Benzyl benzoate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published study of non-standard design but considered to be scientifcally sound and adequate for the assessment of developmental toxicity.

Data source

Reference

Reference Type:

publication

Title:

Safety Evaluation of Chemicals for Use in Household Products (II) Teratological Studies on Benzyl Benzoate and 2-(Morpholinothio)-benzothiazole in Rats

Author:

MORITA S, YAMADA A, OHGAKI S, NODA T & TANIGUCHI S

Year:

1981

Bibliographic source:

Environ. Sci. 43: 90-97

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Pregnant Wistar rats were given commercial powdered diets supplemented with 0.04 or 1.0% benzyl benzoate (BB) from day 0 of gestation to day 21 post-parturition. Various parental reproductive indices and developmental indices, in utero and post-partum were evaluated.

GLP compliance:

not specified

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:

No details provided

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: No data
- Age at study initiation: ~6 weeks
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): Oriental powdered feed M. The test material was administerd by dietary admixture at two inclusion levels, diet given ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-25
- Humidity (%): 45-55
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no details

IN-LIFE DATES: From: no data;n To: no data. Study findings published in 1981.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: powdered diet

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): no details
- Mixing appropriate amounts with (Type of food): Dose levels were selected on basis of a preliminary study (data not reported here), the maximum tolerated dose of benzyl benzoate was 1% dietary inclusion. The lower dose was 0.04%.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No data

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight, from pro-oestrous to oestrous.

- Proof of pregnancy: time sperm observed in the vaginal smear was referred to as Day 0 of pregnancy/gestation.

A vaginal smear was taken from nulliparous females ad examined microscopically to determine stage of oestrous cycling. The females were pair housed with a single male over night from the estimated pro-oestrous to oestrous stages. A second smear was examined each morning after overnight pairing and if sperm were observed in the smear then that was designated day 0 of pregnancy.

Duration of treatment / exposure:

Dams had access to treated diets from Gestation Day 0 to Day 20 and from Day 0 to Day 21 post-partum.

Frequency of treatment:

daily via treated diet

Duration of test:

Gestation day 0 -20
Gestation day 0 to day 21 post partum

No. of animals per sex per dose:

21 females per group
One group of 21 females was split - 14 animals were terminated at Day 20 and 7 retained for a 21 day post-partum phase.

Control animals:

yes, plain diet

Details on study design:

No further details available

Examinations

Maternal examinations:

Pre-partum Study phase
General clinical observations of the condition of pregnant animals were made daily together with body weight recordings and recording of treated or control diet consumption.

Dams were sacrificed on Day 20 of pregnancy by ether anaesthesia, the abdomen was immediately opened, the uterus was removed, the number of implantation sites, number of resorptions and number of foetal deaths were determined, the body weight of live foetuses was measured and the gender and presence of external abnormalities was investigated.

In addition, the number of corpora lutea in the maternal ovaries were determined.

Post-partum Study:
Pregnant females were allowed to give birth naturally and the number of live offspring and number of dead offspring were determined immediately after confirming birth had taken place. Neonates were allowed to suckle from dams until Day 21 postpartum and, during this time, the growth differentiation conditions such as the gender, the number of live newborn, presence of external abnormalities, pinnae detachment, emergence of hair and opening of eyelids as well as general conditions were observed. Body weights were measured 7, 14 and 21 days postpartum. Surviving neonates were sacrificed by ether anaesthesia up to Day 21 post-partum and a skeletal examination was conducted using soft X rays. In addition, dams were sacrificed in the same way and the number of implantation sites was investigated.

Ovaries and uterine content:

Dams were sacrificed on Day 20 of pregnancy by ether anaesthesia, the abdomen was immediately opened, the uterus was removed, the number of implantation sites, number of resorptions and number of foetal deaths were determined, the body weight of live foetuses was measured and the gender and presence of external abnormalities was investigated.

In addition, the number of corpora lutea in the maternal ovaries were determined.

Fetal examinations:

For the pre-partum treatment groups, skeletal staining was conducted in approximately half the live foetuses using Alizarin red S according to the method of Dawson following fixation in 90% ethanol. The presence of skeletal abnormalities and the degree of ossification were investigated. The remaining foetuses had abnormalities in internal organs determined according to the method of Wilson, following fixation in Bouin's solution.

