Trimanganese tetraoxide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 December 2016 to 22 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau.

Version / remarks:

November 24, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

trimanganese tetraoxide

IUPAC Name:

trimanganese tetraoxide

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 70 days of age.
- Weight at study initiation: 220 to 319 g.
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatisation and gestation periods. Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing. During acclimatisation up to four animals were housed together. During pairing, the animals were housed as one (stock) male and one female. During gestation one female was housed per cage.
- Diet: SDS VRF1 Certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Food was available ad libitum.
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Water was available ad libitum.
- Acclimation period: Five days.

ENVIRONMENTAL CONDITIONS
- Temperature: Monitored and maintained within the range of 20 - 24 ºC.
- Humidity: Monitored and maintained within the range of 40 - 70 %.
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: Artificial lighting, 12 hours light : 12 hours dark.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1.0 % w/v methylcellulose (MC) aqueous solution.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The required amount of test material was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogeniser.
- A series of suspensions/formulations at the required concentrations were prepared by dilution of individual weighings of the test material.
- Frequency of preparation: Weekly
- Storage of formulation: Formulations in the concentration range of 1 to 200 mg/mL are stable at ambient temperature (15 to 25 °C) for up to 1 day; refrigerated (2 to 8 °C) for up to 15 days.
- Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

VEHICLE
- Concentration in vehicle: Constant doses in mg/kg/day, calculated from the most recently recorded scheduled body weight.
- Amount of vehicle: Dose Volume 5 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 200 mg/mL were analysed to assess the stability and homogeneity of the test material in the liquid matrix.
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 3 of treatment were analysed for achieved concentration of the test material.

Preparation of Calibration Standards
A primary standard solution (1 000 μg/mL) was prepared by dissolving an accurately weighed quantity (ca. 50 mg) of the test material in extract solvent (20 mL) which was then made to volume (50 mL) with diluent.
Solutions for instrument calibration were prepared by appropriate dilution of the primary standard using diluent to contain the test material at nominal concentrations of 2 μg/mL, 4 μg/mL, 5 μg/mL, 6 μg/mL, 8 μg/mL and 10 μg/mL.
Calibration solutions were read on the Atomic Absorption Spectrometer, at the beginning of each sample analysis sequence and then standard 3 at an interval of every 10 samples.

Preparation of Test Samples
A representative sample of test formulation (1 mL, accurately weighed) was dissolved using ultrasonic vibration in a suitable volume of extract solvent (20 mL) and made to volume (200 mL) using diluent. The extract was diluted using diluent, to provide a solution containing the test material at an expected concentration within the range 4 μg/mL to 8 μg/mL. The concentration of the test material in the final solution was quantified by Atomic Absorption Spectrometer.

Preparation of Recovery Samples
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (1 % w/v methylcellulose) with known amounts of the test material. The prepared procedural recoveries were analysed in accordance with the analytical procedure.

Instrumentation Parameters
Atomic Absorption Spectrometer (AAS): Perkin Elmer, AAnalyst 200
Lamp: Perkin Elmer Manganese Lumina, 6 mA
Fuel: Acetylene, 2.5 L/min
Oxidant: Air 10 L/min
Wavelength: 279.48 nm
Slit width: 1.8/0.6 mm
Integration time: 3.0 seconds
Number of replicates: 3
Read delay: 5 seconds

Calculations
The response of the test material in each calibration standard was measured. Calibration curves were constructed by quadratic (2nd order) regression of the response versus calibration standard concentration. The response observed for the test material in sample and procedural recovery solution was measured. The concentration of the test material was determined using the following equation:

Analysed concentration, mg/mL = (-b + √(b^2 - 4a (c - Y)) / 2a) x (V / W) x (D / 1 000)

Procedural recovery values were determined using the following equation:

Procedural recovery (%) = (Analysed concentration, mg/mL / Fortified concentration, mg/mL) x 100

Where
Y = Mean absorbance response for the test material in test sample
a,b,c = Coefficients derived from quadratic regression of calibration data
V = Dilution volume of sample (mL)
W = Weight of sample (g)
D = Density of sample (g/mL)
Note: A nominal mass of 1 g for sample weight was used when calculating results for procedural recovery samples.

Validation of the Analytical Procedure
The analytical procedure was validated by determining the following parameters:
- The specificity of the analysis in control samples.
- The linearity of detector response over the calibration standard concentration range.
- The repeatability of the mid-level calibration standard.
- The method accuracy and precision, by determining five procedural recoveries at nominal concentrations of 1 mg/mL and 200 mg/mL during the method validation.

Homogeneity in 1 % w/v Methylcellulose Formulations
The homogeneity of the test material in 1 % w/v methylcellulose formulations were assessed at nominal concentrations of 1 mg/mL and 200 mg/mL, during ambient and refrigerated storage.
Freshly prepared specimen formulations (400 mL) were equally sub divided into 4 amber glass screw top bottles by Pharmacy personnel and submitted for analysis.

Ambient Temperature Storage (15 to 25 °C)
On receipt, the contents of one bottle (Bottle 1) of each formulation were mixed by 20-fold inversion followed by magnetic stirring. After stirring for 20 minutes (representing 0 hour) and 1 and 2 hours, single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the continuously stirred formulation. The remainder of the bottle was stored at ambient temperature and after 1 days storage the contents were remixed and sampled as detailed above.

Refrigerated Storage (2 to 8 °C)
The remaining bottles (Bottles 2, 3 and 4) were refrigerated on receipt and on Day 1, Day 8 and Day 15; the appropriate bottle was removed from storage and equilibrated to ambient temperature. The contents of the bottles were mixed by 20-fold inversion followed by magnetic stirring for 20 minutes and single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the stirred formulation.

Concentration of Dose Formulations
For first and last occasions, six 1 mL samples (accurately weighed), were taken from the middle of the formulation by Pharmacy personnel. Three samples were analysed in accordance with the analytical procedure. The remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved. The contingency samples for first occasion were analysed due to a suspected instrument error during the original analysis.

