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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 December 1998 - 31 December 1998
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
See below ("Principles of method if other than guideline").
Deviations:
yes
Remarks:
Evaluation was made with knowledge of treatment group, as procedures are already in place to minimise bias during these portions of the study.
Qualifier:
according to
Guideline:
other: Ministry of Agriculture, Forestry and Fisheries for Japan, Noh San No. 4200.
Deviations:
no
Qualifier:
according to
Guideline:
other: European Economic Communities, Comission Directive 94/79EEC
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Benzoflex 9-88 (Dipropylene glycol dibenzoate DPGDB)
- Physical state: A clear liquid
- Analytical purity: Dipropylenne glycol dibenzoate - 94.84 Area%
- Impurities (identity and concentrations):
Dipropylene glycol monobenzoate - 2.91 Area%
Propylene glycol dibenzoate 1 - 1.79 Area%
2- (2-Propylenoxy) 1-propyl benzoate - 0.30 Area%
- Lot/batch No.: 56826710
- Storage condition of test material: At ambient temperature
Specific details on test material used for the study:
The substance is mixture of benzoates and it is a clear liquid. The substance can be stored at ambient temparture.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England.
- Age at study initiation: 10 to 11 weeks
- Weight at study initiation: 214 to 273g
- Fasting period before study: Not reported
- Housing: Cages consisting of stainless steel (acclimatisation and mating) or high-density polypropylene (gestation) bodies with lids and floors of stainless steel grid, suspended in batteries over trays covered with absorbent paper
- Diet: Free access to a commercially available pelleted laboratory animal diet
- Water: Tap water from the public supply was freely available.
- Acclimation period: At least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Nominally 21°C (Range 19 - 23°C)
- Humidity (%): Nominally 55% (range 40 - 70%)
- Air changes (per hr): At least 15
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light.


IN-LIFE DATES: From: 2 December 1998 To: 31 December 1998

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the highest required concentration (200 mg/mL) the required amount of DPGDB was weighed out and mixed with a small amount of the vehicle. This mixture was quantitatively transferred to the mixing vessel with further portions of vehicle and adjusted to volume. Homogenisation of the final product was achieved using a mechanical blender such as a Silverson stirrer/emulsifier. Lower concentrations were prepared by serial dilution. The use of equipment containing polar rubber compounds or polyvinyl chloride was avoided during the preparation/dispensing of formulations as much as possible.


VEHICLE
- Justification for use and choice of vehicle (if other than water): DPGDB has poor water solubility
- Concentration in vehicle: 0, 50, 100, and 200 mg/mL (for dose levels 0, 250, 500, and 1000 mg/kg/day, respectively).
- Amount of vehicle (if gavage): 5 mL/kg.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken from formulations prepared for use during the first and last weeks of the dosing period for determination of achieved concentrations of DPGDB. On each occasion of sampling four samples (nominally 1 mL accurately weighed) were taken from each formulation; 2 assays were performed from each test group and one assay for the control group. The remainder of the samples were frozen (nominally -20°C) as contingency for analysis if any result required confirmation. These samples were taken in the Pharmacy department using the types of glass syringes and rubber catheters used to dose the animals. This precaution was taken to provide assurance that contact between the dose formulations and the rubber catheter did not affect the achieved concentrations of DPGDB.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: one-to-one
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: The day on which a sperm positive vaginal smear or at least three copulation plugs were found was designated Day 0 of
gestation.
Duration of treatment / exposure:
13 Days (Days 6 to Day 19 after, inclusive)
Frequency of treatment:
Once per day administration
Duration of test:
20 Days
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
22 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Eighty eight females showing unequivocal evidence of mating were allocated to group and cage position in sequence thus ensuring that animals mated on any one day were evenly distributed amongst the groups

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Pre-dosing, on return of the animal to home cage, after dosing each group, 1 to 2 hours after completion of dosing, and as late as possible during the working day.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least twice daily (as above)


BODY WEIGHT: Yes
- Time schedule for examinations: Weighed on days 0, 3, 6 to 17 inclusive and 20 after mating