For the postpartum phase, pregnant females were allowed to give birth naturally and the number of live offspring and number of dead offspring were determined immediately after confirming birth had taken place.

The neonates were raised naturally and allowed to suckle from dams until Day 21 postpartum. During this period growth differentiation conditions were monitored - gender, number of live newborn, presence of external abnormalities, pinnae detachment, emergence of hair and opening of eyelids. The general condition of the pups was also observed through this period. Pup body weights were recorded 7, 14 and 21 days postpartum.

Surviving neonates were sacrificed by ether anaesthesia on Day 21 postpartum and a skeletal examination was conducted using soft X rays. The dams were also sacrificed on Day 21 and the number of implantation sites was determined.

Statistics:

Significant differences between the control group and administration groups were investigated using the χ2 test, t-test or the rank sum test. Furthermore, data handling was conducted with reference to the standard method in 'The Joint Parallel Study Relating to Teratogenicity of LAS by The Ministry of Health, Labour and Welfare 1974 group'.

Historical control data:

no information available

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Benzyl benzoate effects on dams in the pre-partum phase.

Benzyl benzoate ingestion via treated diet at 0.04 and 1% had little effect on body weight progression for pregnant female rats. The daily food intake for treated and control groups showed no marked variations between Gestation days 0 and 20.

Benzyl benzoate ingested was calculated in terms of mg/rat over the entire period of gestation (Day 0-20). The low group (0.04%) mean total consumption was 153.4 mg/rat, equivalent to 7.7 mg benzyl benzoate per day. For the high dose group (1%), total consumption was 3886.7 mg/rat and 194.3 mg per day.

There were no noteworthy changes in the general conditions for either treated groups over the gestation period. Benzyl benzoate caused no macroscopic changes attributable to treatment.

Effect levels (maternal animals)

Dose descriptor:

dose level: 194.3 mg/rat per day.

Remarks on result:

other: no effect on pregnant females

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Benzyl benzoate effects on foetuses
Foetal effects on Day 20 of pregnancy are presented in Tables 2 and 3 attached.
There were few differences between control and treated groups in relation to the numbers of corpora lutea incidence of pregnancy, the number of implantations, the number of foetal deaths (either early stage or late stage), the number of live foetuses, the pup sex ratio and the body weights of live foetuses.

From the external observations on Gestation Day 20, one foetus in the control group had an umbilical hernia. In the benzyl benzoate 1% treated groups there was one foetus each with mandibular defects, absence of a tongue or a cleft palate but there was no significant difference in incidence compared with controls.
No effects were apparent in the low dose treated group.
the skeletal observations revealed no malformations in any group. The occurrence of foetuses with incomplete ossification of sternebral centres decreased in the high dose group, benzy benzoate 1.0%. The visceral observations revealed one foetus in the high dose group, 1% benzyl benzoate had bilateral heterotaxiabut there was no significant difference in incidence of findings between treated and control groups.

Other abnormalities noted but not showing any treatment relationship, or statistically significant difference in incidence, included dilation of the renal pelvis (seen in one foetus in the low dose 0.04% benzyl benzoate group), dilation of the renal pelvis (two foetuses) and bisection of the apex (one foetus) were observed in the high dose group, 1% benzyl benzoate.

Effects on neonates during post-partum phase
Neonate body weights for the benzyl benzoate groups a small suppression of weight gain and by Day 14 both male and female pups from treated dams had slightly lower bodyweight than controls and a similar effect was also present on day 21 although the effect was not dose dependent. No changes of note in general condition occurred during the 21 day post-partum phase for any group.

Postnatal growth at parturition and for the neonates is presented in Table 4, (attached below). There were no biologically significant differences in the pup indices between any group in the gestation period. The number of implantations, the number of live offspring, the number of dead offspring, pup sex ratio, suckling rate and growth differentiation were similar for all groups. No external or skeletal abnormalities were seen in any of the groups

Effect levels (fetuses)

Dose descriptor:

dose level: 194.3 mg/rat per day.