RESULTS
Method Validation
The analytical procedure was successfully validated for the test material with respect to the specificity of analysis, the linearity of detector response, repeatability, method accuracy and precision.

The specificity of the AA assay was demonstrated by the absence of a response for the test material in the control sample.
Linearity was confirmed over the nominal concentration range 2 μg/mL to 10 μg/mLwith a coefficient of determination >0.992. The repeatability was <5 % for six replicate readings of a standard solution containing the test material at a nominal concentration of 5 μg/mL. A mean procedural recovery value of 99.8 % (CV=2.44 %, n=5) was obtained for 1 mg/mL and 101.4 % (CV=0.79 %, n=5) was obtained for 200 mg/mL.

Homogeneity of Dose Formulations
The homogeneity of the test material in 1 % w/v methylcellulose formulations were assessed with respect to the level of concentration at nominal concentrations of 1 mg/mL and 100 mg/mL. Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1 day and refrigeration for up to 15 days. At each time-point, the mean analysed concentration for the three samples remained within 9 % of the initial time zero value and the coefficient of variation was less than 7.5 %.
Recovery results during the trial remained within ± 7.5 % of the mean recovery found during validation showing the continued accuracy of the method. This is with the exception of 2 recoveries which were excluded in accordance with SOPs.

Concentration of Dose Formulations
The mean concentrations of the test material in test formulations analysed during the study and the deviation of the mean result from the nominal value were assessed. For the last occasion the mean concentrations were within 6 %, confirming the accuracy of formulation. Coefficient of variation values remained within 3 %, confirming the accuracy of analysis. For the first occasion samples the mean concentrations were within 23 % and the coefficient of variation values were within 15 %.

For the last occasion, procedural recovery results remained within ± 7.5 % of the mean recovery found during validation showing the continued accuracy of the method. For the first occasion the procedural recovery results were variable.

Clarification of Study Conduct
The first and last occasion samples were analysed outside of their confirmed stability/homogeneity period. Atomic absorption is not a stability indicating method and in this case only measures the amount of manganese present in a sample. Manganese will not be unstable therefore the fact that the samples were analysed outside of their confirmed stability period had no impact upon the results. The last occasion samples were all within acceptable limits, confirming that the animals were dosed the correct amount of test material, and also showing that no degradation has occurred. The first occasion Group 2 samples were all within acceptable limits. Group 3 and Group 4 had a high % CV and Group 4 also had a high mean concentration. Of the six samples analysed for Groups 3 and 4, three samples had a concentration of >15 % of nominal concentration, with the highest being +40 % of nominal. The procedural recoveries for the first occasion were not within acceptable limits. It is suspected that an analytical error occurred with these samples, although this could not be confirmed. As samples were not corrected for procedural recoveries this had no impact upon the data reported

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of analysis, linearity of detector response, repeatability, method accuracy and precision. The homogeneity was confirmed for the test material in 1 % w/v methylcellulose formulations at nominal concentrations of 1 mg/mL and 200 mg/mL during ambient temperature (15 - 25 ºC) for 1 day and refrigerated storage (2 - 8 °C) for up to 15 days. The mean concentrations of the test material in test formulations analysed for the study were within ± 15 % of nominal concentrations confirming accurate formulation, this is with the exception of the first occasion Group 4.

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1:1 with identified stock males. 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.
- Proof of pregnancy: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm. Day 0 of pregnancy when positive evidence of mating was detected. On the day of positive evidence of mating (Day 0) only females showing at least two copulation plugs were allocated.
- Allocation: 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.

Duration of treatment / exposure:

Day 6 to 19 after mating.

Frequency of treatment:

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

Duration of test:

13 days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

83 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses used in this study (0, 83, 250 and 750 mg/kg/day) were selected in conjunction with the Sponsor and were based on the effects of a preliminary embryo-foetal study at this laboratory (Envigo study number JR43JW) which investigated dose levels of 250, 500 or 1 000 mg/kg/day. In that study there were no unscheduled deaths, no signs associated with dosing and no treatment related clinical signs at all dose levels investigated. For females that received 250 or 500 mg/kg/day there were no inter-group differences in body weight performance or food consumption and no findings detected at necropsy of the dams or external examination of the foetuses. Amongst females that received 1 000 mg/kg/day, mean body weight gain and mean food consumption towards the end of gestation was lower than that of control. In addition, the extent of post-implantation loss and late resorptions (with one total litter resorption) was higher than control, the resultant mean number of live young was lower than control and the mean foetal and litter weights were also lower than control for females that received 1 000 mg/kg/day.
The total litter resorption and another litter with a high number of late resorptions at 1 000 mg/kg/day was considered to preclude the use of this dose on this OECD 414 study. Therefore, the high dose level for this study was set at 750 mg/kg/day with low and intermediate dose levels of 83 and 250 mg/kg/day utilising a common ratio of 3 to investigate the dose response of any potential toxicity observed.

Examinations

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes. A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary. 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 acclimatisation period, observations of the animals and their cages were recorded at least once per day.
Detailed observations were recorded daily at the following times in relation to dose administration:
One to two hours after completion of dosing of all groups.
As late as possible in the working day.
- A detailed physical examination was performed on each animal on Days 0, 5, 12, 18, and 20 after mating to monitor general health.
- Clinical observations are presented for each animal that showed signs, providing detail of the type of sign, day of occurrence and information on the duration of the sign applicable.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3, 6-20 after mating.
Group mean weight changes were calculated from the weight changes of individual animals. Weight changes were calculated and plotted graphically with respect to Day 6 of gestation.
Adjusted body weights on Day 20 after mating were calculated from the body weight at termination minus the gravid uterine weight. Body weight change values for the period Day 6-20 were also presented, after being adjusted for the contribution of the gravid uterus.

FOOD CONSUMPTION: Yes
- 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.
Group mean food consumptions and standard deviations for each period were derived from unrounded cage values.

