1,1'-[methylenebis(oxy)]dibutane

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 9 JANUARY 2019 to 06 APRIL 2019

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-[methylenebis(oxy)]dibutane

EC Number:

219-909-0

EC Name:

1,1'-[methylenebis(oxy)]dibutane

Cas Number:

2568-90-3

Molecular formula:

C9H20O2

IUPAC Name:

1-(butoxymethoxy)butane

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd
- Age at study initiation: Approximately 71 days old
- Weight at study initiation: 211 to 281 g
- Fasting period before study: No
- Housing: Acclimatization: up to four animals; During pairing: one (stock) male and one female ; Gestation: one female
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet. ad libitum
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. ad libitum
- Acclimation period: Five days before commencement of pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours dark : 12 hours light

IN-LIFE DATES: From: 16 January 2019 To: 11 to 14 February 2019

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of test material was weighed out into a suitable container. Approximately 50% of the final volume of vehicle was added to the test material and mixed with a magnetic stirrer until homogenous. The solution was then made up to the required volume with vehicle. The formulation was transferred to a final container and mixed with a magnetic stirrer until homogenous.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.


VEHICLE
- Concentration in vehicle: 0, 20, 66, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method involved extraction and dilution in acetone followed by gas chromatographic analysis with flame ionization detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 10 μg/mL to 100 μg/mL.
An accurate volume of internal standard solution (100 μL) was added to all standard and sample solutions (1000 μL) and mixed by vortex to provide a final concentration of ca. 50 μg/mL of tetradecane.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1 with identified stock males
- Proof of pregnancy: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm referred to as day 0 of pregnancy

Duration of treatment / exposure:

Females were treated from Day 6 to Day 19 (inclusive) after mating

Frequency of treatment:

Daily (at approximately the same time each day)

Duration of test:

up to day 20 (necropsy)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

330 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 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: Dose levels of 0, 100, 330 and 1000 mg/kg/day were selected in conjunction with the Sponsor based on findings from a preliminary embryo-fetal development study conducted at these laboratories.
In the preliminary study oral administration of Butylal at 250, 500 and 1000 mg/kg/day was well tolerated with no effects on maternal clinical condition, macropathology, embryo-fetal survival or development. Effects were limited to low maternal body weight gain at 500 and 1000 mg/kg/day and low maternal food consumption at 1000 mg/kg/day.
Based on these findings there was nothing to preclude the use of 1000 mg/kg/day as the high dose level in this study. Dose levels of 100 and 330 mg/kg/day were then selected as low and intermediate dose levels to provide approximate 3-fold dose intervals and to allow assessment of any dose response.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3, 6-20 after mating.

FOOD CONSUMPTION AND COMPOUND INTAKE: 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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: 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.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Number of fetuses live and dead

Fetal examinations:

- External examinations: Yes: Examination of all viable fetuses and placentae
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes

Statistics:

For all adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

Historical control data:

Historical control data on major fetal examinations, skeletal fetal examinations and visceral fetal examinations

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Two animals receiving 1000 mg/kg/day showed increased salivation; this is often associated with dosing and is considered to relate to the palatability of the formulation rather than a direct effect of treatment
Tables 2 and 3

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Overall group mean body weight gain during treatment (Gestation Days 6 to 19) for females receiving 1000 mg/kg/day was low when compared with Controls (p<0.01) and mean body weight at the end of dosing (GD 20) was significantly low when compared with Controls (p<0.05). For females receiving 100 or 330 mg/kg/day mean body weight gain during treatment was similar to the Controls.
At 1000 mg/kg/day the gravid uterine weight was low when compared with Controls (p<0.05) and the maternal body weight gain following adjustment for the gravid uterine weight was also low for females at the high dose (p<0.01); the gravid uterine weight and adjusted maternal weight gain at 100 or 330 mg/kg/day were similar to the Controls.
Tables 4 and 5

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the treatment period group mean food consumption was low for females receiving 1000 mg/kg/day; with the difference attaining statistical significance GD6-9 (p<0.05) and GD10-17 (p<0.01).
Overall food consumption for females receiving 100 or 330 mg/kg/day was similar to the Controls.
Table 6

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Thyroid weight for females on GD20 were unaffected by treatment with Butylal.
Table 7

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination of females on Day 20 after mating did not reveal any findings that could be attributed to treatment.
Table 8

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic examination of the thyroid glands showed no changes that could be related to treatment with Butylal.
Table 9

Other effects:

no effects observed

Description (incidence and severity):

There was no clear effect on serum T3 concentrations and its prohormone T4 in pregnant rats following administration of Butylal.
There was no dose related effect evident on serum TSH concentrations in pregnant rats after the administration of Butylal via oral gavage.

Maternal developmental toxicity

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Table 11

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Table 1

Early or late resorptions:

no effects observed

Description (incidence and severity):

Table 11

Dead fetuses:

no effects observed

Description (incidence and severity):

Tables 10 and 11

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Table 11

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

clinical signs

dead fetuses

early or late resorptions

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Placental, litter or fetal weights were unaffected by maternal treatment.
Table 12

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Tables 10 and 12

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Table 11

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Placental, litter or fetal weights were unaffected by maternal treatment.
Table 12

External malformations:

no effects observed

Description (incidence and severity):

Ano-genital of fetuses on GD20 were unaffected by maternal treatment with Butylal.
Table 13
There was no evidence on an increase in major malformations associated with administration of Butylal treatment
Tables 14, 15,16 and 17

Skeletal malformations:

no effects observed

Description (incidence and severity):

At 330 and 1000 mg/kg/day there was an increased incidence of short supernumerary 14th rib when compared with the concurrent Controls; the fetal and litter incidence at 1000 mg/kg/day was outside the Historical Control data (HCD) range. Studies have shown that this finding does not persist in the rat and therefore in isolation the increased incidence in this variant is considered not to be adverse.
In the intermediate and high dose groups, there appeared to be lower incidences of incompletely ossified cranial centres, hyoid, sternebrae and sacrocaudal vertebrae compared to the Control group and in the low dose group (100 mg/kg/day). However, all incidences were within HCD ranges and therefore it was concluded that these findings at 330 and 1000 mg/kg/day are not adverse.
Tables 18, 19, 20 and 21

Visceral malformations:

no effects observed

Description (incidence and severity):

Tables 22, 23, 24 and 25

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

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

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

	Control	Butylal
Dose group	1	2	3	4
Dose (mg/kg/day)	0	100	330	1000

 

