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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
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
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
-Test substance (name as cited in study report): Eastman TXIB
-Source of test material: Eastman Chemical Company
-Purity: >98%
-Production date: 4/26/07
-Date of receipt: 7/26/07
-Stability: 1 year
-Storage: Room temperature and humidity
-Description: Clear liquid
-Specific gravity: 0.92
Specific details on test material used for the study:
Test item: TXIB.
Test item identity (including alternative names): TXIB.
2,2,4-trimethyl-1,3-pentanediol diisobutyrate.
Intended use: Industrial chemical.
Appearance: Clear colorless liquid.
Storage conditions: At ambient temperature (15 to 25C).
Supplier: Sponsor
Batch number: TD17015102
Retest date: February 2018 (or within 2 years of manufacture).
Purity: 99.2%.

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Rabbit 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 15-21°C and 45-70%. (There were no deviations from these ranges.)
Lighting Artificial lighting, 14 hours light : 10 hours dark.
Alarm systems Activated on ventilation failure and when temperature/humidity limits exceeded.
Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Suspended cages fitted with perforated floor panels and mounted in batteries. Undertrays lined with absorbent paper were changed at least three times a week. Cages were also
fitted with a plastic resting platform.
Cage distribution The cages constituting each group were blocked by group and mounted in batteries.
Number of animals per cage Females were individually housed.

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study and replaced when necessary.
Stainless steel key ring Attached to the cage.

Diet Supply Teklad 2930 Diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Diet Availability Restricted (initially 150 g/animal/day during acclimatization up to one week prior to the onset of mating and 200 g/animal/day thereafter). Should an individual show a significant non-treatment related reduced food consumption, moistened diet (50 g pelleted diet moistened with 50 mL of water) was offered, the consumption was recorded.
In addition to this diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly. Consumption of hay and vegetables were monitored qualitatively but not quantitatively.

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

Supplier Certificates of Analysis
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are provided by the water supplier.
Certificates of analysis were also received from the suppliers of the Aspen chew blocks. 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:
other: 1% methylcellulose
Details on exposure:
Route Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at Constant doses in mg/kg/day.
Volume dose 5 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Control (Group 1) Vehicle at the same volume dose as treated groups.
Frequency Females were treated from Day 1 to Day 28 (inclusive) after mating, once daily at approximately the same time each day.
Formulation 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:
Approximately 30% of the required amount of vehicle was added to the pre-weighed quantity of test item and magnetically stirred until uniformly mixed. It was made up to volume with the remaining vehicle and magnetically stirred for a minimum of 20 minutes, until homogeneous. A series of emulsions at the required concentrations were prepared by dilution of individual weighings of the test item. Dosing material was prepared weekly and stored under refrigeration (2-8 degrees C.). 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.

Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 10 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.Samples of each formulation prepared for administration on Days 1 and 28 of treatment were analyzed for achieved concentration of the test item.

Details on mating procedure:
Male/female ratio 1:1 using identified stock New Zealand White bucks.
Checks Natural mating observed.
After mating Each female was injected intravenously with 25 i.u. luteinizing hormone.
Day 0 of gestation On the day of mating.

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 mating. Where possible only females mating at least twice 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 treated group and to prevent more than one sibling female in each group, where possible.

Identification of animals Each animal was assigned a number and identified uniquely within the study by an ear tag.
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.
Duration of treatment / exposure:
Test subjects were treated from gestation days 1 to 28
Frequency of treatment:
daily
Duration of test:
29 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
Mortality
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary. A complete necropsy was performed in all cases.

Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays 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:

Pre-dose observation
One to two hours after completion of dosing As late as possible in the working day

Clinical Signs
A detailed physical examination was performed on each animal on Days 0, 6, 12, 18, 23 and 29 after mating to monitor general health.

Body Weight
The weight of each adult was recorded weekly during acclimatization and daily from Day 0-29 after mating.

Food Consumption
The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 1-28 after mating.Method of Kill

Method of kill for all adult animals: Intravenous injection of sodium pentobarbitone.
Method of kill for fetuses: Subcutaneous injection of sodium pentobarbitone.

Examinations

Maternal examinations:
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.

Schedule Animals surviving until the end of the scheduled study period were killed on Day 29 after mating.