POST-MORTEM EXAMINATIONS: Yes. A complete necropsy was performed in all cases. 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.
- Sacrifice on gestation day #: Animals surviving until the end of the scheduled study period were killed on Day 20 after mating.

OTHER:
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilisation 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 (as appropriate) were calculated from the individual litter values.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
For females surviving to Day 20 after mating only, the following was recorded:
- Gravid uterus weight: Yes, including cervix and ovaries.
The following were recorded for all animals (including those prematurely sacrificed, where possible) for each ovary/uterine horn:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Apparently non pregnant animals and for apparently empty uterine horns the number of uterine implantation sites were checked after staining with ammonium sulphide.

Fetal examinations:

Examination of all viable foetuses 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 of each foetus was recorded.
Examination of nominally 50 % of foetuses in each litter: Sexed internally and eviscerated.
Fixation: Foetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining foetuses were fixed whole in Bouin’s fluid.
Processing: Bouin’s fixed foetuses were subject to free-hand serial sectioning. IMS fixed foetuses were processed and stained with Alizarin Red.
- Soft tissue examinations: Yes: Serial sections were examined for visceral abnormalities.
- Skeletal examinations: Yes: Assessed for skeletal development and abnormalities.

Mean foetal weights were calculated for each litter. Values were presented for male, female and overall foetal weight. Litter weight was calculated as the sum of all foetal weights. Mean placental weight was also calculated for each litter.
Group mean values and SD were calculated using individual litter mean values.

Detailed Fetal Examination
Findings from external, visceral and skeletal examination of foetuses are tabulated on an individual basis for affected litters and foetuses, linking the results of initial external examinations with subsequent visceral and/or skeletal examinations to foetal weight.
Group incidences of observations on foetuses and litters are summarized in terms of major or minor abnormalities or as skeletal variants. The incidence of structural changes are presented as numeric foetal and litter incidences.
Findings observed were classified, according to severity and incidence, as:
- Major abnormalities: Normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. ventricular septal defect.
- Minor abnormalities: Minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated ureter.
- Variants: Alternative structures or stages of development occurring regularly in the control population, e.g. number of ribs, incomplete ossification of 5th and 6th sternebrae.

In the Foetal examinations appendix, observations on repeated structures like ribs, vertebrae and sternebrae are reported as the first and last affected element, in the form ‘5th 13th bilateral ribs’, which should be interpreted as ‘5th to 13th bilateral ribs’.

Statistics:

Statistical Analysis
The following data types were analysed at each timepoint separately:
Body weight, using absolute values and gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
Litter size and survival indices
Fetal, placental and litter weight

The following comparisons were performed: Group 1 vs 2, 3 and 4

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs seen at physical examinations or at post-dose observation considered related to treatment with the test material.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female (3F 49) was found dead on Day 16 of gestation, having had irregular breathing at recording of clinical signs. Macroscopic examination at necropsy revealed trauma to the oesophagus (although a perforation was not detected), adhesions between the lungs, heart and thorax, clear fluid in the thorax and dark lungs and bronchi. This animal was found to be pregnant with 17 foetuses in utero. This death may have been caused by damage to the oesophagus during the dose administration procedure and not a result of treatment with the test material.

One female (3F 48) was killed on Day 17 of gestation for welfare reasons. This animal had shown signs of underactivity, fast breathing, piloerection, pallor, and partially closed eyelids prior to despatch. Macroscopic examination at necropsy revealed a perforated oesophagus (although partially autolysed, water leakage was visible when testing for damage), adhesions between the lungs, heart and thorax and dark lungs and bronchi. This animal was found to be pregnant with 16 foetuses in utero. This death is considered to have been caused by damage to the oesophagus during the dose administration procedure and not a result of treatment with the test material.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Bodyweight and bodyweight gain up until Day 18 of gestation were similar to that of the Control for females which received 750 mg/kg/day. Bodyweight gain thereafter from Days 18-20 of gestation was lower than that of the Control for females which received 750 mg/kg/day. This is likely a consequence of the slightly low litter size and mean foetal weights in this group.
Bodyweight and bodyweight gain throughout gestation were similar to that of the Control for females which received 83 or 250 mg/kg/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of females receiving 83, 250 and 750 mg/kg/day was similar to that of Controls.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Gravid uterine weights were marginally low for animals which received 750 mg/kg/day when compared to the control group. This is likely a consequence of the slightly low mean foetal weights in this group. Adjusted bodyweight values were unaffected by treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test-material related macroscopic abnormalities detected in the adult females at scheduled termination on Day 20 of gestation.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

The number of dams with abortions was 0.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean number of both implantations and total live young was slightly lower than that of the control for all groups receiving the test material. The extent of mean pre- and post-implantation loss was higher than that of the control for females which received the test material, although a dose response was not apparent and a review of the individual litter values did not suggest an effect of treatment so no effect of treatment was inferred.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

0 litter losses by total resorption were observed.

Early or late resorptions:

no effects observed

Description (incidence and severity):

Resorption in the groups 1, 2 3 and 4 was as follows: 10, 14, 11 and 14. The summary of resorptions as early/late and total is given in Table 4.

Dead fetuses:

no effects observed

Description (incidence and severity):

No stillbirths were observed.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No early deliveries were observed.

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Description (incidence and severity):

One female (Group 3 female 48) was killed for welfare reasons on Day 17 of gestation, and one female (Group 3 female 49) was found dead on Day 16 of gestation. One female (Group 4 female 73) was found to be not pregnant at macroscopic examination. 20, 20, 18 and 19 females in Groups 1, 2, 3 and 4, respectively were found to be pregnant with live young on Day 20 of gestation.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (nominal)

Basis for effect level:

other: No maternal effect at highest dose tested

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg bw/day, slightly reduced.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Table 4 details the numbers of live offspring. The percentages of live offspring were 95.9, 92.6, 94.4 and 92.6 for the 0, 83, 250 and 750 mg/kg bw dose groups, respectively.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

For females which received the test material at 750 mg/kg/day, litter weight and overall foetal weight was marginally lower than that of the control as well as if compared to those animals which received 83 and 250 mg/kg/day.
There was no effect of treatment on litter or foetal weights in animals which received the test material at 83 or 250 mg/kg/day.
None of the offspring were reported as being runts.
There was no effect of treatment on placental weights in animals which received the test material.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The mean number of both implantations and total live young was slightly lower than that of the Control for all groups receiving the test material. The extent of mean pre- and post-implantation loss was higher than that of the Control for females which received the test material, although a dose response was not apparent and a review of the individual litter values did not suggest an effect of treatment so no effect of treatment was inferred.

