Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1960
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Limited, reported, non-GLP, the data are sufficient for evaluation of the reproduction end point
Qualifier:
no guideline followed
Principles of method if other than guideline:
A feeding study was performed, in which 4 generations of rats received the test substance (0.5% or 1%). The first generation was exposed for 8 weeks an then allowed to mate (1/1 for a period of 14 days). Mating was repeated in week 48 to raise a second litter. Survival of the first and second generation was measured. The third generation was terminated after 16 weeks and examined histopathologically. In this generation weights of brain, heart, liver, spleen, kidneys and testes were determined. The fourth generation was terminated after weaning of the pups. Body weights were determined in week 4, 8 and 12 weeks of each generation (week 12 males only). Feeding efficiency was measured in all generations after 2,4, 6 and 8 weeks. Some reproduction parameters were assessed: percentage of infertility, delayed sexual maturation, litter size, total pups, surviving pups. These parameters were assessed for all generations (summed) and forthe first two generations separately
GLP compliance:
no
Limit test:
no
Species:
rat
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
generation I and II mean 785-827 days
generation III 16 weeks
generation IV until weaning of first litter
Frequency of treatment:
continuously in feed
Remarks:
Doses / Concentrations:
0, 0.5 and 1%
Basis:
nominal in diet
No. of animals per sex per dose:
20 animals
Control animals:
yes, plain diet
Positive control:
None stated
Parental animals: Observations and examinations:
Body weights were determined in week 4, 8 and 12 weeks of each generation (week 12 males only).
Reproductive parameters: percentage of infertility, delayed sexual maturation, litter size, total pups
Oestrous cyclicity (parental animals):
None stated
Sperm parameters (parental animals):
None stated
Litter observations:
number of pups, survival of pups, litter size
Postmortem examinations (parental animals):
Histopathology of generation III animals on week 16
Weights of brain, heart, liver, spleen, kidneys and testes in generation III animals
Postmortem examinations (offspring):
none stated
Statistics:
None stated
Reproductive indices:
None stated
Offspring viability indices:
None stated
Clinical signs:
not specified
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
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
1% the test substance was tolerated without unfavourable side-effects on growth, food utilization, duration of life, procreation, weight of organs, histological pattern of organs and fertility.
Addition of 0.5% the test substance to the food resulted in significant prolongation of life.
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Reproductive effects observed:
not specified

The authors include a calculation of the 1% dose related to the saturation of the metabolic system at that concentration. They conclude that no bioaccumulation will occur. In addition, theoretically the conclusion was derived that the no effect level in this study is closed to the maximum tolorated level for the test substance.

Conclusions:
No effects were noted after chronic dietary exposure of rats to 1% the test substance in diet. Furthermore, no effects were observed on fertility in four generations after chronic dietary exposure of rats to 1% the test substance in diet. No effects on reproduction were seen.
Executive summary:

A feeding study was performed, in which 4 generations of rats received the test substance (0.5% or 1%). The first generation was exposed for 8 weeks an then allowed to mate (1/1 for a period of 14 days). Mating was repeated in week 48 to raise a second litter. Survival of the first and second generation was measured. The third generation was terminated after 16 weeks and examined histopathologically. In this generation weights of brain, heart, liver, spleen, kidneys and testes were determined. The fourth generation was terminated after weaning of the pups. Body weights were determined in week 4, 8 and 12 weeks of each generation (week 12 males only). Feeding efficiency was measured in all generations after 2,4, 6 and 8 weeks. Some reproduction parameters were assessed: percentage of infertility, delayed sexual maturation, litter size, total pups, surviving pups. These parameters were assessed for all generations (summed) and forthe first two generations separately No effects were noted after chronic dietary exposure of rats to 1% the test substance in diet. Furthermore, no effects were observed on fertility in four generations after chronic dietary exposure of rats to 1% the test substance in diet. No effects on reproduction were seen.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
chronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Animal data

 

No reproductive toxicity studies are available with the notified substance, 3-PPB. However subacute, subchronic and chronic studies, a 4-generation study, a dominant lethal test and a sex-linked recessive lethal test on read across category members are available by the dermal or oral routes. No effects on reproduction were seen in a 4-generation study with benzoic acid by the oral route (feeding study). Both the dominant lethal test and sex-linked recessive lethal test were found to be negative. No compound related effects on reproductive organs (gross and histopathology examination) could be found in the (sub) chronic studies in rats and mice with benzyl alcohol, phenethyl alcohol, 2-methyl-4-phenylpentanol, 2-methyl-5-phenylpentanol, or sodium benzoate. Although toxic effects on rat testes from subchronic exposure to benzyl benzoate and benzyl acetate were found to be treatment related, these effects of benzyl benzoate and benzyl acetate on testes seem to occur at high dose levels (dermal doses of 2000 mg/kg/day and above and at dietary doses of 900 mg/kg/day and above, respectively. Moreover no effects on the fertility of the rats were observed in a 4-generation reproduction study with benzoic acid and no effects on sperm motility, density or percent abnormality were observed when assessed in a 13-week dietary study with benzyl acetate in any of the doses tested.

