data

Clinical signs:

effects observed, treatment-related

Dermal irritation:

no effects observed

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

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:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
No clinical signs of adverse reaction to dermal application of doses from 0.188 to 1.25 g/kg. In the high dose group three males and 1/3 females died between days 6 and 9. Prior to death the majority of these rats showed anogenital discharge, tremors and ataxia. One of the surviving females showed anogenital discharge and hyperactivity on days 7 to 9 but survived the episode, the second surviving female had tremors on days 6 to 8.

BODY WEIGHT AND WEIGHT GAIN: For rats in the groups dosed at 0.188, 0.301, 0.488 or 0.781 g/kg, there were no effects on bodyweight or weight gain. Bodyweight gains in the group dosed at 1.25 g/kg were slightly lower than the other groups. In the high dose group, 2.0 g/kg, 4/6 rats died and bodyweight gains were not assessed for the remaining rats.

DERMAL REACTIONS: The treated skin sites (seven areas on the dorsum dosed on a 7 day rotation) were not affected by repeated topical application of benzyl benzoate despite the recording of a few instances of slight redness or oedema.

HAEMATOLOGY - No notable differences between groups for any of the blood parameters. White blood cell counts and differential counts in groups 5 and 6 were not significantly different (despite only two rats surviving at the highest dose level). Mean white cell counts for the treated groups 1-6 were 10.4; 11.9; 10.1; 11.2; 8.4 and 8.6 respectively.

CLINICAL CHEMISTRY - no effects on any of the parameters except for a trend for higher uric acid levels in groups treated at the lower dose levels

URINALYSIS - no effects of treatment were detected in any of the urinary parameters examined at any dose level.

GROSS PATHOLOGY: all animals, including the decedents, showed no macroscopic abnormalities during necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC - The histopathologically recognised effects of dermal application of benzyl benzoate to rats are slight squamous epithelial hyperplasia, degeneration of hair follicles and sebaceous glands, subcutaneous fibrosis and thyroid gland hyperplasia.

Dose descriptor:

NOAEL

Effect level:

781 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reduced weight gain at 1250 mg/kg bw/d; mortality at 2000 mg/kg bw/d

Critical effects observed:

yes

Lowest effective dose / conc.:

1 250 mg/kg bw/day

System:

other: Reduced weight gain at 1250 mg/kg bw/d; mortality at 2000 mg/kg bw/d

Organ:

other: Reduced weight gain at 1250 mg/kg bw/d; mortality at 2000 mg/kg bw/d

Treatment related:

yes

Dose response relationship:

yes

Conclusions:

Repeated application of 2000 mg/kg bw/d resulted in signs of toxicity and mortality; however effects at lower dose levels were limited to reduced weight gain at 1250 mg.kg bw/d. A clear NOAEL of 781 mg/kg bw/d can therefore be derived for this study.

Executive summary:

Benzyl benzoate was applied topically to sites on the dorsum of 36 rats (six groups of three males and three females) at doses of 188, 301, 488, 781, 1250 or 2000 mg/kg bw/d. Daily checks for mortality, clinical signs of reaction to treatment and assessment of any dermal changes at treatment sites were completed and bodyweights were recorded at weekly intervals. Blood and urine samples were collected shortly prior to termination and all rats were subject to gross necropsy.

No effects of treatment were apparent for rats dosed at 188, 301, 488 or 781 mg/kg bw/d. A slight reduction in bodyweight gain was evident for rats dosed at 1250 mg/kg bw/d in comparison with other treated groups.

Blood analysis indicated a slight increase in mean uric acid levels for rats dosed at 301 mg/kg bw/d but in the absence of similar effects at higher or lower doses, the biological significance of this change was unclear. No biologically significant macroscopic changes were evident at any dose level. Histopathological examinations revealed squamous epithelium hyperplasia, degeneration of hair follicles and sebaceous glands, subcutaneous fibrosis and thyroid gland hyperplasia. A clear NOAEL of 781 mg/kg bw/d is determined for this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

781 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

One 30-day range-finding rat study and one non-standard 90-day rabbit are available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 to 30 April 1980

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

no

Principles of method if other than guideline:

In principle the methods used are the same as those described in standard regulatory guidelines for subacute determination of dermal toxicity.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: CD SD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, USA
- Age at study initiation: no data
- Weight at study initiation: group mean bodyweights at study initiation was , males : 297-344g and females: 198-225g
- Fasting period before study: not applicable
- Housing: individually housed in stainless steel mesh cages
- Diet (e.g. ad libitum):Purina Rat Chow, supplied by Stover Feed Company, ad libitum
- Water (e.g. ad libitum): ad libitum from automatic watering system
- Acclimation period: 4 weeks