At 750 mg/kg/day there were a number of foetuses with bent scapula(e); bent radius/ulna/fibula; short/bent/and thickened humerus with associated medially thickened/kinked/incompletely ossified ribs. These findings are outside of both concurrent and Historical Control Data (HCD).

At 83 mg/kg/day there was a slightly increased foetal incidence of short 13th rib compared to concurrent control and just outside of HCD but no such effects were seen in the 250 mg/kg/day and the 750 mg/kg/day group. Therefore, this finding was considered incidental due to the lack of dose response.

At 750 mg/kg/day there was an increased incidence of partially undescended lobe of thymus (9 foetuses in 7 litters) compared to concurrent control (4 foetuses in 4 litters) and just outside of HCD (up to 5 foetuses in 4 litters).

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

changes in litter size and weights

other: Skeletal variations

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

750 mg/kg bw/day (nominal)

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

Formulation Analysis

The analytical procedure was successfully validated with respect to specificity of analysis, linearity of detector response, repeatability, method accuracy and precision.

The homogeneity was confirmed for the test material in 1 % w/v methylcellulose formulations at nominal concentrations of 1 mg/mL and 200 mg/mL during ambient temperature (15 - 25 ºC) for 1 day and refrigerated storage (2 - 8 °C) for up to 15 days.

The mean concentrations of the test material in test formulations analysed for the study were within ± 15 % of nominal concentrations confirming accurate formulation, this is with the exception of the first occasion Group 4.

Table 1: Group Mean Body Weight Values (g) During Gestation

Day	Statistical Test		Dose Group (mg/kg/day)
			0	83	250	750
0	Av	Mean SD N	261 24.5 20	259 18.5 20	264 19.3 20	258 19.9 19
3	Av	Mean SD N	276 24.3 20	276 20.9 20	282 18.8 20	275 20.8 19
6	Av	Mean SD N	289 26.2 20	289 19.0 20	295 19.7 20	287 21.5 19
7	Wi	Mean SD N	292 25.9 20	293 20.1 20	299 19.5 20	289 23.1 19
8	Wi	Mean SD N	295 26.0 20	297 19.4 20	303 19.8 20	294 24.6 19
9	Wi	Mean SD N	300 26.4 20	302 21.0 20	307 18.4 20	298 22.0 19
10	Wi	Mean SD N	304 26.3 20	306 20.3 20	313 19.5 20	302 22.9 19
11	Wi	Mean SD N	310 27.9 20	313 21.6 20	322 20.3 20	309 24.2 19
12	Wi	Mean SD N	315 29.5 20	319 22.5 20	328 21.4 20	316 24.6 19
13	Wi	Mean SD N	322 28.5 20	327 22.9 20	337 22.4 20	323 24.3 19
14	Wi	Mean SD N	330 27.6 20	333 22.9 20	342 22.7 20	329 25.1 19
15	Wi	Mean SD N	338 28.9 20	342 22.8 20	351 23.3 20	336 27.0 19
16	Wi	Mean SD N	350 30.8 20	352 22.8 20	361 22.8 20	347 26.7 19
17	Wi	Mean SD N	362 31.4 20	366 23.9 20	374 26.3 19	359 26.7 19
18	Wi	Mean SD N	377 34.5 20	382 27.0 20	393 29.4 18	375 29.3 19
19	Wi	Mean SD N	394 37.3 20	397 27.9 20	409 31.7 18	388 31.1 19
20	Wi	Mean SD N	421 42.3 20	418 26.0 19	432 31.9 18	409 33.4 19

Av: Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests

Wi: Treated groups compared with Control using Williams’ test

 

Table 2: Body Weight Change - Group Mean Values (g) During Gestation

Days	Statistical Test		Dose Group (mg/kg/day)
			0	83	250	750
0 - 3	KW	Mean SD N	15 6.0 20	17 6.3 20	18 5.5 20	17 2.8 19
3 - 6	Av	Mean SD N	13 7.9 20	13 7.4 20	14 5.1 20	12 6.0 19
6 - 7	Wi	Mean SD N	3 7.0 20	3 5.7 20	3 3.7 20	2 4.0 19
7 - 8	Wi	Mean SD N	3 3.7 20	4 4.2 20	4 3.1 20	5 3.1 19
8 - 9	Wi	Mean SD N	5 3.7 20	5 4.4 20	4 3.9 20	4 4.6 19
9 - 10	Wi	Mean SD N	4 5.9 20	4 3.7 20	6 3.4 20	4 3.8 19
10 - 11	Wi	Mean SD N	6 4.6 20	7 5.2 20	9 4.1 20	7 3.9 19
11 - 12	Sh	Mean SD N	5 8.1 20	6 5.8 20	6 4.6 20	7 3.4 19
12 - 13	Sh	Mean SD N	6 9.8 20	7 3.8 20	9 4.4 20	7 5.1 19
13 - 14	Sh	Mean SD N	9 7.1 20	7 4.3 20	5 2.6 20	6 6.0 19
14 - 15	Wi	Mean SD N	8 5.3 20	8 3.9 20	9 3.4 20	7 3.1 19
15 - 16	Sh	Mean SD N	11 6.6 20	10 4.4 20	10 9.1 20	11 3.6 19
16 - 17	Sh	Mean SD N	12 6.4 20	14 4.7 20	13 9.6 19	12 4.8 19
17 - 18	Wi	Mean SD N	15 6.5 20	16 4.9 20	17 6.2 18	16 5.4 19
18 - 19	Wi	Mean SD N	17 5.0 20	15 4.9 20	16 5.4 18	13* 4.6 19
19 - 20	Wi	Mean SD N	27 5.7 20	24 4.4 19	23* 4.4 18	21** 5.5 19
6 - 18	Sh	Mean SD N	88 25.8 20	92 10.3 20	98 15.5 18	88 13.2 19
18 – 20	lWi	Mean SD N	44 9.4 20	39 4.1 19	39 6.1 18	34** 7.0 19
6 - 20	Sh	Mean SD N	132 32.3 20	130 11.3 19	138 18.5 18	122* 18.2 19