Sequence To allow satisfactory inter-group comparison. The tissue samples fixed are detailed as follows:
Abnormalities
Adrenals
Esophagus
Kidneys
Liver
Stomach
Animal ID retained

Tissues were routinely preserved in 10% Neutral Buffered Formalin. This list of tissues preserved was intended to satisfy any possible future requirement for further examination of tissues.
Ovaries and uterine content:
For females surviving to term, the following was recorded: Uterus Gravid uterine weight (including cervix and ovaries). The following were recorded for all animals (including those prematurely sacrificed, where possible):
Corpora lutea.
Implantation sites.
Intrauterine deaths (classified as early or late resorptions).
Fetuses (live and dead).

The absence or number of uterine implantation sites was confirmed.
Fetal examinations:
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, sampled as appropriate and retained in appropriate fixative. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each fetus was also recorded.

Fixation Nominally one half of fetuses were decapitated; heads were initially stored in Bouin’s fluid.

All fetuses and torsos (intact ad decapitated) were eviscerated and fixed in Industrial Methylated Spirit.

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

Industrial Methylated Spirit fixed fetuses and torsos were processed and stained with Alizarin Red.

Fetal Pathology Examination
Bouin’s fixed heads Serial sections were examined for soft tissue abnormalities.
Alizarin Red stained fetuses and torsos Assessed for skeletal development and abnormalities
Statistics:
The following data types were analyzed 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

A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For pretreatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other comparisons the F1
approximate test was applied. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972). The test statistic compares the mean square, NMS, for the deviations of the observed means from the maximum likelihood means, calculated under a constraint of
monotonicity with the usual error mean square, EMS. The null hypothesis is that the true means are monotonically ordered. The test statistic is F1 = NMS/EMS which can be compared with standard tables of the F-distribution with 1 and error degrees of freedom. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the
F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no adverse clinical signs observed at the detailed physical examination that were attributable to treatment with TXIB.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
1F, 8 (control): On Day 10 of gestation this female was found dead in her home cage. No signs of ill health had been observed prior to her death. Macroscopic examination revealed a
small spleen. The female was also found to be pregnant with 7 embryos. This death was considered to be incidental.

1F, 20 (control): On Day 10 of gestation this female was killed for reasons of animal welfare. Clinical signs for this animal consisted of little diet and hay eaten, little water drunk, fast breathing, thin build, pallor whole body and reduced faeces and urine output. At macroscopic examination lungs and bronchi were dark, mass(es) in the thorax, and fluid filled area and soft material adhering to the lung lobes. This female was pregnant with 10 embryos. This death was considered to be incidental.

2F, 37 (100 mg/kg/day): On the morning of Day 8 of gestation (at the first am check), this female was found dead in her home cage. No signs of ill health had been observed prior to her death. Macroscopic examination revealed abnormal contents in the trachea (Dark congealed fluid); in addition, all lung lobes were congested and were unable to collapse fully. This death is considered to be incidental and likely due to an underlying respiratory problem.

4F, 73 (1000 mg/kg/day): On Day 22 of gestation this female was found dead in her home cage. No signs of ill health had been observed prior to her death. Macroscopic examination revealed trauma to the trachea and bruising to the oesophagus, the right lobes of the lungs and bronchi were also dark in appearance. The female was also found to be pregnant with 7 normal fetuses. This death was considered to be incidental and not related to treatment.

4F, 74 (1000 mg/kg/day): On Day 8 of gestation, this female was despatched following a prolonged period of in-appetence and significant body weight loss during the gestation period despite offerings of moistened diet and additional hay. Furthermore, this female produced little faecal and urine output and was noted from Day 4 of gestation, to have red/brown staining on the cage tray paper and to be of thin build and was subsequently despatched due to poor clinical condition. At macroscopic examination, there were no abnormalities observed. This female was pregnant with 9 implantations.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Group mean body weight gain (Day 1 to 28 of gestation) was slightly reduced across all TXIB treated groups (100, 300 or 1000 mg/kg/day) when compared with Controls; the differences did not attain statistical significances.

