Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Pairing commenced: 13 May 2019 Treatment commenced: 20 May 2019 Experimental completion date (fetal pathology) 30 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Test item: Acetylacetone Peroxide (13784-51-5)
Test item identity (including alternative names): Acetylacetone Peroxide
2,4-Pentanedione, peroxide (upper limit: 42% w/w; typical concentration: 30% w/w)
Trigonox 44B
CAS number: 13784-51-5
Intended use: Substance used in industry
Appearance: Clear, colorless liquid
Storage conditions: Ambient temperature 15-25°C in the dark
Supplier: Sponsor
Batch number: 1809428243
Expiry date: 13 June 2021
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 0.5 ml representative sample was taken from the batch of test item. This sample was placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Strain/Species RccHan™:WIST rat.
Supplier Envigo RMS Limited.
Number of animals ordered 90 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization 5 days before commencement of pairing.
Age of the animals at the start of the study (Day 0 of gestation) 77 to 83 days old.
Weight range of the animals at the start of the study (Day 0 of gestation) 176 to 220 g.

Animal Care and Husbandry
Environmental Control
Rodent facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 20-24ºC and 40-70%.
There were no deviations from these ranges.
Lighting Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods.
Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.
Cage distribution The cages constituting each group were blocked by group and mounted in batteries.
Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage
Acclimatization up to four animals
During pairing one (stock) male and one female
Gestation one female

Environmental Enrichment
Aspen gnawing material Provided to each cage throughout the study and replaced when necessary.
Plastic shelter Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

Diet Supply
Diet SDS VRF1 Certified pelleted diet.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted.

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.

Supplier Certificates of Analysis
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen gnawing material.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Formulation
Group Treatment Dose Formulated Dose volume
(mg/kg/day) concentration (mL/kg)
(mg/mL)
1 Vehicle 0 0 10
2 Acetylacetone Peroxide (13784-51-5) 50 5 10
3 Acetylacetone Peroxide (13784-51-5) 150 15 10
4 Acetylacetone Peroxide (13784-51-5) 500 50 10

Correction factor None.

Vehicle Purified water.

Method of preparation Acetylacetone Peroxide was prepared for administration by dilution of individual weighings of the test item. The required amount of test item was weighed out into a suitable container and approximately 50% of the final volume of vehicle was added and mixed with a magnetic stirrer until the formulation appeared visibly homogenous. The solution was then made up to the required volume with vehicle and stirred with a magnetic stirrer until homogenous.

Frequency of preparation Weekly.

Storage of formulation Refrigerated (2-8°C) for up to 15 days and 24 hours at ambient temperature (15-25 C).

Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Stability and homogeneity Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 100 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.

Achieved concentration Samples of each of the first and last preparation formulations were analyzed for achieved concentration of the test item.

Administration
Route Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.

Treated at Constant doses in mg/kg/day.

Dose volume 10 mL/kg body weight.

Individual dose volume Calculated from the most recently recorded scheduled body weight.

Control (Group 1) Vehicle at the same dose volume as treated groups.

Frequency Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.

Formulation Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The samples were analyzed in accordance with the validated Covance Analytical Procedure (DFA/M002/19).

The analytical method involved extraction and dilution in acetonitrile/water 5/95 v/v followed by reverse phase high performance liquid chromatographic analysis with ultra violet detection at 195 nm. Sample concentrations were determined with reference to external standards prepared in the concentration range 40 μg/mL to 400 μg/mL.

Concentration of Dose Formulations
The formulations for Day 6 and Day 19 were sampled, 2 × 3 mL (accurately weighed), from the middle of the formulation by Pharmacy personnel.

Duplicate aliquots from the first sample were analyzed in accordance with the analytical procedure. The remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.
The mean concentrations were within ±5% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 4%, confirming precise analysis.
Details on mating procedure:
Mating
Male/female ratio 1:1 with identified stock males.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
Day 0 of gestation When positive evidence of mating was detected.

A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.

Allocation and Identification
Allocation On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.

Method To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups.

Allocation was controlled to prevent any stock male from providing more than one mated female in each treatment group.

Identification of animals Each animal was assigned a number and identified uniquely within the study by a microchip inserted subcutaneously in the dorsal cervical region.

Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).
Duration of treatment / exposure:
Females Day 6 to 19 after mating
Frequency of treatment:
Once daily
Duration of test:
Day 0-6: Mating
Day 6-19: Treatment
Day 20: Necropsy
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle only
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
20 females per dose hroup
Control animals:
yes, concurrent vehicle
Details on study design:
Animal Model
The rat was chosen as the test species because it is accepted by regulatory agencies. The Han Wistar (RccHan™;WIST) strain was used because of the historical control data available at this laboratory

Route of Administration
The oral route of administration was chosen as it is a possible route of human exposure during manufacture, handling or use of the test item. The test item was administered by gavage.

Rationale for Dose Level Selection
The doses of the main study where based on effects observed in a preliminary study.
In the preliminary study three groups, each comprising 6 females received the test item at doses of 250, 500 or 1000 mg/kg/day at a volume dose of 10 mL/kg, from, from Day 6 to 19.
In this study effects on gravid uterine weight, adjusted body weight change and fetal weight at 500 or 1000 mg/kg/day were possibly due to the observed statistically significant low food consumption.
Gravid uterine weight was low at 250, 500 or 1000 mg/kg/day (83%, 78% or 72% of Control, respectively). Adjusted body weight change was markedly low at 500 or 1000 mg/kg/day (76% or 52% of Control, respectively).
When compared with Control, overall food consumption (Days 6-20) was low in females receiving 500 or 1000 mg/kg/day (86% or 81% of Control, respectively) and slightly correlated with the low body weight gain observed in these animals.
Overall body weight gain (Days 6-20) was slightly low in females receiving 250 mg/kg/day and was low at 500 or 1000 mg/kg/day (84%, 76% or 69% of Control, respectively).
Total litter weight and overall fetal weight (both males and females) were low at 250, 500 or 1000 mg/kg/day (83 and 93%, 76 and 88% or 67 and 79% of Control, respectively).
In the preliminary study there were no macroscopic findings in the adult and no fetal abnormalities attributable to treatment at any dose.
The reduction of in overall mean fetal weight at 1000 mg/kg/day was considered to preclude the use of that dose on this study. Therefore, doses of 0, 50, 150 or 500 mg/kg/day were selected for investigation in this study.

See tables in 'any other information on materials and methods including tables'

Allocation and Identification
Allocation On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.

Method To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups.

Allocation was controlled to prevent any stock male from providing more than one mated female in each treatment group.

Identification of animals Each animal was assigned a number and identified uniquely within the study by a microchip inserted subcutaneously in the dorsal cervical region.

Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Examinations

Maternal examinations:
Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.


During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration:

Immediately before dosing
At the end of dosing each group.
One to two hours after completion of dosing all groups.
As late as possible in the working day.

Clinical Signs
A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

Body Weight
The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

Food Consumption
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive after mating.

Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

The retained tissues were checked before disposal of the carcass.

Schedule Animals were killed on Day 20 after mating.
Sequence To allow satisfactory inter-group comparison.

Ovaries and uterine content:
The following was recorded for all females:
For each ovary/uterine horn

Number of:
Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).

Apparently non pregnant animals and for apparently empty uterine horns
The number of uterine implantation sites were checked after staining with ammonium sulphide (modification of the Salewski staining technique (Salewski, E, 1964)).
Fetal examinations:
Fetal Examination and Processing
Examination of all viable fetuses and placentae
Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. The sex and ano-genital distance of each fetus was recorded.

Examination of nominally 50% of fetuses in each litter
Sexed internally and eviscerated

Fixation
Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS).

Remaining fetuses were fixed whole in Bouin’s fluid.

Processing
Bouin’s fixed fetuses were subject to free-hand serial sectioning.

IMS fixed fetuses were processed and stained with Alizarin Red.

Fetal Pathology Examination
Bouin’s fixed fetuses Serial sections were examined for visceral abnormalities.
Alizarin Red stained fetuses Assessed for skeletal development and abnormalities.