KW: Pre-treatment comparison of all groups using Kruskal-Wallis test followed by pairwise Wilcoxon rank sum tests

Av: Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests

Wi: Treated groups compared with Control using Williams’ test

Sh: Treated groups compared with Control using Shirley’s test

l: Data were log transformed for the statistical analysis

* p < 0.05

** p < 0.01

 

Table 3: Gravid Uterine Weight, Adjusted Body Weight and Adjusted Body Weight Change - Group Mean Values (g) On Day 20 Of Gestation

	Statistical Test		Dose Group (mg/kg/day)
			0	83	250	750
Body weight on day 6	Av	Mean SD N	289 26.2 20	289 19.0 20	294 17.9 18	287 21.5 19
Terminal body weight on day 20	Wi	Mean SD N	419 41.1 20	420 29.0 20	431 31.9 18	410 33.2 19
Body weight change days 6 - 20	Sh	Mean SD N	130 31.6 20	130 12.5 20	137 18.7 18	123* 18.2 19
Gravid uterine weight	Wi	Mean SD N	95 15.8 20	90 15.5 20	91 20.4 18	86 14.0 19
Adjusted bodyweight day 20	Wi	Mean SD N	324 28.8 20	329 21.0 20	339 21.3 18	324 26.5 19
Adjusted body weight change days 6 - 20	Sh	Mean SD N	35 21.8 20	40 11.6 20	45 9.1 18	36 12.3 19

Av: Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests

Wi: Treated groups compared with Control using Williams’ test

Sh: Treated groups compared with Control using Shirley’s test

* p < 0.05

 

Table 4: Litter Data - Group Mean Values on Day 20 of Gestation

		Statistical Test		Dose Group (mg/kg/day)
				0	83	250	750
Corpora Lutea		Du	Mean SD N	18.0 2.63 20	18.8 2.59 20	20.5* 3.01 18	17.9 2.53 19
Implantations		Wi	Mean SD N	17.1 1.99 20	16.2 2.23 20	16.0 3.73 18	16.3 2.14 19
Resorptions	Early	Wc	Mean SD N	0.7   20	1.1   20	0.9   18	1.2   19
	Late	Wc	Mean SD N	0.0   20	0.1   20	0.0   18	0.1   19
	Total	Wc	Mean SD N	0.7   20	1.2   20	0.9   18	1.3*   19
Live young	Male	Wi	Mean SD N	8.7 2.80 20	7.1 2.17 20	6.7 3.01 18	8.5 2.52 19
	Female	Wi	Mean SD N	7.8 2.17 20	7.9 2.43 20	8.4 2.66 18	6.6 2.27 19
	Total	Wi	Mean SD N	16.4 2.19 20	15.0 2.68 20	15.1 3.69 18	15.1 2.25 19
Sex ratio (%M)		Wa	Mean SD N	52.0   20	47.5   20	44.4   18	56.1   19
Implantation Loss (%)	Pre-	Wa	Mean SD N	6.9   20	13.5   20	22.0**   18	9.3   19
	Post-	Wa	Mean SD N	3.9   20	7.4   20	5.5   18	7.8*   19

Du: Treated groups compared with Control using Dunnett’s test

Wi: Treated groups compared with Control using Williams’ test

Wc: Treated groups compared with Control using Wilcoxon rank sum test

Wa: Treated groups compared with Control using Wald’s test

* p < 0.05

** p < 0.01

 

Table 5: Placental, Litter and Foetal Weights - Group Mean Values (g) on Day 20 of Gestation

	Statistical Test		Dose Group (mg/kg/day)
			0	83	250	750
Placental weight	lWi	Mean SD N	0.56 0.040 20	0.58 0.055 20	0.60 0.095 18	0.56 0.073 19
Litter weight	Wi	Mean SD N	58.69 10.993	55.48 11.255 20	56.40 13.907 18	52.56 9.639 19
Litter Size	Wi	Mean SD N	16.40 2.186 20	15.00 2.675 20	15.11 3.692 18	15.05 2.248 19
Male foetal weight	Sh	Mean SD N	3.66 0.375 20	3.81 0.228 20	3.88 0.165 18	3.58 0.274 19
Female foetal weight	Wi	Mean SD N	3.45 0.357	3.57 0.213 20	3.62 0.197 18	3.37 0.281 19
Overall foetal weight	Wi	Mean SD N	3.56 0.366 20	3.69 0.210 20	3.74 0.174 18	3.48 0.261 19

Wi: Treated groups compared with Control using Williams’ test

Sh: Treated groups compared with Control using Shirley’s test

l: Data were log transformed for the statistical analysis

 

Table 6: Foetal Examinations – Major Abnormality Findings – Group Incidences

	Foetuses				Litters
	Dose Group (mg/kg/day)
	0	83	250	750	0	83	250	750
Number Examined	328	300	272	286	20	20	18	19
Total Number Affected	6	0	3	40	3	0	3	10
Cervical/Thoracic- Visceral
Retroesophageal aortic arch	1	0	0	0	1	0	0	0
Right sided aortic arch	1	0	0	0	1	0	0	0
Right sided azygos vein/descending aorta	1	0	0	0	1	0	0	0
Muscular ventricular septal defect	1	0	0	0	1	0	0	0
Large atrium	1	0	0	0	1	0	0	0
Diaphragmatic hernia	0	0	1	0	0	0	1	0
Lumbar (and abdominal)/Sacral/Caudal- Visceral
Omphalocele	0	0	1	0	0	0	1	0
Lumbar (and abdominal)/Sacral/Caudal- Appendicular Skeletal
Bent scapula(e)	4	0	1	37	1	0	1	10
Short/thickened humerus	1	0	0	32	1	0	0	8
Bent humerus	4	0	0	6	1	0	0	5
Bent radius/ulna/fibula	1	0	0	18	1	0	0	5

Note: Individual foetuses/litters may occur in more than one category.