Table 1: Summary of maternal disposition

	Group 1	Group 2	Group 3	Group 4
	Number of animals
Number mated	20	20	20	20
Number not pregnant	0	0	0	0
Number pregnant	20	20	20	20
Number of early deliveries	0	0	0	0
Number of litters with dead fetuses	0	0	0	0
Number of litters with resorptions	8	10	7	9
Number of litters subject to fetal pathology	20	20	20	20

 

 

Table 2: Signs associated with dosing – group distribution of observations during gestation

			Number of animals affected
Category	Observation	Group/Sex: Initial no:	1F 20	2F 20	3F 20	4F 20
Behavior	Salivation		0	0	0	2

 

Table 3: Clinical signs – group distribution of observations during gestation

			Number of animals affected
Category	Observation	Group/Sex: Initial no:	1F 20	2F 20	3F 20	4F 20
Behavior	Salivation Vocalization		0 0	0 0	0 0	2 1
Coat	Hair loss, Forelimbs Hair loss, Hindlimbs Hair loss, Ventral surface		3 1 1	1 0 0	0 0 0	1 0 0
Skin	Encrustation, Ear – right Encrustation, Hindlimb – left		0 1	1 0	0 0	0 0
Staining	Abnormal color, Brown, Ears Abnormal color, Brown, Forelimbs Abnormal color, Brown, Head Abnormal color, Brown, Upper dorsal surface		1 1 3 1	0 0 2 3	0 0 1 1	0 0 0 1

 

 

 

Table 4: Body weight and body weight change - group mean values (g) during gestation

Group /Sex		Day
		0	3	6	7	8	9	10	11	12	13	14	15	16
Statistics test		Av	Av	Av	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi
1F	Mean	245	261	277	278	282	286	291	297	304	307	312	320	331
	SD	17.8	18.6	19.6	18.4	17.6	18.7	18.7	18.9	20.1	20.7	20.7	21.4	22.3
	N	20	20	20	20	20	20	20	20	20	20	20	20	20
2F	Mean	247	265	280	281	285	290	295	302	306	311	317	325	336
	SD	19.3	21.0	22.0	22.6	22.8	22.1	24.1	24.7	25.2	24.7	25.3	26.1	25.8
	N	20	20	20	20	20	20	20	20	20	20	20	20	20
3F	Mean	244	262	277	276	280	285	291	297	301	306	311	317	328
	SD	18.6	19.5	19.7	20.7	20.5	21.5	21.9	22.2	22.4	22.6	22.7	23.7	23.2
	N	20	20	20	20	20	20	20	20	20	20	20	20	20
4F	Mean	244	260	275	271	274	277	284	290	294	298	305	311	319
	SD	14.4	14.8	15.3	14.5	13.7	16.9	14.8	15.0	14.9	14.9	14.3	14.8	16.1
	N	20	20	20	20	20	20	20	20	20	20	20	20	20

 

Table 4 (cont): Body weight and body weight change - group mean values (g) during gestation

Group /Sex		Day 17	18	19	20	Change 0-3	Change 0-6	Change 6-7	Change 6-8	Change 6-9	Change 6-10	Change 6-11	Change 6-12
Statistics test		Wi	Wi	Wi	Wi	Av	Av	Wi	Wi	Sh	Wi	Wi	Wi
1F	Mean	345	360	373	389	16	32	1	4	9	13	20	26
	SD	23.6	24.9	27.8	29.9	6.1	6.9	3.6	5.2	4.4	5.0	4.7	5.7
	N	20	20	20	20	20	20	20	20	20	20	20	20
2F	Mean	350	366	381	396	18	34	0	4	10	14	21	26
	SD	26.5	27.6	29.3	30.2	5.5	6.3	4.2	2.9	4.8	6.0	5.7	6.5
	N	20	20	20	20	20	20	20	20	20	20	20	20
3F	Mean	341	356	368	385	18	33	-1	3	8	14	19	24
	SD	27.1	26.0	27.6	28.1	4.4	5.9	4.4	3.8	4.6	5.0	5.9	6.4
	N	20	20	20	20	20	20	20	20	20	20	20	20
4F	Mean	329*	343*	354*	368*	15	31	-4**	-1**	2**	9*	15**	18**
	SD	15.5	17.4	16.8	19.0	3.4	5.8	5.0	5.0	9.1	5.4	5.7	5.6
	N	20	20	20	20	20	20	20	20	20	20	20	20

 

Table 4 (cont): Body weight and body weight change - group mean values (g) during gestation

Group /Sex		Change 6-13	Change 6-14	Change 6-15	Change 6-16	Change 6-17	Change 6-18	Change 6-19	Change 6-20
Statistics test		Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi
1F	Mean	30	35	43	54	68	83	96	111
	SD	5.1	4.9	5.4	6.5	7.9	9.9	13.6	15.4
	N	20	20	20	20	20	20	20	20
2F	Mean	30	36	44	55	69	86	100	116
	SD	6.0	6.8	8.8	9.4	10.4	11.3	14.0	13.2
	N	20	20	20	20	20	20	20	20
3F	Mean	29	34	40	51	64	79	91	107
	SD	6.1	5.6	7.4	7.6	11.3	11.2	13.5	14.9
	N	20	20	20	20	20	20	20	20
4F	Mean	23**	30**	36**	44**	54**	68**	79**	93**
	SD	6.8	6.4	7.3	8.0	8.9	9.4	11.4	13.1
	N	20	20	20	20	20	20	20	20

 

Table 5: Gravid uterine weight, adjusted body weight and adjusted body weight change - group mean values (g) on Day 20 of gestation

Group /Sex		Body weight Day 6	Terminal body weight Day 20	Body weight change 6-20	Gravid uterine weight	Adjusted body weight Day 20	Adjusted body weight change 6-20
Statistics test		Av	Wi	Wi	Wi	Wi	Wi
1F	Mean	277	389	111	86.4	302	25
	SD	19.6	30.2	15.6	15.01	25.1	8.5
	N	20	20	20	20	20	20
2F	Mean	280	397	117	90.1	307	26
	SD	22.0	30.1	13.6	8.13	25.4	9.7
	N	20	20	20	20	20	20
3F	Mean	277	385	108	83.0	302	24
	SD	19.7	28.2	14.9	11.89	24.6	8.3
	N	20	20	20	20	20	20
4F	Mean	275	368*	93**	78.3*	290	14**
	SD	15.3	19.0	13.0	11.54	14.0	7.5
	N	20	20	20	20	20	20

 