IN-LIFE DATES: From: 1 April 1980 To: 30 Apil 1980

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: not specified - seven shaved areas identified on the dorsum. Treatment of the prepared sites was rotated over each 7 day period
- % coverage: no information
- Type of wrap if used: none used
- Time intervals for shavings or clipplings: dorsum shaved before dosing commenced, no details for interim subsequent shaving occasions

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no details


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): six dose levels - 0.188; 0.301; 0.488; 0.781; 1.25 and 2.0 g/kg bw


USE OF RESTRAINERS FOR PREVENTING INGESTION: no details

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No information

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0.188; 0.301; 0.488; 0.781; 1.25 and 2.0 g/kg bw
Basis:
nominal per unit body weight

No. of animals per sex per dose:

three males and three females per group

Control animals:

no

Details on study design:

- Dose selection rationale: doses selected to provide range-finding information for a 90-day study
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: not applicable

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily assessment

BODY WEIGHT: Yes
- Time schedule for examinations: weekly recording

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to termination, obtained by cardiac puncture
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: all - 3 males and 3 females per group
- Parameters checked : haematocrit; haemoglobin; erythrocytes; total and differential leucocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:immediately prior to termination, obtained by cardiac puncture
- Animals fasted: Yes
- How many animals:all - 3 males and 3 females per group
- Parameters checked : alkline phosphatase AP; lactic dehydrogenase; serum glutamic-oxalacetic transaminase GOT; serum glutamic-pyruvic transaminase GPT; glucose, cholesterol, calcium, phosphorus; uric acid; blood urea nitrogen, total protein, total bilirubin and albumin

URINALYSIS: Yes
- Time schedule for collection of urine: samples collected directly from under housing cages on day prior to or day of terminal sacrifice
- Metabolism cages used for collection of urine: No
- Animals fasted: No
- Parameters checked : appearance, pH, ketones, occult blood, bilirubin, protein, glucose, microscopic examination of sediment .

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
On day 31, all surviving rats were sacrificed following an overdose of ether . All animals including any decedents were examined for gross pathology. The following tissues were processed for histoptholoical examination:

treated and untreated skin
adrenals
heart
lung and bronchi
mesenteric lymph nodes
thyroid
pituitary
brain (3 sections)
sternal bone marrow
spinal cord
testes with epididymus
ovaries
spleen
urinary bladder
nerve with muscle
kidney
gross lesions

Other examinations:

no data

Statistics:

no data

Clinical signs:

effects observed, treatment-related

Dermal irritation:

no effects observed

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

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:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
No clinical signs of adverse reaction to dermal application of doses from 0.188 to 1.25 g/kg. In the high dose group three males and 1/3 females died between days 6 and 9. Prior to death the majority of these rats showed anogenital discharge, tremors and ataxia. One of the surviving females showed anogenital discharge and hyperactivity on days 7 to 9 but survived the episode, the second surviving female had tremors on days 6 to 8.

BODY WEIGHT AND WEIGHT GAIN: For rats in the groups dosed at 0.188, 0.301, 0.488 or 0.781 g/kg, there were no effects on bodyweight or weight gain. Bodyweight gains in the group dosed at 1.25 g/kg were slightly lower than the other groups. In the high dose group, 2.0 g/kg, 4/6 rats died and bodyweight gains were not assessed for the remaining rats.

DERMAL REACTIONS: The treated skin sites (seven areas on the dorsum dosed on a 7 day rotation) were not affected by repeated topical application of benzyl benzoate despite the recording of a few instances of slight redness or oedema.

HAEMATOLOGY - No notable differences between groups for any of the blood parameters. White blood cell counts and differential counts in groups 5 and 6 were not significantly different (despite only two rats surviving at the highest dose level). Mean white cell counts for the treated groups 1-6 were 10.4; 11.9; 10.1; 11.2; 8.4 and 8.6 respectively.

CLINICAL CHEMISTRY - no effects on any of the parameters except for a trend for higher uric acid levels in groups treated at the lower dose levels

URINALYSIS - no effects of treatment were detected in any of the urinary parameters examined at any dose level.

GROSS PATHOLOGY: all animals, including the decedents, showed no macroscopic abnormalities during necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC - The histopathologically recognised effects of dermal application of benzyl benzoate to rats are slight squamous epithelial hyperplasia, degeneration of hair follicles and sebaceous glands, subcutaneous fibrosis and thyroid gland hyperplasia.