Statistics:
Where appropriate, group mean values, each with standard deviation (SD), were calculated from individual data. Summary tabulated data was normally restricted to data derived from females/litters with live young at Day 20 after mating. Group mean values and standard deviations were frequently calculated using a greater number of decimal places than presented in the appendices. It is, therefore, not always possible to derive exact group values from the data presented in the appendices.
Indices:
Reproductive Assessment
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:
Pre-implantation loss (%) = (Number of corpora lutea - Number of implantations) / Number of corpora lutea x 100

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).
Post-implantation loss was calculated from the formula:
Post-implantation loss (%) = (Number of implantations - Number of live fetuses) / Number of implantations x 100

All group values and SD were calculated from the individual litter values.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs attributable to treatment and no signs associated with administration.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Overall body weight and adjusted body weight gains (Days 6-20) were unaffected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by treatment.
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:
no effects observed
Description (incidence and severity):
Thyroid weight was unaffected by treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination revealed no test item related lesions. The incidence and distribution of all other findings were considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The microscopic examination revealed no test item related lesions. The incidence and distribution of all other findings were considered to be unrelated to treatment.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Thyroid Hormone Analysis

Thyroid Stimulating Hormone (TSH)
Mean serum TSH concentrations were high, when compared with Control at 150 or 500 mg/kg/day (139% or 195%, respectively) and attained statistical significance at 500 mg/kg/day.
Mean serum T3 concentrations were similar to Control at 50, 150 or 500 mg/kg/day.
Mean serum T4 concentration were low and attained statistical significance at 500 mg/kg/day (71%). Mean serum T4 concentration were unaffected by treatment at 50 or 150 mg/kg/day.
PLEASE REFER TO TABLES IN SECTION "ANY OTHER INFORMATON ON RESULTS"

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The number of implantations, resorptions, pre- and post-implantation losses were unaffected by treatment.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
> 500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No effects observed

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Placental and total litter weights were unaffected by treatment.

Overall fetal weight (male and female) at 500 mg/kg/day was marginally, but statistically significantly low (89% of Control). Overall fetal weights at 50 or 150 mg/kg bwt/day were unaffected by treatment.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Fetal Pathology
There was an increase, when compared with Control, in the incidence of unossified/incompletely ossified sternebrae along with a decrease in incidence of ossified cervical vertebral centra at 500 mg/kg/day that were outside the Historical Control Data range.
The overall pattern of skeletal ossification is consistent with a generalized delay. Generalized delays have a common “fingerprint”, characterized by reduced ossification of bones that normally exhibit rapid ossification during the last few days of gestation, such as the phalanges, sternebrae (especially numbers 5 and 6), calvarium and the cervical, thoracic, sacral and caudal vertebral centra (Carney and Kimmel 2007). There are no indications of a deviations from the normal pattern of development, normal cartilage is present. These minor skeletal variants are related to decreased fetal body weight and of low significance and are therefore considered non-adverse.

Incidental findings:
The only major abnormalities were seen in a single fetus (Dam/Litter No. 59) at 150 mg/kg/day were considered a spontaneous occurrence and clearly not related to maternal treatment.
Visceral malformations:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Thyroid hormone analysis

Text table 1     Mean serumTSH concentrations (pg/mL) on Day 20 of gestation

Group

Treatment

Dose

(mg/kg/day)

 

Day 20

of gestation

1

Control

0

Mean

734

%CV

60.8

N

18

2

Acetylacetone Peroxide (13784-51-5)

50

Mean

792

%CV

44.3

N

19

3

Acetylacetone Peroxide (13784-51-5)

150

Mean

1020

%CV

59.8

N

20

4

Acetylacetone Peroxide (13784-51-5)

500

Mean

1430***

%CV

61.2

N

19

*** p< 0.001

Triiodothyronine (T3) and Thyroxine (T4)

Text table 2     Mean serum T3 concentrations(pg/mL)

Group

Treatment

Target Dose

(mg/kg/day)

Terminal Females

1

Control

(Vehicle)

0

Mean

458

SD

111

CV %

24.2

N

18

2

Acetylacetone Peroxide

(13784-51-5)

50

Mean

449

SD

82

CV %

18.2

N

19

3

Acetylacetone Peroxide

(13784-51-5)

150

Mean

507

SD

115

CV %

22.7

N

20

4

Acetylacetone Peroxide

(13784-51-5)

500

Mean

492

SD

109

CV %

22.2

N

19

Text table 3     Mean serum T4 concentrations (pg/mL)

Group

Treatment

Target Dose

(mg/kg/day)

Terminal Females

1

Control

(Vehicle)a

0

Mean

18500

SD

5020

CV %

27.1

N

18

2

Acetylacetone Peroxide

(13784-51-5)

50

Mean

17800

SD

4240

CV %

23.8

N

19

3

Acetylacetone Peroxide

(13784-51-5)

150

Mean

19100

SD

4430

CV %

23.2

N

20

4

Acetylacetone Peroxide

(13784-51-5)

500

Mean

13700*

SD

5200

CV %

38.0

N

19

*p<0.05(Williams' test)

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the maternal NOAEL is 500 mg/kg/day.

