1-isopropyl-2,2-dimethyltrimethylene diisobutyrate

Developmental toxicity / teratogenicity

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

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Propanoic acid, 2-methyl-, 1,1'-[2,2-dimethyl-1-(1-methylethyl)-1,3-propanediyl] ester

IUPAC Name:

Propanoic acid, 2-methyl-, 1,1'-[2,2-dimethyl-1-(1-methylethyl)-1,3-propanediyl] ester

Constituent 2

Reference substance name:

1-isopropyl-2,2-dimethyltrimethylene diisobutyrate

EC Number:

229-934-9

EC Name:

1-isopropyl-2,2-dimethyltrimethylene diisobutyrate

Cas Number:

6846-50-0

Molecular formula:

C16H30O4

IUPAC Name:

trimethoxy[3-(oxiran-2-ylmethoxy)propyl]silane

Constituent 3

Reference substance name:

2,2,4-Trimethylpentanediol-1,3-diisobutyrate

IUPAC Name:

2,2,4-Trimethylpentanediol-1,3-diisobutyrate

Constituent 4

Reference substance name:

Texanol isobutyrate; TXIB

IUPAC Name:

Texanol isobutyrate; TXIB

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 or test system 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 / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 4

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:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

maternal abnormalities

mortality

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

1

Abnormalities:

not specified

2

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.