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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
It is a study that has been published in a peer reviewed journal. The restriction is also due to the use of the read across approach: the test was performed not with 3-PPB but with benzyl benzoate, a substance which has been demonstrated to be very similar in structure, physical/chemical properties and toxicological profile.

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

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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
No details provided

Test animals

Species:
rat
Strain:
Wistar
Details on test animals 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 To: no data. Study findings published in 1981Administration / exposureRoute of administration

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
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.04 and 1%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
26 and 646 mg/kg bw/day
Basis:
actual ingested
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)

open allclose all
Dose descriptor:
NOAEL
Effect level:
646 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
646 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

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

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

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

   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  
 BB ingested (mg/rat)  18.3  19.2  19.3
 Total (GD 0 -20)  0  153.4  3886.7
Per day       

     

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

   Control  BB 0.04%  BB 1.0%
 Number of females inseminated 20  19   22
 Number of pregnant females  14  14  14
 Number of dams with live foetuses  14  14  14
 Mean number of corpora lutea 202  201  202
  Mean number of implants  175  175  167
 Number of dead foetuses  0  0  1 {0.6}
 Sex ratio (male:female pups)  1.13 (79/70)  1.00 (75/75)  1.13(78/69)
Mean body weight oflive foetuses       
 Male  3.90  4.03  3.98
Female   3.76  3.70  3.91
       

Table 3: External, skeletal and visceral observations of foetuses 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)
 Malformaiton type (no. of foetuses affected)  umbilical hernia (1)  0  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)
 Split or bifurcation of 1st or 2nd cervical vertebral arch  0  0  0
 Extra lumbar vertebrae (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 (5)  27 (29.4)  28 (36.3)  15 (18.6*)
 Mean number of proximal and middle phalangesforepawhindpaw  5.04.0  5.44.0 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)
 Types (number of foetuses) --  --  Heteroataxia (1)
 Number of foetuses with other anomalies (5)  --  Dialation of renal pelvis(1)  Dilation of renal pelvis (2)Bifid apex of heart (1)
       

Table 4 Post natal observations for offspring from dams fed BB in diet

   Control  BB 0.04%  BB 1.0%
 Number of females inseminated  11  13  13
 Nmber of pregnant females  10  10  11
 Number of dams that delivered pups  10  10  11
 Number of lactating dams  7  7  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  2.5  2.3  2.8
 hair growth  6.7  8.6  9.3
 opening of eyelids  14.9  14.2  14.6
 Number of pups with external anomalies  0  0  0
 Number of pups with skeletal anomalies  0  0  0

## Number of live pups at 21 days post partum/number of live pups at birth x 100

Applicant's summary and conclusion

Conclusions:
In this developmental toxicity study, benzyl benzoate administered in diet to pregnant rats at inclusion levels of 0.04% and 1.0% from Gestation Day 0 to Day 21 post-partum produced no evidence of maternal or developmental toxicity. The maternal and developmental NOAEL is 1.0% (eq.to 646 mg/kg/day).