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



WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: The organs of the reproductive tract, complete with ovaries.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (Uterus with cervix)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: The number and distribution of fetuses in each uterine horn
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
Statistical tests employing analysis of variance followed by an inter group comparison with the Control were performed on the following parameters:
Bodyweight change, bodyweight change adjusted for gravid uterine weight, food consumption, litter data, litter weight, fetal weight and placental
weight.
Dependant on the heterogeneity of variance between treatment groups, parametric tests (analysis of variance, Snedecor and Cochran 1967) followed by Williams' test (Williams 1971/2) or non parametric tests (Kruskal-Wallis, Hollander and Wolfe 1973) followed by Shirley's test (Shirley 1977) were used to analyse these data, as appropriate.
For litter data (excluding fetal, litter and placental weights) and implantation loss, due to the preponderance of non-normal distributions, non-parametric tests are generally the most consistent and were routinely used.
All significant (i.e. p<0.05) inter-group differences from the Control are reported only where supported by a significant analysis ofvariance (i.e. p<0.05).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
one animal showed salivation after the second dose (Day 7) at 1000 mg/kg/d but by the fifth dose (Day 10) the majority of animals were affected. The pattern was similar at 500 mg/kg/d except that the day of first occurence was Day 10 (fifth dose). At 250 mg/kg/d, the occurrence of salivation was much more transitory, with no animals being affected on more than four cosecutive day.
Mortality:
no mortality observed
Description (incidence):
There were no deaths observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of treatment on absolute or adjusted bodyweight gain.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect on treatment on food intake
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No treatment related effects were observed.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no effects of treatment were indicated by the extent of pre and post implantaion loss. The mean percentage value for pre implantaion loss for control group was 7.1, 6.6 for Group 2, 5.2 for Group 3 and 5.4 for Group 4. No statistical significance values were acheived. The Group 1 is control (0 mg/kg/d) and group 2 is 250 mg/kg/d and group 3 is 500 mg/kg/d and group 4 is 1000 mg/kg/d dose group.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no effects of treatment were indicated by resorption. The mean percentages for total resorption was 0.9, 0.8, 0.8 and 1.0 for Group 1,2,3 and 4. The Group 1 is control (0 mg/kg/d) and group 2 is 250 mg/kg/d and group 3 is 500 mg/kg/d and group 4 is 1000 mg/kg/d dose group.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no effects of treatment were indicated by early or late resorption. The mean percentages for early resorption was 0.9, 0.7, 0.8 and 1.0 for Group 1,2,3 and 4. The mean percentages for early resorption was 0.0, 0.0, 0.0 and 0.0 for Group 1,2,3 and 4. The Group 1 is control (0 mg/kg/d) and group 2 is 250 mg/kg/d and group 3 is 500 mg/kg/d and group 4 is 1000 mg/kg/d dose group.
Dead fetuses:
no effects observed
Description (incidence and severity):
No effects of treatment on prenatal survival and the number of live fetuses were observed. The mean group values for live fetus were similar in all groups.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
22 pregnant females per group were evaluted after treatment of females from day 6 to day 19.
Description (incidence and severity):
1 (0 mg/kg/d) 2(250 mg/kg/d)3(500 mg/kg/d)4 (1000 mg/kg/d)

pregnant females 22 22 22 22
evalated pregnant females 22 22 22 22


The group mean values for litters were

1 2 3 4
Corpora lutea 16.8 16.3 16.5 16.0
Implantations 15.4 15.4 15.6 15.1
Resorptions 0.9 0.8 0.8 1.0
Live fetus 14.5 14.6 14.9 14.1
Sex ratio (%) male 49.7 51.5 49.6 49.1
female 50.3 48.5 50.4 50.9
weight of fetus (g) male 3.88 3.94 3.87 3.84
female 3.71 3.75 3.68 3.66
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Clinical signs associated with treatment were restricted to transient post-dosing salivation.
There was no effect of treatment on absolute or adjusted bodyweight gain.
There was no effect of treatment on food intake.
There were no findings considered to be related to treatment.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
clinical signs

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The overall fetal body weights were comparable to control and were not statistically significant in any group in comparison to control. The overall mean (g) weight was 3.79 in control 1 and 3.85, 3.77 and 3.74 gm in Group 2, 3 and 4. The Group 1 is control (0 mg/kg/d) and group 2 is 250 mg/kg/d and group 3 is 500 mg/kg/d and group 4 is 1000 mg/kg/d dose group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio was 49.7, 51.5, 49.6 and 49.1% in Group 1, 2, 3 and 4. The Group 1 is control (0 mg/kg/d) and group 2 is 250 mg/kg/d and group 3 is 500 mg/kg/d and group 4 is 1000 mg/kg/d dose group.
Description (incidence and severity):
There were no effects of treatment were indicated in litter weights. The mean (g) litter weight was 54. 94 in control 1 and 56.14, 56.01 and 52.64 gm in Group 2, 3 and 4. The Group 1 is control (0 mg/kg/d) and group 2 is 250 mg/kg/d and group 3 is 500 mg/kg/d and group 4 is 1000 mg/kg/d dose group.
Description (incidence and severity):
The live young male and female percentage in Group 1,2,3 and 4 were 7.2, 7.4, 7.4 and 7 and 7.3, 7.2,7.5 and 7.1. The treated groups were comparable to the control group.
External malformations:
no effects observed
Description (incidence and severity):
There were no effect of treatment on the incidence of external malformations were observed.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
at 1000 mg/kg/d, the skeletal examination revealed a small but definite increase in the incidence of cervical ribs compared with the controls and the background historical control data. Therefore the findings of this skeletal anomaly is considered to be related to treatment. At 500 and 250 mg/kg/d, the incidence of fetuses with cervical ribs was within the recent background control data and there was no clear dosage relationship, their occurence at these dosages is likely to be coincidental and unrelated to the treatment.