Overall fetal weight (male and female) at 500 mg/kg/day was marginally, but statistically significantly low (89% of Control), in the absence of any reduction in maternal body weight gain/food consumption. Overall fetal weights at 50 or 150 mg/kg bw/day were unaffected by treatment. The developmental NOAEL is 150 mg/kg/day.

Executive summary:

Summary

The purpose of this study was to assess the influence of Acetylacetone Peroxide (13784‑51‑5), a substance used in industry, on embryo-fetal survival and development when administered orally, by gavage, to Han Wistar rats during the organogenesis and fetal growth phases of pregnancy. Three groups, each comprising 20 females received the test item at doses of 50, 150 or 500 mg/kg/day at a volume dose of 10 mL/kg, from, from Day 6 to 19 after mating and a similarly constituted Control group received the vehicle (purified water) at the same dose volume. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

In the adult, clinical observations, body weight, food consumption, organ weights, thyroid hormones, macropathology and histopathology investigations were undertaken. The fetuses were examined macroscopically, the ano‑genital distance was measured and all fetuses underwent a detailed internal visceral or skeletal examination.

Results

There were no clinical signs and no signs associated with administration.

Overall body weight, gravid uterine weight and gravid uterine adjusted body weight gain and food consumption were unaffected by treatment.

There were no macroscopic findings in the adult related to treatment.

Adult serum thyroid stimulating hormone concentrations were high at 150 or 500 mg/kg/day and serum thyroxine concentrations were low at 500 mg/kg/day. Serum triiodothyronine concentrations were unaffected by treatment.

Thyroid weight was unaffected by treatment and there was no pathological change in the thyroid gland. The reduction in adult serum T4 levels at 500 mg/kg/day, together with the clear increase in serum TSH levels suggests a minor perturbation of thyroid function that did not result in any detectable microscopic changes in the thyroid glands. The slight increase in serum TSH at 150 mg/kg/day did not attain statistical significance and occurred in isolation with no supporting findings and is of uncertain biological significance and relationship to treatment. In a 90 -day study no effects on T4, T3 or TSH where observed.

Implantation, resorption, pre- and post-implantation losses and the number of live young and the ratio of male to female fetuses and placental weight were unaffected by treatment.

At 500 mg/kg/day, fetal weight was 89% of Control,and there was an increased incidence of unossified/incompletely ossified sternebrae and a decreased incidence of ossified cervical vertebral centra: these findings were considered to represent a retardation in fetal development associated with the retarded fetal growth. The overall pattern of skeletal ossification is consistent with a generalized delay. Generalized delays have a common “fingerprint”, characterized by reduced ossification of bones that normally exhibit rapid ossification during the last few days of gestation, such as the phalanges, sternebrae (especially numbers 5 and 6), calvarium and the cervical, thoracic, sacral and caudal vertebral centra (Carney and Kimmel 2007). There are no indications of a deviations from the normal pattern of development, normal cartilage is present. These minor skeletal variants are related to decreased fetal body weight and of low significance and are therefore considered non-adverse.

Fetal ano-genital distancewas unaffected by treatment.

Conclusion

Based on the results of this study, the maternal NOAEL is 500 mg/kg/day.

In the range finding study when compared with control, overall food consumption was low in females receiving 500 or 1000 mg/kg/day and slightly correlated with the low body weight gain observed in these animals. Overall body weight gain (Days 6-20) was slightly low in females receiving 250 mg/kg/day and was low at 500 or 1000 mg/kg/day. Total litter weight and overall fetal weight (both males and females) were low at 250, 500 or 1000 mg/kg/day (83 and 93%, 76 and 88% or 67 and 79% of Control, respectively).

Overall fetal weight (male and female) at 500 mg/kg/day was marginally, but statistically significantly low (89% of Control), in the absence of any reduction in maternal body weight gain/food consumption. Overall fetal weights at 50 or 150 mg/kg bw/day were unaffected by treatment. The developmental NOAEL is 150 mg/kg/day.