 

Table 7: Foetal Examinations – Minor Skeletal Abnormality and Variants Findings – Group Incidences

	Foetuses				Litters
	Dose Group (mg/kg/day)
	0	83	250	750	0	83	250	750
Number Examined	164	148	137	142	20	20	18	19
Minor skeletal abnormalities- Cranial
Fissure(s)	1	0	1	0	1	0	1	0
Interparietal fissure(s)	1	0	0	1	1	0	0	1
Minor skeletal abnormalities- Ribs
Medially thickened / kinked / marked	4	1	3	49	1	1	3	11
Minor skeletal abnormalities- Sternebrae
Misaligned ossification sites	1	1	0	0	1	1	0	0
Minor skeletal abnormalities- Appendicular
Misshapen scapula	0	0	0	1	0	0	0	1
Total Minor skeletal abnormalities
Total affected by one or more	7	2	4	50	4	2	4	12
Rib and vertebral configuration- Cervical rib
Short supernumerary	1	1	3	3	1	1	3	3
Full supernumerary	0	1	0	0	0	1	0	0
Rib and vertebral configuration- 13th Rib
Short with/without costal cartilage	1	5	0	1	1	3	0	1
Rib and vertebral configuration- Number of 14th Ribs
Short supernumerary	9	6	3	4	7	3	2	3
Full supernumerary	0	0	1	0	0	0	1	0
Total	9	6	4	4	7	3	3	3
Pelvic girdle
Unilateral caudal shift	0	1	0	0	0	1	0	0
Delayed/Incomplete ossification/unossified- Cranial
Cranial centres/marked	26	13	19	30	11	7	11	12
Presphenoid	2	0	1	0	1	0	1	0
Hyoid	36	18	22	12	14	9	11	6
Delayed/Incomplete ossification/unossified- Sternebrae
5th and/or 6th	110	104	77	91	20	20	17	19
Other	22	6	8	9	9	6	6	5
Total	111	105	78	93	20	20	17	19
Delayed/Incomplete ossification/unossified- Vertebrae
Cervical	7	2	1	4	4	2	1	3
Thoracic	17	16	16	16	9	11	10	10
Lumbar	2	0	1	1	1	0	1	1
Sacrocaudal	26	13	12	10	8	8	8	6
Caudal	2	1	0	2	2	1	0	2
Delayed/Incomplete ossification/unossified- Ribs
Any/marked	2	0	1	5	1	0	1	3
Delayed/Incomplete ossification/unossified- Appendicular
Pelvic Bones	21	10	13	10	7	7	7	8
Clavicles	0	0	0	1	0	0	0	1
Metacarpals	3	1	1	2	2	1	1	2
Metatarsals	2	1	1	3	2	1	1	3

Note: Individual foetuses/litters may occur in more than one category.

 

Table 8: Foetal Examinations - Minor Visceral Abnormality and Necropsy Findings - Group Incidences

	Foetuses				Litters
	Dose Group (mg/kg/day)
	0	83	250	750	0	83	250	750
Number Examined	164	152	135	144	20	20	18	19
Total Number Affected	28	26	15	23	16	15	10	13
Visceral abnormalities- Brain
Dilated interventricular foramen	0	1	2	0	0	1	1	0
Visceral abnormalities- Eyes
Variation in lens shape	1	0	2	0	1	0	2	0
Visceral abnormalities- Thyroid
Absent lobe	0	1	0	0	0	1	0	0
Visceral abnormalities- Thymus
Partially undescended lobe	4	1	1	9	4	1	1	7
Thymic remnant	1	0	0	0	1	0	0	0
Visceral abnormalities- Oesophagus
Right sided	1	0	0	0	1	0	0	0
Visceral abnormalities- Caudal vena cava
Anomalous confluence with left hepatic vein	1	0	0	0	1	0	0	0
Visceral abnormalities- Diaphragm
Thinning with liver protrusion	0	3	1	3	0	3	1	3
Visceral abnormalities- Liver
Folded posterior caudate lobe	0	2	0	0	0	2	0	0
Small posterior caudate lobe	0	0	0	1	0	0	0	1
Visceral abnormalities- Ureter(s)
Dilated	1	0	0	0	1	0	0	0
Visceral abnormalities- Testis(es)
Undescended	1	1	0	0	1	1	0	0
Malpositioned	1	1	1	1	1	1	1	1
Visceral abnormalities- Umbilical artery
Left	3	1	0	0	3	1	0	0
Haemorrhages -Head
Brain	4	6	5	3	4	4	5	3
Haemorrhages- Neck/Thorax
Thoracic cavity	1	0	0	0	1	0	0	0
Haemorrhages- Abdomen
Abdominal cavity	7	11	5	6	5	6	4	5
Liver lobes	6	1	3	1	4	1	3	1

Note: Individual foetuses/litters may occur in more than one category.