Taking into account the available data on the reproductive toxicity of the members of the category 3-PPB is not expected to be classified as a reprotoxicant.


Justification for selection of Effect on fertility via oral route:
This endpoint has been evaluated by a weight of evidence approach which includes this 4-generation study and subacute, subchronic and chronic studies with read across analogues of 3-PPB (part of the benzyl derivatives category). Although this study is limited and non-GLP, the data are sufficient for evaluation of the reproduction end point. It was performed with benzoic acid which is the final metabolite 3-phenyl propyl benzoate. Repeated dose studies in rats and mice are available with the alcohol analogues of 3-phenyl-1-propanol, benzyl alcohol, phenethyl alcohol, 2-methyl-4-phenylpentanol and 2-methyl-5-phenylpentanol and showed that no there were no compound related effects on reproductive organs (see read across justification document in section 13 (Jupe, 2015)) .

Effects on developmental toxicity

Link to relevant study records
Reference
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.
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
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
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
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
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
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.
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
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
Abnormalities:
not specified
Developmental effects observed:
not specified

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

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).
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
646 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Animal data

 

The teratogenicity of benzyl benzoate has been investigated in Pregnant Wistar rats given commercial flour diets supplemented with 0.04 or 1.0% benzyl benzoate, from day 0 to day 20 of gestation (Morita et al., 1980). These dietary levels correspond to the average daily intake of 26 and 646 mg/kg bw. There were no harmful effects of benzyl benzoate on the foetuses with respect to external, skeletal and visceral anomalies. In addition, benzyl acetate and sodium benzoate were tested for developmental toxicity in teratology studies and only produced adverse effects on the foetus at maternally toxic concentrations. At a dose not maternally toxic, phenylethyl alcohol did not affect litter values in a teratology study. However, there were slight differences from control values in respect of some of the morphological changes observed at this dosage which cannot be discounted entirely. The authors suggested that the relevance of the slight differences from control values observed at the non-maternally toxic dose was debatable and it would be best considered as a threshold dosage for developmental toxicity in the rat.

In a recently published prenatal toxicity study, benzyl benzoate treatment did not induce external malformations in the skeleton (Koçkaya and Kiliç, 2011). However, the authors described increased resorption and effects on liver, kidney and heart tissues and differences in the immunolocalisation of vascular endothelial growth factor (VEGF) in pregnant Wistar rats treated with a daily oral dose of 25 and 100 mg/kg benzyl benzoate. This study was not deemed valid to use for 3-PPB classification and risk assessment as it has seriuos flaws such as the low number of animals used, poor experimental detail and the lack of control data.

 

Human data

 

In a retrospective controlled cohort study, Myttonet al. looked at the effects of topically applied 25% benzyl benzoate on women's

pregnancy outcome (2007). Overall only 10.9% of treatments were in the first trimester, 42.2% in the second and 46.9% in the third. The overall median gestation of exposure was 24.5 weeks. They found no evidence of adverse effects on pregnancy outcome, no significant increased risk of abortion, stillbirth, congenital abnormality, neonatal death, reduction in birthweight or gestational age or proportion of premature infants.

 

Based on all this data, 3-PPB is not expected to be classified as a reprotoxicant.


Justification for selection of Effect on developmental toxicity: via oral route:
This endpoint has been evaluated by a weight of evidence approach which includes developmental toxicitystudies with read across analogues of 3-PPB (part of the benzyl derivatives categorysee read across justification document in section 13 (Jupe, 2015)) . Although this study is a published study of non-standard design, it is considered to be scientifically sound and adequate for the assessment of developmental toxicity.

Justification for classification or non-classification

Developmental toxicity studies, subacute, subchronic, chronic studies, 4-generation study and epidemiological data performed with the benzyl derivatives category indicate that these compounds are not reprotoxic or developmental toxicants.

Taking into account the available data on reprotoxicity and developmental toxicity of the members of this category it is considered likely that 3-PPB is not a significant reprotoxicant and should not be classified as such under CLP EU Regulation 1272/2008.

CLP EU Regulation 1272/2008.
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