On Day 29 of gestation, group mean gravid uterine weight was low in females treated with TXIB when compared with Controls however the magnitude of difference was slight and showed no relationship to dose level. Overall body weight change, when adjusted for the weight of the gravid uterus was comparable to controls at all dose levels (100, 300 or 1000 mg/kg/day).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group mean food consumption was slightly reduced in females treated at 1000 mg/kg/day, until day 24 of gestation; thereafter it was comparable to, or greater than Controls.

In females receiving 100 or 300 mg/kg/day food intake were comparable to 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:
no effects observed
Gross pathological findings:
no effects observed
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
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
On Day 29 of gestation, six females were confirmed not pregnant however the distribution showed no correlation to treatment.

Mean numbers of corpora lutea and implantations were considered to be unaffected by treatment with TXIB.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
Mean numbers of early resorptions and thus post-implantation losses, were high in females treated at 1000 mg/kg/day when compared with Controls and when compared with historical control data ranges; as a result, the mean live litter size was slightly low compared with controls. A relationship to treatment cannot be ruled out.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
gross pathology
maternal abnormalities

Results (fetuses)

Fetal body weight changes:
no effects observed
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:
no effects observed
Visceral malformations:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
At 100, 300 or 1000 mg/kg/day, an increase in the incidence of short supernumerary cervical ribs was observed when compared with Controls however the distribution showed no relationship to treatment. The numbers of litters involved in the 300 mg/kg/day or 1000 mg/kg/day groups were within the historical control data ranges so no effect of treatment is inferred.

At 1000 mg/kg/day or 300 mg/kg/day there was also a slight increase in incidence of delayed
ossification of 1st to 4th and 6th sternebrae when compared with control litters however the
incidence and distribution were found to be within historical control data ranges and as such a
relation to treatment is not inferred.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

open allclose all
Abnormalities:
not specified
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other:
Description (incidence and severity):
Higher numbers of early resorptions and post-implantation losses were observed in females treated at 1000 mg/kg/day and consequently total live young were marginally reduced however in surviving fetuses examined on Day 29 of gestation, there were no effects on fetal growth or development at any level.

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Litter Data
    Corpora Lutea implantations Early Resorptions  Late Resorptions  Total Resorptions  Live Male Live female Total Sex% Male Preimplantation loss (%) Post implantation loss (%)
1F
0 mg/kg
mean 10.1 9 0.6 0.3 0.8 4.6 3.5 8.2 52.6 13.7 8.9
SD 1.94 2.81       2.29 1.35 2.65      
  N 19 19 19 19 19 19 19 19 19 19 19
                         
2F
100 mg/kg
mean 8.9 7.5 0.7 0.2 0.9 3.6 3.1 6.6 58.1 18.5 11.6
SD 2.01 2.77       1.82 2.26 2.7      
  N 18 18 18 18 18 18 18 18 18 18 18
                         
3F
300 mg/kg
mean 10.7 8.8 1.2 0.3 1.5 3.8 3.5 7.3 54.3 18.1 16.4
SD 1.8 2.68       1.63 1.91 2.67      
  N 21 21 21 21 21 21 21 21 21 21 21
                         
4F
1000 mg/kg
mean 10.2 8.8 1.5* 0.4 1.9* 4.2 2.7 6.9 61.3 13.8 22.5**
SD 2.18 2.59       2.14 1.87 2.61      
  N 20 20 20 20 20 20 20 20 20 20 20

Placental, litter and fetal weights - group mean values (g) on Day 29 of gestation
Group/Sex Placental Weight Litter Weight Litter Size Male fetal weight Female fetal weight Male fetal weight Overall Fetal Weight
1F
0 mg/kg
Mean 5.24 338.25 8.16 41 42.67 42.28
SD 0.94 103.025 2.651 3.42 5.2 4.114
  N 19 19 19 18 19 19
               
1F
100 mg/kg
Mean 5.45 277.98 6.61 44.17 41.3 43.21
SD 0.981 103.79 2.704 5.636 4.564 5.152
  N 18 18 18 18 16 18
               
1F
300 mg/kg
Mean 5.2 293.77 7.33 41.58 40.15 41.15
SD 0.752 97.042 2.671 5.546 3.964 4.754
  N 21 21 21 21 20 21
               