Table 6: Food consumption - group mean values (g/animal/day) during gestation

Group /Sex		Day 0-2	3-5	6-9	10-13	14-17	18-19	Total 6-19	As % Control
Statistics test		Av	Av	Wi	Wi	Wi	Wi
1F	Mean	20	22	19	21	24	21	298	-
	SD	1.9	1.7	1.9	1.9	1.6	3.0
	N	20	20	20	20	20	20
2F	Mean	22	24	20	22	24	22	309	103
	SD	2.9	2.6	2.6	2.5	2.7	3.0
	N	20	20	20	20	20	20
3F	Mean	21	23	20	21	23	21	298	100
	SD	2.1	1.9	2.1	2.1	2.4	2.8
	N	20	20	20	20	20	20
4F	Mean	21	23	17*	19**	20**	19	262	88
	SD	1.6	2.0	1.9	1.8	1.7	2.5
	N	20	20	20	20	20	20

 

 

 

 

Table 7: Organ weights - group mean absolute and adjusted values (g) on Day 20 of gestation

Group /Sex		Terminal Body weight	Thyroids and Parathyroids
Statistics test		Wi
1F	Mean	389	0.015
	SD	30	0.002
	N	20	20
2F	Mean	397	0.014
	SD	30	0.003
	N	20	20
3F	Mean	385	0.015
	SD	28	0.003
	N	20	20
4F	Mean	368*	0.013
	SD	19	0.003
	N	20	20

 

		Thyroids and Parathyroids
Statistics test		Wi
1F	Adjusted Mean	0.015
2F	Adjusted Mean	0.013
3F	Adjusted Mean	0.015
4F	Adjusted Mean	0.013

 

Table 8: Macropathology - group distribution of findings on Day 20 of gestation

		Number of animals affected
Tissue/Organ and Findings	Group/Sex No. of animals	1F 20	2F 20	3F 20	4F 20
Vagina Abnormal contents		0	0	1	0

 

Table 9: Histopathology - group distribution of findings on Day 20 of gestation

		Number of animals affected
Tissue/Organ and Findings	Group/Sex No. of animals	1F 20	2F 20	3F 20	4F 20
Thyroids Cyst(s), Follicular	No. examined Minimal Total	20 0 0	20 0 0	20 1 1	20 0 0

 

Table 10: Litter data - summary

Live offspring compared to	0 mg/kg/day	100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
Dead fetuses (%)	100	100	100	100
Concurrent controls (%)	100	107	97	95
Number of implantations (%)	100	95	96	94

 

 

Table 11: Litter data - group mean values on Day 20 of gestation

Group /Sex		Corpora lutea	Implantations	Resorption			Implantation loss (%)		Implantation loss#		Live young			Sex ratio (%M)
				Early	Late	Total	Pre-	Post-	Pre-	Post-	Male	Female	Total
Statistics test		Wi	Sh				Wi	Wi					Wi	Wi
1F	Mean	16.6	15.2	0.6	0.0	0.6	10.0	3.9	1.4	0.6	7.6	7.0	14.6	50.8
	SD	2.65	3.17	0.88	0.00	0.88	14.60	5.52			2.78	1.97	3.03	13.48
	N	20	20	20	20	20	20	20	20	20	20	20	20	20
2F	Mean	17.1	16.3	0.8	0.0	0.8	5.3	4.6	0.9	0.8	7.7	7.8	15.5	49.5
	SD	1.37	1.29	0.91	0.00	0.91	6.93	5.67			1.98	1.77	1.54	10.86
	N	20	20	20	20	20	20	20	20	20	20	20	20	20
3F	Mean	17.2	14.7	0.6	0.0	0.6	14.0	3.5	2.5	0.6	7.2	6.9	14.1	50.7
	SD	2.92	2.48	0.94	0.00	0.94	11.55	5.54			2.24	1.80	2.25	11.92
	N	20	20	20	20	20	20	20	20	20	20	20	20	20
4F	Mean	15.9	14.8	0.9	0.0	0.9	7.7	6.1	1.2	0.9	7.1	6.8	13.9	50.5
	SD	2.02	2.31	1.21	0.00	1.21	10.31	8.63			2.59	2.17	2.37	15.74
	N	20	20	20	20	20	20	20	20	20	20	20	20	20

# Calculated using the sum of individual values within each group/number of litters per group

 

Table 12: Placental, litter and fetal weights - group mean values (g) on Day 20 of gestation

Group /Sex		Placental weight	Total litter weight	Male fetal weight	Female fetal weight	Overall fetal weight
Statistics test		Wi	Wi	Wi	Wi	Wi
1F	Mean	0.50	54.29	3.90	3.61	3.75
	SD	0.060	10.113	0.240	0.231	0.224
	N	20	20	20	20	20
2F	Mean	0.51	56.75	3.80	3.56	3.68
	SD	0.048	5.861	0.267	0.223	0.231
	N	20	20	20	20	20
3F	Mean	0.52	52.48	3.78	3.67	3.73
	SD	0.057	8.020	0.256	0.255	0.247
	N	20	20	20	20	20
4F	Mean	0.50	50.69	3.76	3.57	3.68
	SD	0.060	8.117	0.225	0.261	0.247
	N	20	20	20	20	20

 

Table 13: Ano-genital distance - group mean absolute and adjusted values for fetuses

Group /Sex		Fetal weight (g)	Ano-genital distance (mm)
Statistics test		Wi
1M	Mean	3.9	4.1
	SD	0.24	0.22
	N	20	20
2M	Mean	3.8	4.1
	SD	0.27	0.15
	N	20	20
3M	Mean	3.7	4.1
	SD	0.28	0.27
	N	20	20
4M	Mean	3.8	4.1
	SD	0.23	0.17
	N	20	20

 

		Ano-genital distance
Statistics test		Wi
1M	Adjusted Mean	4.1
2M	Adjusted Mean	4.1
3M	Adjusted Mean	4.1
4M	Adjusted Mean	4.1

 

Table 13 (cont): Ano-genital distance - group mean absolute and adjusted values for fetuses

Group /Sex		Fetal weight (g)		Ano-genital distance (mm)
Statistics test		Wi
1F	Mean	3.6		2.5
	SD	0.23		0.19
	N	20		20
2F	Mean	3.6		2.5
	SD	0.22		0.21
	N	20		20
3F	Mean	3.7		2.5
	SD	0.26		0.18
	N	20		20
4F	Mean	3.6		2.5
	SD	0.26		0.16
	N	20		20
			Ano-genital distance
Statistics test			Wi
1F	Adjusted Mean		2.5
2F	Adjusted Mean		2.5
3F	Adjusted Mean		2.5
4F	Adjusted Mean		2.5

 