Dose descriptor:

NOAEL

Effect level:

781 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reduced weight gain at 1250 mg/kg bw/d; mortality at 2000 mg/kg bw/d

Critical effects observed:

yes

Lowest effective dose / conc.:

1 250 mg/kg bw/day

System:

other: Reduced weight gain at 1250 mg/kg bw/d; mortality at 2000 mg/kg bw/d

Organ:

other: Reduced weight gain at 1250 mg/kg bw/d; mortality at 2000 mg/kg bw/d

Treatment related:

yes

Dose response relationship:

yes

Conclusions:

Repeated application of 2000 mg/kg bw/d resulted in signs of toxicity and mortality; however effects at lower dose levels were limited to reduced weight gain at 1250 mg.kg bw/d. A clear NOAEL of 781 mg/kg bw/d can therefore be derived for this study.

Executive summary:

Benzyl benzoate was applied topically to sites on the dorsum of 36 rats (six groups of three males and three females) at doses of 188, 301, 488, 781, 1250 or 2000 mg/kg bw/d. Daily checks for mortality, clinical signs of reaction to treatment and assessment of any dermal changes at treatment sites were completed and bodyweights were recorded at weekly intervals. Blood and urine samples were collected shortly prior to termination and all rats were subject to gross necropsy.

No effects of treatment were apparent for rats dosed at 188, 301, 488 or 781 mg/kg bw/d. A slight reduction in bodyweight gain was evident for rats dosed at 1250 mg/kg bw/d in comparison with other treated groups.

Blood analysis indicated a slight increase in mean uric acid levels for rats dosed at 301 mg/kg bw/d but in the absence of similar effects at higher or lower doses, the biological significance of this change was unclear. No biologically significant macroscopic changes were evident at any dose level. Histopathological examinations revealed squamous epithelium hyperplasia, degeneration of hair follicles and sebaceous glands, subcutaneous fibrosis and thyroid gland hyperplasia. A clear NOAEL of 781 mg/kg bw/d is determined for this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

Study duration:

subacute

Species:

rat

Quality of whole database:

One 30-day range-finding rat study and one non-standard 90-day rabbit are available

Additional information

The cytostatic activity and general systemic toxicity of benzyl benzoate was investigated following administration of 200 or 800 mg/kg bw/d 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/d.

 

In a subacute dermal toxicity investigation benzyl benzoate was applied topically to sites on the dorsum of 36 rats (six groups of three males and three females) at doses of 188, 301, 488, 781, 1250 or 2000 mg/kg bw/d over a four week period.

Clinical signs and mortality were observed at the highest dose level; the lowest effect level was 1250 mg/kg bw/d - rats gained slightly less weight than the remaining groups. A NOAEL of 781 mg/kg bw/d is therefore derived for this study.

No biologically significant macroscopic changes were evident at any dose level. There were no observations of local dermal irritation which were clearly attributable to treatment. Histopathological examinations revealed squamous epithelium hyperplasia, degeneration of hair follicles and sebaceous glands, subcutaneous fibrosis and thyroid gland hyperplasia in all treated groups; effects on the skin did not exhibit any dose-response relationship. Similar effects were not apparent on untreated skin, however it is unclear whether the histopathological effects observed in the treated skin were due to the test material, the dressing and/or the repeated shaving of the skin at the application site.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

In a 13 week study male and female rats received once daily on 5 days/week 0, 50, 100, 200, 400, 800 mg/kg bw benzyl alcohol via gavage. Based on clinical signs and reduced body weight development in males and females and histopathological changes in the brain at 800 mg/kg bw the NOAEL was considered to be 400 mg/kg bw/day (NTP TR 343, 1989).

 

Benzoic acid (BA) was administered in the diet to male and female Sprague Dawley Crl:CD(SD) rats in an OECD Test Guideline 443 Extended One-Generation Reproductive Toxicity (EOGRT) study to test for effects that may occur as a result of pre- and postnatal exposure. The study included cohorts of F1 offspring to evaluate potential effects of benzoic acid on reproduction, the developing immune system, and the developing neurological system with the inclusion of learning and memory assessments. Benzoic acid was incorporated in the diet at concentrations of 0, 7,500, 11,500, and 15,000 mg/kg diet (ppm). These concentrations were selected based on the results of preliminary studies, and, based on average food consumption, were intended to supply BA doses of approximately 0, 500, 750, and 1000 mg/kg bw/day. To avoid exceeding these target dose levels, the dietary
The highest dietary concentration (15,000 ppm), providing a dosage of approximately 1000 mg/kg bw/day, was the NOAEL for benzoic acid in this EOGRT study.

 

The NOAEL of 400 mg/kg bw/day from the 13 week study with benzyl alcohol is used for the derivation of the systemic DNELs.

Justification for classification or non-classification

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.