1F
1000 mg/kg
Mean 5.39 280.43 6.9 41.27 41.9 41.56
SD 0.746 95.252 2.614 3.804 5.229 4.159
  N 20 20 20 20 20 20

Fetal examinations - major abnormality findings - group incidences
              Fetuses       Litters
Group 1 2 3 4 1 2 3 4
Number Examined 155 119 154 138 19 18 21 20
Total number Affected             0 2 0 1 0 2 0 1
                                   
Head
Visceral
Partially open eye lid(s) 0 0 0 1 0 0 0 1
Cervical/Thoracic
Skeletal
      Multiple thoracic vertebral/rib abnormalities     0 2 0 0       0 2 0 0
Note: Individual fetuses/litters may occur in more than one category.                              

Fetal examinations - minor skeletal abnormality findings - group incidences                      
                           
      Fetuses       Litters
Group 1 2 3 4 1 2 3 4
Number Examined 155 119 154 138 19 18 21 20
Number Intact     73 55 69 64       19 18 21 20
Minor skeletal abnormalities
Cranial
sutural bone(s)   0 1 0 0       0 1 0 0
  fissure(s) 0 1 0 0 0 1 0 0
  additional suture(s) 1 1 1 1 1 1 1 1
  bent cornu(a) of hyoid 0 0 3 0 0 0 3 0
Vertebral element abnormalities thoracic 0 2 1 0 0 2 1 0
Ribs branched 0 1 2 1 0 1 2 1
  absent 0 2 0 0 0 2 0 0
  partially fused 0 1 0 0 0 1 0 0
  additional 0 0 1 0 0 0 1 0
  absent articulating surface 0 0 1 0 0 0 1 0
Sternebrae partially fused 1 2 1 2 1 2 1 2
  bipartite ossified 0 0 1 0 0 0 1 0
  misaligned hemicentres 0 1 0 0 0 1 0 0
  supernumerary site 1 2 4 1 1 2 3 1
  misshapen 0 2 1 0 0 2 1 0
Costal cartilage partially fused 0 0 1 1 0 0 1 1
  misaligned 0 1 1 1 0 1 1 1
  branched 0 2 0 0 0 2 0 0
  additional 0 1 2 1 0 1 2 1
  7th not connected to sternum 15 16 12 15 7 6 7 10
Minor skeletal abnormalities  
Costal cartilage hole in xiphoid 0 0 1 1 0 0 1 1
Total affected by one or more of the above 17 24 23 22 9 11 13 12
Rib and vertebral configuration  

Cervical rib
short supernumerary 2 10 6 7 1 7 3 4
  full supernumerary 0 0 1 1 0 0 1 1
12th rib short/with costal cartilage 0 0 0 1 0 0 0 1
Number of 13th ribs short supernumerary 55 32 55 35 16 12 19 15
  full supernumerary 26 31 34 29 10 10 13 16
  total 70 54 78 52 17 13 19 18
Thoracolumbar vertebrae 18 1 1 1 0 1 1 1 0
  20 8 4 7 10 6 2 5 7
Pelvic girdle unilateral caudal shift 4 2 4 3 3 1 4 3
Delayed/incomplete ossification/unossified  
Cranial large anterior fontanelle 0 1 0 0 0 1 0 0
  large posterior fontanelle 1 1 0 1 1 1 0 1
Sternebrae 5th 18 21 18 21 8 8 9 12
  other 7 8 11 15 3 6 5 8
  total 25 25 25 32 9 11 10 15
Vertebrae cervical (includes odontoid process) 0 0 2 1 0 0 2 1
  thoracic 2 0 0 0 2 0 0 0
Appendicular pubes 0 0 0 1 0 0 0 1
  epiphyses 9 7 10 9 3 5 6 6
  talus 0 0 0 1 0 0 0 1
  metacarpals/phalanges 7 6 5 8 4 4 4 5
  metatarsals/ phalanges 1 1 1 4 1 1 1 3
Increased ossification  
Cranial partially fused jugal to maxilla   2 2 2 0       1 2 2 0
Note: Individual fetuses/litters may occur in more than one category.

