Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 December 1998 - 07 January 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with Japanese, US EPA, OECD and EC test guidelines, and in compliance with GLP.

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)
Version / remarks:
2nd Draft Guideline, March 1998
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See below ("Principles of method if other than guideline").
Qualifier:
according to
Guideline:
other: European Economic Communities, Comission Directive 94/79EEC
Deviations:
no
Qualifier:
according to
Guideline:
other: Ministry of Agriculture, Forestry and Fisheries for Japan, Noh San No. 4200.
Deviations:
no
Principles of method if other than guideline:
Deviation from EPA OPPTS 870.3700 guideline: The Guideline states that, "Evaluation of the dams during caesarian section and subsequent fetal analyses should be conducted without knowledge of treatment group in order to minimise bias". Evaluation was made with knowledge of treatment group, as procedures are already in place to minimise bias during these portions of the study. These procedures include routine reviews of necropsy technicians evaluation skills and scientific peer review of at least 25% of the raw data of the fetal analyses including examination of serial sections for visceral anomalies and examination of fetal skeletons.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Benzoflex 2-45 (Diethylene glycol dibenzoate DEGDB)
- Physical state: Clear colourless liquid but solidified to an opaque white crystalline solid after receipt
- Storage condition of test material: Room temperature, dessicated

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: Approximately 10 to 11 weeks of age.
- Weight at study initiation: 214 to 265g
- Fasting period before study: Not reported.
- Housing: Cages consisting of stainless steel (acclimatisation and mating) or high density polypropylene (gestation period) bodies with lids and floors of stainless steel grid, suspended in batteries over trays covered with adsorbent paper.
- Diet (e.g. ad libitum): Free access to a commercially available pelleted laboratory animal diet.
- Water (e.g. ad libitum): Tap water from the public supply was freely available to the animals via polythene or polycarbonate bottles with sipper tubes.
- Acclimation period: A minimum of 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Nominally 21°C (Range 19 - 23°C); achieved 20 - 21°C
- Humidity (%): Nominally 55% (range 40 - 70%); achieved 46 - 57%
- Air changes (per hr): At least 15
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light.

IN-LIFE DATES: From: 14 December 1998 (Animals paired for mating) To: 07 January 1999 (completion of necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For each concentration the required amount of DEGDB was weighed out, following warming in a water bath at approximately 40 - 45°C and mixed with the required amount of warmed vehicle. Homogenisation of the final product was achieved using a mechanical blender such as a Silverson stirrer emulsifier. The use of equipment containing polar rubber compounds or polyvinyl chloride was avoided during the preparation dispensing of formulations as much as possible.

VEHICLE
- 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 also taken from formulations prepared for use during the first and last weeks of the dosing period for determination of achieved concentrations of DEGDB. 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 DEGDB.
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 19 after mating, inclusive).
Frequency of treatment:
Once per day administration
Duration of test:
19 days after mating.
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.

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): 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, treatment-related
Description (incidence and severity):
Clinical signs associated with treatment were restricted to transient post-dosing salivation.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Bodyweight and bodyweight gain at all dosages were generally similar to the Control group throughout and overall bodyweight gain from Day 6 adjusted for the input of the gravid uterus did not indicate any obvious adverse effect of treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption during gestation was similar in all groups and did not indicate any obvious adverse effect of treatment.
Food efficiency:
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
The incidence of findings at necropsy was low and did not indicate any obvious adverse effect of DEGDB.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Maternal developmental toxicity

Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
Post-implantation loss was higher in all treated groups compared to the concurrent Control, differences attaining statistical significance at 500 and 1000 mg/kg/day. However, values were comparable with recent background control data and it is considered that the test groups were disadvantaged by a particularly high survival rate in the Control. It was concluded that in utero survival had not been adversely affected by treatment since live litter size was unaffected and was similar in all groups.
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.
Bodyweight and bodyweight gain at all dosages were generally similar to the Control group throughout and overall bodyweight gain from Day 6 adjusted for the input of the gravid uterus did not indicate any obvious adverse effect of treatment.
Food consumption during gestation was similar in all groups and did not indicate any obvious adverse effect of treatment.
The incidence of findings at necropsy was low and did not indicate any obvious adverse effect of DEGDB.