 

Table 9: Control Incidence Major Abnormalities - Crl:CD(SD) Rat

	Study Number
	1		2		3		4		5		6
Number of foetuses/litters examined	317	20	319	20	300	21	304	19	282	20	322	22
Cervical/Thoracic
Diaphragmatic hernia	-	-	-	-	-	-	-	-	-	-	-	-
Lumbar (and abdominal)/sacral/caudal
Omphalocele	-	-	-	-	-	-	-	-	-	-	-	-
Appendicular
Bent scapula(e )	1	1	-	-	-	-	-	-	2	2	-	-
Short/thickened humerus	-	-	-	-	-	-	-	-	-	-	-	-
Bent humerus	-	-	-	-	-	-	-	-	-	-	-	-
Bent radius/ulna/fibula	-	-	-	-	-	-	-	-	-	-	-	-
	Study Number
	7		8		9		10		11
Number of foetuses/litters examined	296	20	310	20	331	21	291	20	305	20
Cervical/Thoracic
Diaphragmatic hernia	-	-	-	-	-	-	-	-	-	-
Lumbar (and abdominal)/sacral/caudal
Omphalocele	-	-	-	-	-	-	-	-	-	-
Appendicular
Bent scapula(e )	-	-	-	-	-	-	-	-	-	-
Short/thickened humerus	-	-	-	-	-	-	-	-	-	-
Bent humerus	-	-	-	-	-	-	-	-	-	-
Bent radius/ulna/fibula	1	1	-	-	-	-	-	-	-	-
	Current Study - Dose Group (mg/kg)
	0		83		250		750
Number of foetuses/litters examined	328	20	300	20	272	18	286	19
Cervical/Thoracic
Diaphragmatic hernia	-	-	-	-	1	1	-	-
Lumbar (and abdominal)/sacral/caudal
Omphalocele	-	-	-	-	1	1	-	-
Appendicular
Bent scapula(e )	4	1	-	-	1	1	37	10
Short/thickened humerus	1	1	-	-	-	-	32	8
Bent humerus	3	1	-	-	-	-	3	3
Bent radius/ulna/fibula	1	1	-	-	-	-	18	5

 

Table 10: Control Incidence Minor Skeletal abnormalities - Crl:CD(SD) Rat

	Study Number
	1		2		3		4		5		6
Number of foetuses/litters examined	160	20	160	20	138	21	152	19	141	20	163	22
Skeletal abnormalities
Ribs - medially thickened/kinked	2	2	-	-	-	-	-	-	1	1	2	1
Appendicular - misshapen scapula	-	-	-	-	-	-	-	-	-	-	-	-
Cervical rib - full supernumerary	-	-	-	-	-	-	-	-	-	-	-	-
13th rib - short with/without costal cartilage	3	3	3	3	1	1	3	2	-	-	-	-
14th rib - full supernumerary	-	-	-	-	-	-	-	-	-	-	-	-
Pelvic girdle - unilateral caudal shift	1	1	-	-	-	-	-	-	-	-	-	-
Delayed/Incomplete ossification/unossified
Ribs - any	1	1	-	-	-	-	-	-	-	-	-	-
Appendicular - clavicle	-	-	-	-	-	-	-	-	-	-	-	-
	Study Number
	7		8		9		10		11
Number of foetuses/litters examined	149	20	153	20	166	21	147	20	153	20
Skeletal abnormalities
Ribs - medially thickened/kinked	2	1	1	1	-	-	-	-	1	1
Appendicular - misshapen scapula	-	-	-	-	-	-	-	-	-	-
Cervical rib - full supernumerary	-	-	-	-	-	-	1	1	-	-
13th rib - short with/without costal cartilage	-	-	1	1	1	1	2	2	2	2
14th rib - full supernumerary	-	-	-	-	-	-	-	-	-	-
Pelvic girdle - unilateral caudal shift	1	1	-	-	-	-	-	-	1	1
Delayed/Incomplete ossification/unossified
Ribs - any	1	1	1	1	-	-	-	-	-	-
Appendicular - clavicle	-	-	-	-	-	-	-	-	-	-
	Current Study - Dose Group (mg/kg)
	0		83		250		750
Number of foetuses/litters examined	164	20	148	20	137	18	142	19
Skeletal abnormalities
Ribs - medially thickened/kinked	4	1	1	1	3	3	49	11
Appendicular - misshapen scapula	-	-	-	-	-	-	1	1
Cervical rib - full supernumerary	-	-	1	1	-	-	-	-
13th rib - short with/without costal cartilage	1	1	5	3	-	-	1	1
14th rib - full supernumerary	-	-	-	-	1	1	-	-
Pelvic girdle - unilateral caudal shift	-	-	1	1	-	-	-	-
Delayed/Incomplete ossification/unossified
Ribs - any	2	1	-	-	1	1	5	3
Appendicular - clavicle	-	-	-	-	-	-	1	1

 

Table 11: Control Incidence Minor Visceral Abnormalities - Crl:CD(SD) Rat

	Study Number
	1		2		3		4		5		6
Number of foetuses/litters examined	157	20	159	20	136	21	152	19	141	20	159	22
Fixed visceral observations
Brain - dilated interventricular foramen	-	-	-	-	-	-	-	-	-	-	-	-
Thyroid - absent	-	-	-	-	-	-	-	-	-	-	-	-
Thymus - partially undescended lobe	4	3	2	2	-	-	1	1	2	2	4	3
Diaphragm - thinning with liver protrusion	1	1	1	1	3	3	-	-	-	-	1	1
Liver - folded posterior caudate lobe	-	-	-	-	1	1	-	-	-	-	-	-
Liver - small posterior caudate lobe	-	-	-	-	-	-	-	-	-	-	-	-
	Study Number
	7		8		9		10		11
Number of foetuses/litters examined	147	20	157	20	165	21	144	20	152	20
Fixed visceral observations
Brain - dilated interventricular foramen	1	1	-	-	1	1	-	-	-	-
Thyroid - absent	-	-	-	-	-	-	-	-	-	-
Thymus - partially undescended lobe	5	4	4	4	2	2	4	4	2	2
Diaphragm - thinning with liver protrusion	-	-	1	1	-	-	3	3	1	1
Liver - folded posterior caudate lobe	-	-	-	-	-	-	-	-	1	1
Liver - small posterior caudate lobe	-	-	-	-	-	-	-	-	-	-
	Current Study - Dose Group (mg/kg)
	0		83		250		750
Number of foetuses/litters examined	164	20	152	20	135	18	144	19
Fixed visceral observations
Brain - dilated interventricular foramen	-	-	1	1	2	1	-	-
Thyroid - absent	-	-	1	1	-	-	-	-
Thymus - partially undescended lobe	4	4	1	1	1	1	9	7
Diaphragm - thinning with liver protrusion	-	-	3	3	1	1	3	3
Liver - folded posterior caudate lobe	-	-	2	2	-	-	-	-
Liver - small posterior caudate lobe	-	-	-	-	-	-	1	1

 

Discussion

In this study, treatment with the test material at 83 or 250 mg/kg/day was well tolerated by pregnant females and there were no effects of treatment on the body weight or food consumption of the parental females, or on placental, litter or foetal weights.