Table 14: Fetal examinations - major abnormality findings - group incidences

		Fetuses				Litters
Group		1	2	3	4	1	2	3	4
Number Examined		291*	310**	282***	277	20	20	20	20
Total Number Affected		1	3	1	1	1	2	1	1
Head Skeletal External	Brachygnathia Cleft palate Protruding tongue	0 0 0	0 0 0	0 0 0	1 1 1	0 0 0	0 0 0	0 0 0	1 1 1
Cervical/Thoracic Skeletal Visceral	Partially split sternum Retroesophageal aortic arch Dorsally displaced pulmonary trunk Muscular ventricular septal defect Malrotated heart Thoracic situs inversus	0 1 0 0 1 0	1 0 1 1 1 0	0 0 0 1 0 1	0 0 0 0 0 0	0 1 0 0 1 0	1 0 1 1 1 0	0 0 0 1 0 1	0 0 0 0 0 0
Lumbar (and abdominal)/Sacral/Caudal Skeletal External     Visceral	Termination vertebral column sacral region Gastroschisis Threadlike tail Imperforate anus Abdominal situs inversus	0 0 0 0 0	1 1 1 1 1	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	1 1 1 1 1	0 0 0 0 0	0 0 0 0 0

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

*Litter 13, 7 fetuses heads only examined, included in tabulated data.

**Litter 26 head only examined for one fetus, included in tabulated data.

***Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated.

Table 14 (cont): Fetal examinations - major abnormality findings - group incidences

		Fetuses				Litters
Group		1	2	3	4	1	2	3	4
Number Examined		291*	310**	282***	277	20	20	20	20
Total Number Affected		1	3	1	1	1	2	1	1
Appendicular Skeletal	Short/bent/thickened long bones Clavicle bent/incompletely ossified	0 0	0 0	0 0	1 1	0 0	0 0	0 0	1 1

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

*Litter 13, 7 fetuses heads only examined, included in tabulated data.

**Litter 26 head only examined for one fetus, included in tabulated data.

***Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated

 

Table 15: Fetal examinations - major abnormality findings - group percentage incidences

		Fetuses				Litters
Group		1	2	3	4	1	2	3	4
Number Examined		291*	310**	282***	277	20	20	20	20
Total Number Affected		1	3	1	1	1	2	1	1
Head Skeletal External	Brachygnathia Cleft palate Protruding tongue	0.0 0.0 0.0	0.0 0.0 0.0	0.0 0.0 0.0	0.7 0.4 0.4	0.0 0.0 0.0	0.0 0.0 0.0	0.0 0.0 0.0	5.0 5.0 5.0
Cervical/Thoracic Skeletal Visceral	Partially split sternum Retroesophageal aortic arch Dorsally displaced pulmonary trunk Muscular ventricular septal defect Malrotated heart Thoracic situs inversus	0.0 0.7 0.0 0.0 0.7 0.0	0.6 0.0 0.6 0.6 0.6 0.0	0.0 0.0 0.0 0.7 0.0 0.7	0.0 0.0 0.0 0.0 0.0 0.0	0.0 5.0 0.0 0.0 5.0 0.0	5.0 0.0 5.0 5.0 5.0 0.0	0.0 0.0 0.0 5.0 0.0 5.0	0.0 0.0 0.0 0.0 0.0 0.0
Lumbar (and abdominal)/Sacral/Caudal Skeletal External     Visceral	Termination vertebral column sacral region Gastroschisis Threadlike tail Imperforate anus Abdominal situs inversus	0.0 0.0 0.0 0.0 0.0	0.6 0.3 0.3 0.3 0.6	0.0 0.0 0.0 0.0 0.0	0.0 0.0 0.0 0.0 0.0	0.0 0.0 0.0 0.0 0.0	5.0 5.0 5.0 5.0 5.0	0.0 0.0 0.0 0.0 0.0	0.0 0.0 0.0 0.0 0.0
Appendicular Skeletal	Short/bent/thickened long bones Clavicle bent/incompletely ossified	0.0 0.0	0.0 0.0	0.0 0.0	0.7 0.7	0.0 0.0	0.0 0.0	0.0 0.0	5.0 5.0

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

*Litter 13, 7 fetuses heads only examined, included in tabulated data.

**Litter 26 head only examined for one fetus, included in tabulated data.

***Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated

Table 16: Fetal examinations - Major Historical Control Data - incidence

Species/strain/source	Rat/ Crl:CD(SD) / Charles River UK
Necropsy date range	Aug 2018– Apr 2019
Number of studies	8
Study types	Main

 

		GB01PV								HCD Range
		Fetuses				Litters				Fetuses	Litters
Group		1	2	3	4	1	2	3	4
Number Examined		291	310	282	277	20	20	20	20	2098	141
Head
Skeletal	Brachygnathia	0	0	0	1	0	0	0	1	0-1	0-1
External	Cleft palate	0	0	0	1	0	0	0	1	0-0	0-0
	Protruding tongue	0	0	0	1	0	0	0	1	0-1	0-1
Cervical/Thoracic
Skeletal	Partially split sternum	0	1	0	0	0	1	0	0	0-1	0-1
Visceral	Dorsally displaced pulmonary trunk	0	1	0	0	0	1	0	0	0-0	0-0
	Muscular ventricular septal defect	0	1	1	0	0	1	1	0	0-0	0-0
	Thoracic situs inversus	0	0	1	0	0	0	1	0	0-0	0-0
Lumbar (and abdominal)/Sacral/Caudal
Skeletal	Termination vertebral column sacral region	0	1	0	0	0	1	0	0	0-0	0-0
External	Gastroschisis	0	1	0	0	0	1	0	0	0-0	0-0
	Threadlike tail	0	1	0	0	0	1	0	0	0-0	0-0
	Imperforate anus	0	1	0	0	0	1	0	0	0-0	0-0
Visceral	Abdominal situs inversus	0	1	0	0	0	1	0	0	0-0	0-0
Appendicular
Skeletal	Short/bent/thickened long bones	0	0	0	1	0	0	0	1	0-1	0-1
	Clavicle bent/incompletely ossified	0	0	0	1	0	0	0	1	0-1	0-1

 

Table 17: Fetal examinations - Major Historical Control Data - percentage (%)

Species/strain/source	Rat/ Crl:CD(SD) / Charles River UK
Necropsy date range	Aug 2018– Apr 2019
Number of studies	8
Study types	Main

 

