Refractories, fibers, aluminosilicate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on OECD421

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25th January 2010 to June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with no deviations

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

oxo[(oxoalumanyl)oxy]alumane; silanedione

EC Number:

604-314-4

Cas Number:

142844-00-6

Molecular formula:

amorphous glass Si(n)O(3n+1) polymeric anions bonded to Zr and Al(3+)

IUPAC Name:

oxo[(oxoalumanyl)oxy]alumane; silanedione

Details on test material:

Name of test material( as cited in study report): RCF Covance
Substance type: Inorganic
- Physical state: solid
- Analytical purity: 100%
- Purity test date: 14.12.09
- Lot/batch No.: BG-10-X99-2982
- Expiration date of the lot/batch: 31.1.2020
- Stability under test conditions: stable
- Storage condition of test material: sealed container room temp

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River laboratories ,Margate ,UK
- Age at study initiation: 10-12weeks old
- Weight at study initiation: (P) Males: 286.2- 353.2 g; Females: 186.0-208.6 g
- Fasting period before study: No
- Housing:Cages conforming to "code of practice for the housing and care of animals used in scientific procedures" (home office,Lndon 1989)
- Diet - SQC rat and mouse breeder diet number 3 (special diet services' limited, Witham)
- Water:ad libitum
- Acclimation period:2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 10th February 2010 To:6th April 2010

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: methyl cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Daily

VEHICLE
- Justification for use and choice of vehicle (if other than water): Viscous solution to maintain suspension of test article
- Concentration in vehicle: see table 1
- Amount of vehicle (if gavage): 10ml/kg

Formulations of the test article in the vehicle were prepared daily.
The test article was formulated as a suspension in 1% (w/v) methylcellulose following dispensary SOPs and the formulation method 8215_686_O_01D as maintained in the study data

Details on mating procedure:

- M/F ratio per cage:1:1
- Length of cohabitation: 15 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged : individually
- Any other deviations from standard protocol:none

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

XRF

Duration of treatment / exposure:

12weeks

Frequency of treatment:

daily

Doses / concentrations

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Basis:
nominal conc.

No. of animals per sex per dose:

10 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The test and control articles were administered orally, by gavage, daily to the males for two weeks prior to pairing, during the pairing period and until the day before necropsy and daily to the females for two weeks prior to pairing, during the pairing period and until Day 4 post-partum, inclusive. The females were allowed to litter and rear their offspring to Day 4 post-partum. The males were treated for eight weeks prior to necropsy.

The dose levels were based on the results of previous inhalation studies and the physical properties of the test articles. Due to the potential risk of physical interference with gut motility, a high dose of between 50 and 100 mg/rat/day (250 mg/kg/day) was considered to be the limiting dose for this study

Examinations

Parental animals: Observations and examinations:

CAGE SIDE AND CLINICAL OBSERVATIONS : Yes
- Time schedule: Twice daily: beginning and end (nominal) of the working day
- Cage side observations checked
All animals were examined at least once daily for signs of ill health or overt toxicity. Any abnormalities of appearance or behaviour or other signs of reaction to treatment or ill health were recorded and a detailed individual record was maintained of the clinical condition of each animal on the days of body weight recording. Clinical signs for females during the gestation and lactation periods, were recorded more frequently that weekly due to days of body weight recording. This deviation was considered not to have affected the outcome or integrity of the study.
In addition, the animals were observed immediately on return to the home cage after dosing and at 0.5, 1, 2 and 4 hours post dose for signs of reaction to treatment for the first two weeks of dosing. As no signs were observed the animals were observed immediately on return to the home cage after dosing and at 0.5 and 1 hour post dose only from the start of the pairing period until the end of dosing.
On Days 4 and 13 of dosing, the 2 hour post-dose observations were recorded approximately 24 minutes late in error. This deviation did not affect the outcome or integrity of the study.



BODY WEIGHT: Yes
- Time schedule for examinations:males :Day -7 (randomisation body weight check)
Weekly .Day of (prior to) necropsy
females -Day -7 (randomisation body weight check)
Weekly prior to pairing and until confirmation of mating
Days 0, 7, 14 and 20 of gestation
Days 1 and 4 post partum



FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Pre-pairing: Over the same intervals as the body weights prior to pairing (males and females)
Gestation:
Day 0 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17 and 17 to 20 of gestation (females only)
Post-partum:
Day 1 to 4 post-partum (females only)




WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:


OTHER:

Oestrous cyclicity (parental animals):

During the pairing procedure one male was housed with one female. Mating was confirmed by the presence of a vaginal plug in situ or sperm in a vaginal washing. On confirmation of mating vaginal washing was discontinued and the male was removed and rehoused as detailed in the section Environment. The day on which mating was confirmed was designated Day 0 of gestation.

Litter observations:

The females were allowed to litter and the date of parturition and the duration of gestation were recorded.
The following data were recorded for each litter to Day 4 post partum:
• number of pups born (live and dead)
• daily live litter size and sex (reported on Days 1 and 4)
• daily clinical observations
• individual pup weights on Days 1 and 4 post partum
• necropsy findings of dead pups where condition permitted

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals [after assessment of females
- Maternal animals: All females with live pups were sent to necropsy on Day 5 post-partum,


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY / ORGAN WEIGHTS

Organ weights
Animals were weighed before being sent to necropsy. Testes and epidiymides and ovaries were dissected free from fat and other contiguous tissues and weighed before fixation.

Tissue retention
The following tissues from all terminal adult animals were retained, where appropriate:
ovaries (with oviducts) testes
uterus epididymides
cervix seminal vesicles
vagina prostate
pituitary coagulating gland
gross lesions animal identification

All tissues were preserved in relevant preservatives.