Bodyweight performance of females receiving the test material at 750 mg/kg/day was also unaffected by treatment with the test material up until Day 18 of gestation, however, bodyweight gain between Days 18-20 of gestation was lower than that of the control, and the gravid uterine weight of females which received the test material at 750 mg/kg/day was lower than that of the control. These differences were considered to be as a consequence of the slightly lower litter size and foetal weights in this group, since there was no difference from Control in the bodyweight gain of females on Day 20 of gestation when adjusted for the weight of the gravid uterus. Food consumption was considered unaffected. 

Embryo-foetal survival (as indicated by the extent of pre- and post-implantation losses and the number of total live young) was marginally lower than that of the Control for females which received the test material although there was no dose response and review of the individual litter values did not suggest an effect of treatment.

Foetal pathology examination at 750 mg/kg/day revealed a number of foetuses with bent scapula(e); bent radius/ulna/fibula; short/bent/and thickened humerus with associated medially thickened/kinked/incompletely ossified ribs which are outside of both concurrent and Historical Control Data (HCD). Since these occurred in a high frequency in this group only, these findings are considered related to treatment with test material.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the NOAEL for maternal toxicity was 750 mg/kg/day (bodyweight gain and gravid uterine weight) and the NOAEL for embryo-foetal toxicity was concluded to be 250 mg/kg/day due to litter size and mean foetal weights that were slightly reduced. Embryo-foetal survival was considered unaffected by treatment, but foetal development was adversely affected with bent scapula(e); bent radius/ulna/fibula; short/bent/and thickened humerus with associated medially thickened/kinked/incompletely ossified ribs.

Executive summary:

The developmental toxicity of the test material was investigated in accordance with the standardised guidelines OECD 414 under GLP conditions.

The purpose of this study was the assessment of the influence of the test material (an industrial chemical) on embryo-foetal survival and development when administered during the organogenesis and foetal growth phases of pregnancy in the rat.

Three groups of 20 females received the test material at doses of 83, 250 or 750 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, 1.0 % w/v methylcellulose (MC) aqueous solution at the same volume dose as treated groups and for the same duration. Animals were killed on Day 20 after mating for reproductive assessment and foetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and the gravid uterus weight recorded. All foetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination. 

The mean concentrations of the test material in test formulations analysed for the study were within ±15 % of nominal concentrations confirming accurate formulation, this is with the exception of the first occasion Group 4.

There were two deaths in the 250 mg/kg/day group but these were considered unrelated to treatment.

There were no signs seen at physical examinations or at post-dose observation considered related to treatment with the test material.

Bodyweight and bodyweight gain throughout gestation were similar to that of the Control for females which received 83 or 250 mg/kg/day.

Bodyweight and bodyweight gain up until Day 18 of gestation were similar to that of the Control for females which received 750 mg/kg/day. Bodyweight gain thereafter from Days 18-20 of gestation was lower than that of the Control for females which received 750 mg/kg/day. This is likely a consequence of the slightly low litter size and mean foetal weights in this group.

Gravid uterine weights were marginally low for animals which received 750 mg/kg/day when compared to the control group. This is likely a consequence of the slightly low mean foetal weights in this group.  Adjusted bodyweight values were unaffected by treatment.

Food consumption of females receiving 83, 250 and 750 mg/kg/day was similar to that of Controls.

There were no test-material related macroscopic abnormalities detected in the adult females at scheduled termination on Day 20 of gestation.

One female (Group 4 female 73) was found to be not pregnant at macroscopic examination. 20, 20, 18 and 19 females in Groups 1, 2, 3 and 4, respectively were found to be pregnant with live young on Day 20 of gestation.

Embryo-foetal survival was considered to be unaffected by treatment.

For females which received the test material at 750 mg/kg/day, litter weight and overall foetal weight was marginally lower than that of the control as well as if compared to those animals which received 83 and 250 mg/kg/day.

There was no effect of treatment on litter or foetal weights in animals which received the test material at 83 or 250 mg/kg/day.

There was no effect of treatment on placental weights in animals which received the test material.

Foetal pathology examination at 750 mg/kg/day revealed there were a number of foetuses with bent scapula(e); bent radius/ulna/fibula; short/bent/and thickened humerus with associated medially thickened/kinked/incompletely ossified ribs. These findings are outside of both concurrent and Historical Control Data (HCD). However, some of these findings have been reported in published literature to be reversible in quantity and severity post-natally but because these occurred in a high frequency in this group only, these findings are considered related to treatment with the test material.

In this study, treatment with the test material at 83 or 250 mg/kg/day was generally well tolerated.

At 750 mg/kg/day, maternal body weight performance was unaffected by treatment with the test material up until Day 18 of gestation, however, bodyweight gain between Days 18-20 of gestation was lower than that of the control, and the gravid uterine weight of females which received the test material at 750 mg/kg/day was lower than that of the control. Litter size and mean foetal weights were slightly reduced. Embryo-foetal survival was considered unaffected by treatment, but foetal development was adversely affected with bent scapula(e); bent radius/ulna/fibula; short/bent/and thickened humerus with associated medially thickened/kinked/incompletely ossified ribs.

Therefore, the No-Observed-Adverse-Effect-Level (NOEL) for maternal toxicity was 750 mg/kg/day and the No-Observed-Adverse-Effect-Level (NOAEL) for embryo-foetal toxicity was concluded to be 250 mg/kg/day.