		GB01PV								HCD Range
		Fetuses				Litters				Fetuses	Litters
Group		1	2	3	4	1	2	3	4
Number Examined		291	310	282	277	20	20	20	20	2098	141
Head
Skeletal	Brachygnathia^*	0.0	0.0	0.0	0.7	0.0	0.0	0.0	5.0	0.0-0.7	0.0-5.3
External	Cleft palate	0.0	0.0	0.0	0.4	0.0	0.0	0.0	5.0	0.0-0.0	0.0-0.0
	Protruding tongue	0.0	0.0	0.0	0.4	0.0	0.0	0.0	5.0	0.0-0.3	0.0-5.3
Cervical/Thoracic
Skeletal	Partially split sternum^*	0.0	0.6	0.0	0.0	0.0	5.0	0.0	0.0	0.0-1.0	0.0-16.7
Visceral	Dorsally displaced pulmonary trunk^**	0.0	0.6	0.0	0.0	0.0	5.0	0.0	0.0	0.0-0.0	0.0-0.0
	Muscular ventricular septal defect^**	0.0	0.6	0.7	0.0	0.0	5.0	5.0	0.0	0.0-0.0	0.0-0.0
	Thoracic situs inversus^!	0.0	0.0	0.7	0.0	0.0	0.0	5.0	0.0	0.0-0.0	0.0-0.0
Lumbar (and abdominal)/Sacral/Caudal
Skeletal	Termination vertebral column sacral region^*	0.0	0.6	0.0	0.0	0.0	5.0	0.0	0.0	0.0-0.0	0.0-0.0
External	Gastroschisis	0.0	0.3	0.0	0.0	0.0	5.0	0.0	0.0	0.0-0.0	0.0-0.0
	Threadlike tail	0.0	0.3	0.0	0.0	0.0	5.0	0.0	0.0	0.0-0.0	0.0-0.0
	Imperforate anus	0.0	0.3	0.0	0.0	0.0	5.0	0.0	0.0	0.0-0.0	0.0-0.0
Visceral	Abdominal situs inversus	0.0	0.6	0.0	0.0	0.0	5.0	0.0	0.0	0.0-0.0	0.0-0.0
Appendicular
Skeletal	Short/bent/thickened long bones^*	0.0	0.0	0.0	0.7	0.0	0.0	0.0	5.0	0.0-0.7	0.0-5.3
	Clavicle bent/incompletely ossified^*	0.0	0.0	0.0	0.7	0.0	0.0	0.0	5.0	0.0-0.7	0.0-5.3

* Findings noted at skeletal examination, therefore % was calculated from number of skeletons examined 

^** Findings noted at visceral examination, therefore % was calculated from number of fixed fetuses.

 

 

Table 18: Fetal examinations - minor skeletal abnormality and variants findings - group incidences

		Fetuses				Litters
Group		1	2	3*	4	1	2	3	4
Number Examined		144	154	141	138	20	20	20	20
Minor skeletal abnormalities Cranial Vertebral element abnormality Ribs Sternebrae   Costal cartilage     Total affected by one or more of the above	Sutural bone(s) Thoracic Medially thickened/kinked Misaligned ossification sites Misshapen Partially fused Misaligned 7^th not connected to sternum	0 0 0 1 0 1 1 0 2	0 1 1 0 1 0 0 0 3	0 0 0 0 0 0 0 0 0	1 1 2 1 0 0 0 1 5	0 0 0 1 0 1 1 0 2	0 1 1 0 1 0 0 0 3	0 0 0 0 0 0 0 0 0	1 1 2 1 0 0 0 1 5
Rib and vertebral configuration Cervical rib 1^st rib 13^th rib Number of 14^th ribs   Thoracolumbar vertebrae Pelvic girdle	Short supernumerary Short with costal cartilage Short with/without costal cartilage Short supernumerary Total 20 Unilateral cranial shift	0 1 5 3 3 1 0	0 0 1 6 6 0 1	0 0 4 14 14 0 0	1 0 0 23 23 1 0	0 1 4 3 3 1 0	0 0 1 6 6 0 1	0 0 3 8 8 0 0	1 0 0 12 12 1 0

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

*Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated.

Table 18 (cont): Fetal examinations - minor skeletal abnormality and variants findings - group incidences

		Fetuses				Litters
Group		1	2	3*	4	1	2	3	4
Number Examined		144	154	141	138	20	20	20	20
Delayed/Incomplete ossification/unossified Cranial     Sternebrae     Vertebrae         Ribs Appendicular       Increased ossification Cervical vertebral centra	Large nasofrontal suture Cranial centres Hyoid 5^th and/or 6^th Other Total Cervical Thoracic Lumbar Sacrocaudal Caudal Any Scapula(e) Pelvic bones Metacarpals Metatarsals   All ossified	0 15 17 92 14 93 4 10 1 13 1 0 0 8 3 2   0	1 10 17 113 22 115 2 5 1 14 2 0 0 10 1 1   2	0 4 8 71 7 73 0 5 0 4 3 0 0 5 0 0   0	0 1 0 50 8 52 1 17 1 1 1 1 1 1 0 0   0	0 8 7 20 9 20 4 9 1 9 1 0 0 5 2 2   0	1 5 11 20 11 20 2 5 1 7 2 0 0 5 1 1   2	0 2 6 18 3 18 0 5 0 2 1 0 0 3 0 0   0	0 1 0 18 8 18 1 11 1 1 1 1 1 1 0 0   0

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

*Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated.

 

Table 19: Fetal examinations - minor skeletal abnormality and variants findings - group percentage incidences

		Fetuses				Litters
Group		1	2	3*	4	1	2	3	4
Number Examined		144	154	141	138	20	20	20	20
Minor skeletal abnormalities Cranial Vertebral element abnormality Ribs Sternebrae   Costal cartilage     Total affected by one or more of the above	Sutural bone(s) Thoracic Medially thickened/kinked Misaligned ossification sites Misshapen Partially fused Misaligned 7^th not connected to sternum	0.0 0.0 0.0 0.7 0.0 0.7 0.7 0.0 1.4	0.0 0.6 0.6 0.0 0.6 0.0 0.0 0.0 1.9	0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0	0.7 0.7 1.4 0.7 0.0 0.0 0.0 0.7 3.6	0.0 0.0 0.0 5.0 0.0 5.0 5.0 0.0 10.0	0.0 5.0 5.0 0.0 5.0 0.0 0.0 0.0 15.0	0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0	5.0 5.0 10.0 5.0 0.0 0.0 0.0 5.0 25.0
Rib and vertebral configuration Cervical rib 1^st rib 13^th rib Number of 14^th ribs   Thoracolumbar vertebrae Pelvic girdle	Short supernumerary Short with costal cartilage Short with/without costal cartilage Short supernumerary Total 20 Unilateral cranial shift	0.0 0.7 3.5 2.1 2.1 0.7 0.0	0.0 0.0 0.6 3.9 3.9 0.0 0.6	0.0 0.0 2.8 9.9 9.9 0.0 0.0	0.7 0.0 0.0 16.7 16.7 0.7 0.0	0.0 5.0 20.0 15.0 15.0 5.0 0.0	0.0 0.0 5.0 30.0 30.0 0.0 5.0	0.0 0.0 15.0 40.0 40.0 0.0 0.0	5.0 0.0 0.0 60.0 60.0 5.0 0.0

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

*Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated.