Fetal examinations - minor visceral abnormality and necropsy findings - group incidences
            Fetuses       Litters
Group 1 2 3 4 1 2 3 4
Number Examined 155 119 154 138 19 18 21 20
Number of Heads Examined at Detailed Visceral Examination         82 64 85 74       18 17 21 20
   
Head abnormalities (fixed visceral)  
Brain                                                subdural haemorrhage 2 0 0 2 2 0 0 2
Head                                                supernumerary minor upper incisor 0 0 0 1 0 0 0 1
Total affected by one or more of the above 2 0 0 3 2 0 0 3
Necropsy observations (fresh visceral)  
Heart                                                pericardium contains fluid 0 1 0 0 0 1 0 0
Total affected by one or more of the above                                                                            0 1 0 0 0 1 0 0
Necropsy observations (external)  
Limb(s) flexure forepaw(s) 0 1 0 3 0 1 0 3
Total affected by one or more of the above                                                                                      0 1 0 3       0 1 0 3

Applicant's summary and conclusion

Conclusions:
Oral administration of TXIB at dose levels of 100, 300 or 1000 mg/kg/day from Day 1 to 28 of gestation was generally well tolerated with no adverse effects on general condition body weight gain, food consumption or macropathology in the maternal New Zealand White rabbit.

Higher numbers of early resorptions and post-implantation losses were observed in females treated at 1000 mg/kg/day and consequently total live young were marginally reduced however in surviving fetuses examined on Day 29 of gestation, there were no effects on fetal growth or development at any level.

Based on these results, it is concluded that dose levels up to and including 1000 mg/kg/day did not adversely affect maternal performance or fetal development and growth, however a reduction in embryo-fetal survival at 1000 mg/kg/day cannot be discounted as an effect of treatment. Therefore, the No observed adverse effect level (NOAEL), based on embryo-fetal survival is considered to be 300 mg/kg/day.
Executive summary:

This study was designed to assess the influence of TXIB (an industrial chemical) on embryo-fetal survival and development when administered during the organogenesis and fetal

growth phases of pregnancy (Days 1-28 after mating) in the New Zealand White rabbit. Three groups of 22 females received TXIB at doses of 100, 300 or 1000 mg/kg/day by oral

gavage administration, from Day 1 to 28 after mating. A similarly constituted Control group received the vehicle, 1% methyl cellulose, at the same volume dose as the treated groups.

Animals were killed on Day 29 after mating for reproductive assessment and fetal examination. Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

Results

On Day 8 of gestation, one female (4F, 74) treated at 1000 mg/kg/day was despatched following a prolonged period of in-appetence, significant body weight loss and reduced

faecal and urine output; at macroscopic examination, there were no abnormalities observed. as this premature death was of an isolated incidence, there is no clear correlation to treatment with TXIB. All other premature deaths were incidental and not associated with treatment. There were no signs observed that were attributable to treatment and macroscopic examination revealed no treatment related findings. Group mean body weight gain during the treatment period (Day 1 to 28 of gestation) was slightly low in all treated groups when compared with Controls and food consumption was slightly reduced until Day 24 of gestation in females treated at 1000 mg/kg/day. On Day 29 of gestation, group mean gravid uterine weight was low in females treated with TXIB when compared with Controls however the magnitude of difference was slight and showed no relationship to dose level. Overall body weight change, when adjusted for the weight of the gravid uterus was comparable to controls at all dose levels (100, 300 or 1000 mg/kg/day).

At macroscopic examination, six females were confirmed not pregnant however the distribution showed no correlation to treatment. Mean numbers of corpora lutea and

implantations were considered to be unaffected by treatment with TXIB. Mean numbers of early resorptions and thus post-implantation losses, were high in females treated at 1000 mg/kg/day when compared with Controls and when compared with historical control data ranges; as a result the mean live litter size was slightly low compared with Controls. A relationship to treatment cannot be ruled out. Group mean placental, fetal and litter weights were unaffected by treatment. At 100, 300 or 1000 mg/kg/day, an increase in the incidence of short supernumerary cervical ribs was observed when compared with Controls however the distribution showed no relationship to treatment. At 1000 mg/kg/day or 300 mg/kg/day there was also a slight increase in incidence of delayed ossification of 1st to 4th and 6th sternebrae when compared with control litters however the incidence and distribution were found to be within historical background data ranges and as such a relation to treatment is not inferred.