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

 

Table 1 Food intake and amount of benzyl benzoate (BB) ingested for pregnant rats administered BB in diet

	Control	BB     0.04%	BB 1.0%
Bodyweight (g)
Gestation Day 0	267	274	274
Gestation Day 20	366	378	377
Food intake (g/rat)
Total (GD 0-20)	365	384	389
Per day	18.3	19.2	19.3
BB ingested (mg/rat)
Total (GD 0-20)	0	153.4	3886.7
Per day	0	7.7	194.3

 

 

Table 2 Effect of dietary BB given to pregnant rats on the foetuses

	Control	BB     0.04%	BB 1.0%
Number of females inseminated Number of pregnant females Number of dams with live foetuses	20 14 14	19 14 14	22 14 14
Mean number of corpora lutea	202	201	202
Mean number of implants	175	175	167
Number of dead fetuses (early) [%] Number of dead fetuses (late)	26  [16.3] 0	25  [13.0] 0	19  [11.2] 1  [0.6]
Mean number of live fetuses	149	150	147
Sex ratio (male:female pups)	1.13  [79/70]	1.00  [75/75]	1.13  [78/69]
Mean bodyweight of live fetuses Male Female	3.90 3.76	4.03 3.70	3.98 3.91

 

 

Table 3  External, skeletal and visceral observations of fetuses from dams fed BB in diet

	Control	BB     0.04%	BB 1.0%
External observations
Number of foetuses examined	149	150	147
Number of foetuses with malformations (%)	1 (1.0)	0	1 (1.8)
Malformation type (no. of fetuses affected)	Umbilical hernia (1)	--	Submaxilla defect (1) Tongue defect (1) Cleft palate (1)
Skeletal observations
Number of foetuses examined	82	82	79
Number of foetuses with malformations	0	0	0
Number of foetuses with variations (%)
Asymmetry of sternebrae	2 (3.0)	1 (1.0)	2 (2.2)
Cervical rib	8 (7.3)	6 (6.7)	3 (3.9)
Lumbar rib	1 (1.2)	1 (0.9)	0
Split or bifurcation of 1^stor 2^ndcervical vertebral arch	0	0	0
Extra lumbar vertebra (L7)	1 (1.2)	0	0
Separation of thoracic vertebral centra	1 (1.2)	0	0
Degree of ossification
Number of foetuses with incomplete sternebrae (%)	27 (29.4)	28 (36.3)	15 (18.6*)
Mean number of proximal and middle phalanges forepaw hindpaw	5.8 4.0	5.4 4.0	5.6 4.0
Mean number of sacral and caudal vertebrae	7.9	7.8	8.0
Visceral observations
Number of foetuses examined	67	68	68
Number of foetuses with malformations (%)	0	0	1 (1.2)
Type (number of foetuses)	--	--	Heteroataxia (1)
Number of foetuses with other anomalies (%)	0	1 (1.2)	3 (3.8)
Type (number of foetuses)	--	Dilatation of renal pelvis (1)	Dilatation of renal pelvis (2) Bifid apex of heart (1)

 

 

Table 4 Postnatal observations for offspring from dams fed BB in diet

	Control	BB     0.04%	BB 1.0%
Number of females inseminated Number of pregnant females Number of dams that delivered pups Number of lactating dams	11 10 10 7	13 10 10 7	13 11 11 7
Mean duration of gestation (days)	21.7	22.0	21.7
Mean number of implants	79	71	81
Mean number of live newborns	68	61	74
Mean number of dead newborns	1	0	0
Sex ratio (male/female	1.3 (39/30)	1.18 (33/28	1.11 (39/35)
Viability at weaning, %  ##	83.1	92.1	88.5
External differentiation (mean no. days) detachment of ears hair growth opening of eyelids	2.5 8.7 14.9	2.3 8.6 14.2	2.8 9.3 14.6
Number of pups with external anomalies	0	0	0
Number of pups with skeletal anomalies	0	0	0

 

## No of live pups at 21 days postpartum / no of live pups at birth x 100

 

Applicant's summary and conclusion

Conclusions:

No evidence of teratogenicity or developmental toxicity was observed under the conditions of this assay.

Executive summary:

In this study, pregnant female rats were administered benzyl benzoate throughout gestation, with treatment continuing to Day 21 post-partum in some animals. Benzyl benzoate administered in diet to pregnant rats at inclusion levels of 0.04% and 1.0% . The effects of benzyl benzoate administration to the pregnant females, on foetuses and neonates were examined.

Benzyl benzoate administration had little effect on foetuses and neonates and no teratogenic effects of benzyl benzoate were evident.