An association between treatment at 1000 and 500 mg/kg bw/day and the greater number of fetuses with incomplete ossification of the 5th and or 6th sternebrae cannot be discounted particularly since a delay in ossification would be expected to be the most sensitive marker of an effect on pre-natal development where treatment has continued through to the day before sacrifice (treatment period: Days 6 to 19 of gestation). The assessment of fetal ossification on Day 20 of gestation represents a snapshot in time as the ossification will continue as the animals grow and mature. Although the relationship of these findings to treatment is uncertain they are considered to be transient in nature rather than representing permanent structural changes and therefore are considered to be of no long-term toxicological importance.
The increase in cervical ribs at 1000 mg/kg bw/day is considered to be of greater toxicological significance as it occurred at a dosage which has not produced any detectable signs of maternal toxicity however cervical ribs were only found in a small number of fetuses (10/155) at the limit dosage of 1000 mg/kg bw/day and there was no concomitant change in vertebral configuration.

Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
There were treatment related minor visceral anomalies were obsreved.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
An association between treatment at 1000 and 500 mg/kg/day and the greater number of fetuses with incomplete ossification of the 5th and or 6th sternebrae cannot be discounted particularly since a delay in ossification would be expected to be the most sensitive marker of an effect on pre-natal development where treatment has continued through to the day before sacrifice (treatment period: Days 6 to 19 of gestation). The assessment of fetal ossification on Day 20 of gestation represents a snapshot in time as the ossification will continue as the animals grow and mature. Although the relationship of these findings to treatment is uncertain they are considered to be transient in nature rather than representing permanent structural changes and therefore are considered to be of no long-term toxicological importance.

The increase in cervical ribs at 1000 mg/kg/day is considered to be of greater toxicological significance as it occurred at a dosage which has not produced any detectable signs of maternal toxicity however cervical ribs were only found in a small number of fetuses (10/155) at the limit dosage of 1000 mg/kgl/day and there was no concomitant change in vertebral configuration.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Sex:
male/female
Basis for effect level:
skeletal malformations

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The no-adverse-effect-level for maternal toxicity was concluded to be 1000 mg/kgbw/day
The no-observed adverse effect level for all aspects of pre natal development was 500 mg/kgbw/day.
Executive summary:

A pre-natal development study in rats was conducted to determine the effect of the test material DPGDB when administered during and beyond the organogenesis phase of gestation. The study was conducted according to Japanese, US EPA and OECD test guidelines, and in compliance with GLP.

Groups of 22 female rats were selected after mating, and were dosed by oral gavage with corn oil fortified with the test material between day 6 and day 19 of gestation. Dose levels examined were 0 (vehicle control), 250, 500, and 1000 mg/kg bw/day.

An association between treatment at 1000 and 500 mg/kg bw/day and the greater number of fetuses with incomplete ossification of the 5th and or 6th sternebrae cannot be discounted particularly since a delay in ossification would be expected to be the most sensitive marker of an effect on pre-natal development where treatment has continued through to the day before sacrifice (treatment period: Days 6 to 19 of gestation). The assessment of fetal ossification on Day 20 of gestation represents a snapshot in time as the ossification will continue as the animals grow and mature. Although the relationship of these findings to treatment is uncertain they are considered to be transient in nature rather than representing permanent structural changes and therefore are considered to be of no long-term toxicological importance.

The increase in cervical ribs at 1000 mg/kg bw/day is considered to be of greater toxicological significance as it occurred at a dosage which has not produced any detectable signs of maternal toxicity however cervical ribs were only found in a small number of fetuses (10/155) at the limit dosage of 1000 mg/kg bw/day and there was no concomitant change in vertebral configuration.

Salivation after dosing was observed at all dosages of benzoflex, the incidence was dose related but this finding was not considered to be of toxicological importance. At 1000 mg/kg/d, there were no detectable signs of maternal toxicity, there were no maternal deaths and all females had a live litter scarifice. It was concluded that the 1000 mg/kg/d is the NOAEL for maternal toxicity.There were no treatment related effects observed at prenatal survival or growth. At 1000 mg/kg/d, treatment related small but definite increase in the number of fetus with cervical ribs were observed. The no-observed adverse effect level for all aspects of pre-natal development is concluded to be 500 mg/kg bw/day.