Table 19 (cont): Fetal examinations - minor skeletal abnormality and variants findings - group percentage incidences

		Fetuses				Litters
Group		1	2	3*	4	1	2	3	4
Number Examined		144	154	141	138	20	20	20	20
Delayed/Incomplete ossification/unossified Cranial     Sternebrae     Vertebrae         Ribs Appendicular       Increased ossification Cervical vertebral centra	Large nasofrontal suture Cranial centres Hyoid 5^th and/or 6^th Other Total Cervical Thoracic Lumbar Sacrocaudal Caudal Any Scapula(e) Pelvic bones Metacarpals Metatarsals   All ossified	0.0 10.4 11.8 63.9 9.7 64.6 2.8 6.9 0.7 9.0 0.7 0.0 0.0 5.6 2.1 1.4   0.0	0.6 6.5 11.0 73.4 14.3 74.7 1.3 3.2 0.6 9.1 1.3 0.0 0.0 6.5 0.6 0.6   1.3	0.0 2.8 5.7 50.4 5.0 51.8 0.0 3.5 0.0 2.8 2.1 0.0 0.0 3.5 0.0 0.0   0.0	0.0 0.7 0.0 36.2 5.8 37.7 0.7 12.3 0.7 0.7 0.7 0.7 0.7 0.7 0.0 0.0   0.0	0.0 40.0 35.0 100.0 45.0 100.0 20.0 45.0 5.0 45.0 5.0 0.0 0.0 25.0 10.0 10.0   0.0	5.0 25.0 55.0 100.0 55.0 100.0 10.0 25.0 5.0 35.0 10.0 0.0 0.0 25.0 5.0 5.0   10.0	0.0 10.0 30.0 90.0 15.0 90.0 0.0 25.0 0.0 10.0 5.0 0.0 0.0 15.0 0.0 0.0   0.0	0.0 5.0 0.0 90.0 40.0 90.0 5.0 55.0 5.0 5.0 5.0 5.0 5.0 5.0 0.0 0.0   0.0

 

Table 20: Fetal examinations - Skeletal Historical Control Data - incidence

Species/strain/source	Rat/ Crl:CD(SD) / Charles River UK
Necropsy date range	Aug 2018– Apr 2019
Number of studies	8
Study types	Main

 

		GP01PV								HCD Range
		Fetuses				Litters				Fetuses	Litters
Group		1	2	3	4	1	2	3	4
Number Examined		144	154	141	138	20	20	20	20	1105	141
Minor skeletal abnormalities
Cranial	sutural bone(s)	0	0	0	1	0	0	0	1	0-0	0-0
Vertebral element abnormality	thoracic	0	1	0	1	0	1	0	1	0-1	0-1
Ribs	medially thickened/kinked	0	1	0	2	0	1	0	2	0-1	0-1
Sternebrae	misshapen	0	1	0	0	0	1	0	0	0-0	0-0
Costal cartilage	7th not connected to sternum	0	0	0	1	0	0	0	1	0-1	0-1
Rib and vertebral configuration
Cervical rib	short supernumerary	0	0	0	1	0	0	0	1	0-2	0-1
Number of 14^thribs	short supernumerary	3	6	14	23	3	6	8	12	1-14	1-8
Pelvic girdle	unilateral cranial shift	0	1	0	0	0	1	0	0	0-1	0-1
Delayed/Incomplete ossification/unossified
Cranial	large nasofrontal suture	0	1	0	0	0	1	0	0	0-3	0-2
	cranial centres	15	10	4	1	8	5	2	1	1-20	1-11
	hyoid	17	17	8	0	7	11	6	0	10-29	4-13
Sternebrae	5th and/or 6th	92	113	71	50	20	20	18	18	42-99	6-20
	other	14	22	7	8	9	11	3	8	4-11	1-9
	total	93	115	73	52	20	20	18	18	45-99	6-20
Vertebrae	sacrocaudal	13	14	4	1	9	7	2	1	0-18	0-8
Ribs	any	0	0	0	1	0	0	0	1	0-1	0-1
Appendicular	Scapula	0	0	0	1	0	0	0	1	0-0	0-0
Increased ossification
Cervical vertebral centra	all ossified	0	2	0	0	0	2	0	0	0-0	0-0

 

 

Table 21: Fetal examinations - Skeletal Historical Control Data - percentage (%)

	Species/strain/source	Rat/ Crl:CD(SD) / Charles River UK
	Necropsy date range	Aug 2018– Apr 2019
	Number of studies	8
	Study types	Main

 

		GP01PV								HCD Range
		Fetuses				Litters				Fetuses	Litters
Group		1	2	3	4	1	2	3	4
Number Examined		144	154	141	138	20	20	20	20	1105	141
Minor skeletal abnormalities
Cranial	sutural bone(s)	0.0	0.0	0.0	0.7	0.0	0.0	0.0	5.0	0.0-0.0	0.0-0.0
Vertebral element abnormality	thoracic	0.0	0.6	0.0	0.7	0.0	5.0	0.0	5.0	0.0-0.7	0.0-5.3
Ribs	medially thickened/kinked	0.0	0.6	0.0	1.4	0.0	5.0	0.0	10.0	0.0-0.7	0.0-5.3
Sternebrae	misshapen	0.0	0.6	0.0	0.0	0.0	5.0	0.0	0.0	0.0-0.0	0.0-0.0
Costal cartilage	7th not connected to sternum	0.0	0.0	0.0	0.7	0.0	0.0	0.0	5.0	0.0-0.7	0.0-5.3
Rib and vertebral configuration
Cervical rib	short supernumerary	0.0	0.0	0.0	0.7	0.0	0.0	0.0	5.0	0.0-1.3	0.0-5.6
Number of 14^thribs	short supernumerary	2.1	3.9	9.9	16.7	15.0	30.0	40.0	60.0	0.7-10.9	5.3-50.0
Pelvic girdle	unilateral cranial shift	0.0	0.6	0.0	0.0	0.0	5.0	0.0	0.0	0.0-0.7	0.0-5.3
Delayed/Incomplete ossification/unossified
Cranial	large nasofrontal suture	0.0	0.6	0.0	0.0	0.0	5.0	0.0	0.0	0.0-2.2	0.0-11.1
	cranial centres	10.4	6.5	2.8	0.7	40.0	25.0	10.0	5.0	1.0-13.7	15.8-55.0
	hyoid	11.8	11.0	5.7	0.0	35.0	55.0	30.0	0.0	7.5-20.4	31.6-66.7
Sternebrae	5th and/or 6th	63.9	73.4	50.4	36.2	100.0	100.0	90.0	90.0	41.6-67.8	88.9-100.0
	other	9.7	14.3	5.0	5.8	45.0	55.0	15.0	40.0	2.7-7.7	16.7-45.0
	total	64.6	74.7	51.8	37.7	100.0	100.0	90.0	90.0	44.6-67.8	88.9-100.0
Vertebrae	sacrocaudal	9.0	9.1	2.8	0.7	45.0	35.0	10.0	5.0	0.0-13.4	0.0-44.4
Ribs	any	0.0	0.0	0.0	0.7	0.0	0.0	0.0	5.0	0.0-0.7	0.0-5.6
Appendicular	scapula	0.0	0.0	0.0	0.7	0.0	0.0	0.0	5.0	0.0-0.0	0.0-0.0
Increased ossification
Cervical vertebral centra	all ossified	0.0	1.3	0.0	0.0	0.0	10.0	0.0	0.0	0.0-0.0	0.0-0.0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 22: Fetal examinations - minor visceral abnormality and necropsy findings - group incidences