Effect levels (maternal animals)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day mean fetal weights, and consequently litter weight, were slightly lower than Control, combined fetal weight and female fetal weightattaining statistical significance. Placental weight was comparable with Control.
At 250 and 500 mg/kg/day litter weight, mean placental and fetal weights were similar or superior to the concurrent Control values.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio assessed by percentage males was similar in all groups.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were 4, 1, 3, and 2 fetuses (4, 1, 3, and 1 litters affected) showing malformations in Groups 1 to 4 respectively. Neither the type, incidence nor distribution of these findings indicated any obvious association with treatment.
Other effects:
no effects observed
Description (incidence and severity):
The incidence and distribution of visceral anomalies did not indicate any obvious adverse effect of treatment.
At 1000 mg/kg/day 4 fetuses (3 litters affected) showed cervical ribs, this incidence being higher than the concurrent Control and marginally outside the current background control data. Although the incidence of this finding was relatively low it is considered that a treatment relationship could not be ruled out. There was a clearer increase in the incidence of incomplete ossification, principally affecting the cranial centres, sacrocaudal vertebral arches, 5th/6th sternebral centres and pelvic bones compared with the concurrent Control.
At 500 mg/kg/day the incidence and distribution of skeletal anomalies did not indicate any obvious adverse effect of treatment. There was a slight increase in the incidence of incomplete ossification of the 5th/6th sternebral centres, however the incidence was within that seen for the background data and any relationship to treatment was considered equivocal.
The incidence and distribution ofskeletal anomalies and variants at 250 mg/kg/day did not indicate any obvious adverse effect of treatment.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Post-implantation loss was higher in all treated groups compared to the concurrent Control, differences attaining statistical significance at 500 and 1000 mg/kg/day. However, values were comparable with recent background control data and it is considered that the test groups were disadvantaged by a particularly high survival rate in the Control. It was concluded that in utero survival had not been adversely affected by treatment since live litter size was unaffected and was similar in all groups.
Sex ratio assessed by percentage males was similar in all groups.
At 1000 mg/kg/day mean fetal weights, and consequently litter weight, were slightly lower than Control, combined fetal weight and female fetal weightattaining statistical significance. Placental weight was comparable with Control.
At 250 and 500 mg/kg/day litter weight, mean placental and fetal weights were similar or superior to the concurrent Control values.
There were 4, 1, 3, and 2 fetuses (4, 1, 3, and 1 litters affected) showing malformations in Groups 1 to 4 respectively. Neither the type, incidence nor distribution of these findings indicated any obvious association with treatment.
The incidence and distribution of visceral anomalies did not indicate any obvious adverse effect of treatment.
At 1000 mg/kg/day 4 fetuses (3 litters affected) showed cervical ribs, this incidence being higher than the concurrent Control and marginally outside the current background control data. Although the incidence of this finding was relatively low it is considered that atreatment relationship could not be ruled out. There was a clearer increase in the incidence of incomplete ossification, principally affecting the cranial centres, sacrocaudal vertebral arches, 5th/6th sternebral centres and pelvic bones compared with the concurrent Control.
At 500 mg/kg/day the incidence and distribution of skeletal anomalies did not indicate any obvious adverse effect of treatment. There was a slight increase in the incidence of incomplete ossification of the 5th/6th sternebral centres, however the incidence was within that seen for the background data and any relationship to treatment was considered equivocal.
The incidence and distribution ofskeletal anomalies and variants at 250 mg/kg/day did not indicate any obvious adverse effect of treatment.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The no-effect-level (NOEL) for maternal toxicity was concluded to be 1000 mg/kg/day.
The no-adverse-effect-level (NOAEL) for fetal growth and development was 500 mg/kgbw/day and the no-observed-effect-level was 250 mg/kgbw/day.
Executive summary:

A pre-natal development study in rats was conducted to determine the effect of the test material DEGDB when administered during and beyond the organogenesis phase of gestation. The study was conducted according to Japanese, US EPA, OECD, and EC 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/kgbw/day.

Clinical signs related to treatment were limited to post-dosing salivation; a dose-response effect was seen, but was thought to be due to the palatability of the test material. All animals survived to termination and were pregnant with live young in utero.

A slight reduction in mean fetal weights and an increased incidence in the number of fetuses showing cervical ribs was seen in the 1000 mg/kgbw/day group when compared to the control. In each case the effects seen were slight, but could not be ruled out as being evidence of a toxicological effect. A slight increase in the incidence of incomplete ossification was seen in the 500 mg/kgbw/day level, however when compared to the backgroud data a relationship to treatment was considered equivocal. There were no obvious indications of an adverse effect of treatment in the 250 mg/kg/day level.

The no-effect-level for maternal toxicity was concluded to be 1000 mg/kgbw/day. The no-adverse-effect-level for fetal growth and development was 500 mg/kgbw/day and the no-observed-effect-level was 250 mg/kgbw/day.