Histopathology
Testes and epididymides from all terminal adult animals, as appropriate, were embedded in paraffin wax and sectioned at a nominal thickness of 5 μm. Sections allocated for testicular staging were stained with periodic acid Schiff (PAS); all other sections were stained with haemotoxylin and eosin.
Tissues from Groups 1, 2 and 3 were microscopically examined by the study pathologist.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 5 days of age.
- These animals were subjected to postmortem examinations macroscopic examination .

The following data were recorded for each litter to Day 4 post partum:
• number of pups born (live and dead)
• daily live litter size and sex (reported on Days 1 and 4)
• daily clinical observations
• individual pup weights on Days 1 and 4 post partum
• necropsy findings of dead pups where condition permitted



GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

Statistics:

Statistical analysis: Data were processed, where appropriate, to give litter mean values, group mean values and standard deviations

Reproductive indices:

Mating Index, Female and Male Fecundity indices, male and female fertility indices, Median precoital time

Offspring viability indices:

Post implantation survival index, viability index, gestation index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

No adverse effects were seen

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

> 250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other:

Remarks:

no effect observed

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

not specified

Details on results (F1)

No adverse effects were seen

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

no NOAEL identified Generation: P and F1

Generation:

F1

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

Remarks on result:

other:

Remarks:

no NOAEL identified Generation: P and F1

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

A selection of results on health , reproduction and litter condition are given below.

Table 2 Summary of female performance

Test article	Control	AES Covance	RCF Covance
Group	1	2	3
Level (mg/kg/day)	0	250	250

Number of animals:	Group 1	Group 2	Group 3
Males
Number in group	10	10	10
Number died/killed after pairing	0	0	0
Number inducing pregnancy	9	10	9
Females
Number in group	10	10	10
Number died/killed	0	0	0
Number not pregnant	1	0	1
Number pregnant (%)	9 (90.0)	10 (100.0)	9 (90.0)
Number with total embryo/foetal loss	0	0	0
Gestation index (%)	90.0	100.0	90.0
Number with total litter loss	0	1	0
Number with live pups on Day 4post-partum	9	9	9

Table3 Summary of clinical observations

Clinical observations  Males
Observation	Group	Number of animals with sign in Week:								Total
	Number	1	2	3#	4#	5	6	7	8T	affected
Number of animals examined	1	10	10	10	10	10	10	10	10
	2	10	10	10	10	10	10	10	10
	3	10	10	10	10	10	10	10	10
Skin and hair:
Sores/lesions	1
	2	1	1							1
	3				1	1	1	1	1	1
Thinning fur	1
	2	1	2	2	2				1	2
	3
# pairing period                   T = terminal week

Table 4 Clinical observations females pre pairing

Clinical observations  Females
Observation	Group number	Number of animals with sign in Week:		Total affected
		1	2
Number of animals examined	1	10	10
	2	10	10
	3	10	10
Skin and hair:
Thinning fur	1	1	2		2
	2
	3	1	2		2

Table 5 - Clinical observations female gestation

Clinical observations Females
Observation	Group number		Number of animals with sign on Day of gestation:														Total affected
			0		7		14		20		21		22		23
Number of animals examined+	1	9		9		9		9		9		3		0
	2	10		10		10		10		10		2		1
	3	9		9		9		9		9		3		0
Littered	1									6		3
	2									8		1		1
	3									6		3
Skin and hair:
Hairloss	1
	2
	3			1		1		1								1
Staining	1
	2					1		1								1
	3			1		1		1								1
Thinning fur	1
	2	1		1		1		1								1
	3	1		1		1										1
+ excludes non-pregnant females

Table 6 Group Mating Data

Mating indices
	Group 1	Group 2	Group 3
Number of paired males	10	10	10
Number of paired females	10	10	10
Number of mated males	10	10	10
Number of mated females	10	10	10
Number of males inducing pregnancy	9	10	9
Number of pregnant females	9	10	9
Mating index %	100.0	100.0	90.9
Fertility index % : male	90.0	100.0	90.0
: female	90.0	100.0	90.0
Fecundity index % : male	90.0	100.0	90.0
: female	90.0	100.0	90.0

Table 7 Group mean litter data

Pup numbers – females with live pups at Day 4post-partum
	Group 1	Group 2	Group 3
Number of females with live pups at Day 4post-partum	9	9	9
Mean duration of gestation (days)	22.3	22.1	22.3
Mean number of implantation sites	11.8	12.4	12.7
Mean number of pups born	10.8	12.1	11.4
Mean number of pups alive Day 1	10.8	12.1	11.4
Mean % male pups Day 1	42.4	42.8	47.3
Mean number of pups alive Day 4	10.4	12.0	11.4

Table 8 - Group Litter Data - Necropsy

Summary necropsy data – pups surviving to Day 5 necropsy
	Group 1	Group 2	Group 3
Number of animals examined	93	108	103
Number of animals with finding:
Not remarkable (%)	78 (83.9)	100 (92.6)	90 (87.4)
Animal - cannibalised			1
Kidney - small	1
Liver - pale			1
- pale area	1		1
- large			1
- small			1
Lung – red focus	1
Testis - dark			1
Ureter - distension	1		2
Urinary bladder - distension	11	8	9
- abnormal contents		1

Applicant's summary and conclusion

Conclusions:

At what was regarded as the maximum usable dose there were no adverse reproductive effects seen with RCF administered by gavage.

Executive summary:

There are two possible activities of fibres , those mediated by their chemistry and those due to their morphology. When inhaled most fibres are cleared to the gut by the mucocilliary escalator or macrophage activity. Thus any reproductive effects would have to mediated through adsorbtion via the gut. The most likely source of any toxic activity would be through the chemistry of the material.

Unsurprisingly orally administered fibres at many times any dose that would arrive from inhalation had no effect on the reproductive efficiency or health of rats.