		Fetuses				Litters
Group		1	2	3	4	1	2	3	4
Number Examined		147*	156**	141***	139	20	20	20	20
Total Number Affected		31	20	24	26	16	13	14	14
Visceral abnormalities Brain Eyes     Thymus   Lungs Caudal vena cava Diaphragm Liver Adrenal(s) Ureter(s) Testis(es)   Umbilical artery	Dilated interventricular foramen Folded retina Dilated orbital sinus Variation in lens shape Partially undescended lobe Thymic remnant Absent accessory lobe Anomalous confluence/left hepatic vein Thinning with liver protrusion Fissured posterior caudate lobe Dark Dilated Undescended Malpositioned Left	0 1 1 1 5 0 0 2 2 1 0 1 0 2 2	0 0 0 0 5 0 0 0 0 0 0 0 0 1 3	0 2 1 0 1 1 1 0 0 0 1 0 1 3 1	1 0 0 0 5 0 0 0 0 0 0 0 1 1 2	0 1 1 1 4 0 0 1 2 1 0 1 0 2 2	0 0 0 0 4 0 0 0 0 0 0 0 0 1 3	0 2 1 0 1 1 1 0 0 0 1 0 1 3 1	1 0 0 0 3 0 0 0 0 0 0 0 1 1 2

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

*Litter 13, 7 fetuses heads only examined, included in tabulated data.

**Litter 26 head only examined for one fetus, included in tabulated data.

***Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated

Table 22 (cont): Fetal examinations - minor visceral abnormality and necropsy findings - group incidences

		Fetuses				Litters
Group		1	2	3	4	1	2	3	4
Number Examined		147*	156**	141***	139	20	20	20	20
Total Number Affected		31	20	24	26	16	13	14	14
Haemorrhages Head     Neck/Thorax   Abdomen   General	Brain Vitreous humour eye Eye lid(s) Dorsal fat pad Thoracic cavity Abdomen cavity Liver lobes Subcutaneous	3 1 1 1 0 12 1 1	5 0 0 0 0 6 0 0	3 0 0 1 0 5 0 2	6 0 0 1 1 6 0 1	3 1 1 1 0 8 1 1	5 0 0 0 0 4 0 0	2 0 0 1 0 3 0 2	6 0 0 1 1 5 0 1
Necropsy observations (external) Skin   Head Tail	Shiny Subcutaneous edema Short snout Curled	1 0 0 0	0 0 0 0	3 1 0 0	0 0 1 1	1 0 0 0	0 0 0 0	2 1 0 0	0 0 1 1

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

*Litter 13, 7 fetuses heads only examined, included in tabulated data.

**Litter 26 head only examined for one fetus, included in tabulated data.

***Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated

 

 

Table 23: Fetal examinations - minor visceral abnormality and necropsy findings – group percentage incidences

		Fetuses				Litters
Group		1	2	3	4	1	2	3	4
Number Examined		147*	156**	141***	139	20	20	20	20
Total Number Affected		31	20	24	26	16	13	14	14
Visceral abnormalities Brain Eyes     Thymus   Lungs Caudal vena cava Diaphragm Liver Adrenal(s) Ureter(s) Testis(es)   Umbilical artery	Dilated interventricular foramen Folded retina Dilated orbital sinus Variation in lens shape Partially undescended lobe Thymic remnant Absent accessory lobe Anomalous confluence/left hepatic vein Thinning with liver protrusion Fissured posterior caudate lobe Dark Dilated Undescended Malpositioned Left	0.0 0.7 0.7 0.7 3.4 0.0 0.0 1.4 1.4 0.7 0.0 0.7 0.0 1.4 1.4	0.0 0.0 0.0 0.0 3.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.6 1.9	0.0 1.4 0.7 0.0 0.7 0.7 0.7 0.0 0.0 0.0 0.7 0.0 0.7 2.1 0.7	0.7 0.0 0.0 0.0 3.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.7 0.7 1.4	0.0 5.0 5.0 5.0 20.0 0.0 0.0 5.0 10.0 5.0 0.0 5.0 0.0 10.0 10.0	0.0 0.0 0.0 0.0 20.0 0.0 0.0 5.0 10.0 5.0 0.0 5.0 0.0 10.0 10.0	0.0 10.0 5.0 0.0 5.0 5.0 5.0 0.0 0.0 0.0 5.0 0.0 5.0 15.0 5.0	5.0 0.0 0.0 0.0 15.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.0 5.0 10.0

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

*Litter 13, 7 fetuses heads only examined, included in tabulated data.

**Litter 26 head only examined for one fetus, included in tabulated data.

***Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated

Table 23 (cont): Fetal examinations - minor visceral abnormality and necropsy findings – group percentage incidences

		Fetuses				Litters
Group		1	2	3	4	1	2	3	4
Number Examined		147*	156**	141***	139	20	20	20	20
Total Number Affected		31	20	24	26	16	13	14	14
Haemorrhages Head     Neck/Thorax   Abdomen   General	Brain Vitreous humour eye Eye lid(s) Dorsal fat pad Thoracic cavity Abdomen cavity Liver lobes Subcutaneous	2.0 0.7 0.7 0.7 0.0 8.2 0.7 0.7	3.2 0.0 0.0 0.0 0.0 3.8 0.0 0.0	2.1 0.0 0.0 0.7 0.0 3.5 0.0 1.4	4.3 0.0 0.0 0.7 0.7 4.3 0.0 0.7	15.0 5.0 5.0 5.0 0.0 40.0 5.0 5.0	25.0 0.0 0.0 0.0 0.0 20.0 0.0 0.0	10.0 0.0 0.0 5.0 0.0 15.0 0.0 10.0	30.0 0.0 0.0 5.0 5.0 25.0 0.0 5.0
Necropsy observations (external) Skin   Head Tail	Shiny Subcutaneous edema Short snout Curled	0.3 0.0 0.0 0.0	0.0 0.0 0.0 0.0	1.1 0.4 0.0 0.0	0.0 0.0 0.4 0.4	5.0 0.0 0.0 0.0	0.0 0.0 0.0 0.0	10.0 5.0 0.0 0.0	0.0 0.0 5.0 5.0

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

*Litter 13, 7 fetuses heads only examined, included in tabulated data.

**Litter 26 head only examined for one fetus, included in tabulated data.

***Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated

Table 24: Fetal examinations - Visceral Historical Control Data - incidence

Species/strain/source	Rat/ Crl:CD(SD) / Charles River UK
Necropsy date range	Aug 2018– Apr 2019
Number of studies	8
Study types	Main

 

		GB01PV								HCD Range
		Fetuses				Litters				Fetuses	Litters
Group		1	2	3	4	1	2	3	4
Number Examined		147	156	141	139	20	20	20	20	1094	141
Visceral abnormalities
Brain	dilated interventricular foramen	0	0	0	1	0	0	0	1	0-3	0-3
Thymus	thymic remnant	0	0	1	0	0	0	1	0	0-1	0-1
Lungs	absent accessory lobe	0	0	1	0	0	0	1	0	0-0	0-0
Adrenal(s)	dark	0	0	1	0	0	0	1	0	0-0	0-0
Testis(es)	undescended	0	0	1	1	0	0	1	1	0-1	0-1
Haemorrhages
Neck/Thorax	thoracic cavity	0	0	0	1	0	0	0	1	0-0	0-0
Necropsy observations (external)
Skin	subcutaneous edema	0	0	1	0	0	0	1	0	0-0	0-0
Head	short snout	0	0	0	1	0	0	0	1	0-1	0-1
Tail	curled	0	0	0	1	0	0	0	1	0-0	0-0

 

Table 25: Fetal examinations - Visceral Historical Control Data - percentage (%)

 

Species/strain/source	Rat/ Crl:CD(SD) / Charles River UK
Necropsy date range	Aug 2018– Apr 2019
Number of studies	8
Study types	Main

 

 

		GB01PV								HCD Range
		Fetuses				Litters				Fetuses	Litters
Group		1	2	3	4	1	2	3	4
Number Examined		147	156	141	139	20	20	20	20	1094	141
Visceral abnormalities
Brain	dilated interventricular foramen	0.0	0.0	0.0	0.7	0.0	0.0	0.0	5.0	0.0-2.2	0.0-15.8
Thymus	thymic remnant	0.0	0.0	0.7	0.0	0.0	0.0	5.0	0.0	0.0-0.6	0.0-5.0
Lungs	absent accessory lobe	0.0	0.0	0.7	0.0	0.0	0.0	5.0	0.0	0.0-0.0	0.0-0.0
Adrenal(s)	dark	0.0	0.0	0.7	0.0	0.0	0.0	5.0	0.0	0.0-0.0	0.0-0.0
Testis(es)	undescended	0.0	0.0	0.7	0.7	0.0	0.0	5.0	5.0	0.0-0.7	0.0-5.3
Haemorrhages
Neck/Thorax	thoracic cavity	0.0	0.0	0.0	0.7	0.0	0.0	0.0	5.0	0.0-0.0	0.0-0.0
Necropsy observations (external)
Skin	subcutaneous edema	0.0	0.0	0.4	0.0	0.0	0.0	5.0	0.0	0.0-0.0	0.0-0.0
Head	short snout	0.0	0.0	0.0	0.4	0.0	0.0	0.0	5.0	0.0-0.3	0.0-5.3
Tail	curled	0.0	0.0	0.0	0.4	0.0	0.0	0.0	5.0	0.0-0.0	0.0-0.0

 

Applicant's summary and conclusion

Conclusions:

Oral administration of Butylal to pregnant Sprague-Dawley rats during organogenesis and the fetal growth phase at 100, 330 and 1000 mg/kg/day was well tolerated with no effects on maternal clinical condition, thyroid weight and associated hormones or macropathology. Embryo-fetal survival, fetal weight and ano-genital distance were unaffected by maternal treatment.
Maternal effects were limited to low body weight gain, adjusted maternal weight gain and food consumption at 1000 mg/kg/day. In addition the gravid uterine weight for females at 1000 mg/kg/day was low but in the absence of any effect on litter size or fetal weight this was not considered to be toxicologically significant.
Fetal effects were limited to a non-adverse increase in the fetal/litter incidence of the skeletal variant of short supernumerary 14th rib at 1000 mg/kg/day.
Based on the findings in this study the no observed adverse effect level for both maternal animals and embryo-fetal development was considered to be 1000 mg/kg/day.

Executive summary:

The purpose of this study was to assess of the influence of Butylal (an industrial chemical) on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Sprague-Dawley rat.

Three groups of 20 females received Butylal at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating.  A similarly constituted Control group received the vehicle, corn oil at the same volume dose as treated groups.  Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded.  On Day 20 after mating adult females were subject to macroscopic and microscopic pathology investigation and the gravid uterine weight and thyroid weight recorded.  Ano-genital distance were measured for all fetuses and were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Results and conclusion

Oral administration of Butylal to pregnant Sprague-Dawley rats during organogenesis and the fetal growth phase at 100, 330 and 1000 mg/kg/day was well tolerated with no effects on maternal clinical condition, thyroid weight and associated hormones or macropathology. Embryo-fetal survival, fetal weight and ano-genital distance were unaffected by maternal treatment.

Maternal effects were limited to low body weight gain, adjusted maternal weight gain and food consumption at 1000 mg/kg/day.  In addition the gravid uterine weight for females at 1000 mg/kg/day was low but in the absence of any effect on litter size or fetal weight this was not considered to be toxicologically significant.

Fetal effects were limited to a non-adverse increase in the fetal/litter incidence of the skeletal variant of short supernumerary 14th rib at 1000 mg/kg/day.

Based on the findings in this study the no observed adverse effect level for both maternal animals and embryo-fetal development was considered to be 1000 mg/kg/day.