Man-made vitreous (silicate) fibres with random orientation with alkaline oxide and alkali earth oxide (Na2O+K2O+CaO+MgO+BaO) content greater than 18 % by weight

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26th January 2010 to 6th April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully GLP compliant and audited

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)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

amporphous glass fibre formed from the melting and fiberisation of predominately slilcon dioxide, calcium oxide, magnesium oxide

EC Number:

610-130-5

Cas Number:

436083-99-7

Molecular formula:

Amorphous glass consisting of SinO(3n-1)2(n-1) Polymeric anions ionically bonded to Ca2+ and Mg2+ cations or other alkaline earth cations

IUPAC Name:

amporphous glass fibre formed from the melting and fiberisation of predominately slilcon dioxide, calcium oxide, magnesium oxide

Details on test material:

- Name of test material (as cited in study report): AES Covance
- Substance type: Inorganic
- Physical state: Solid
- Analytical purity: >99%
- Impurities (identity and concentrations): trace impurities
- Purity test date: 14/12/09
- Lot/batch No.: BG-10-X99-2981
- Expiration date of the lot/batch: 31/12/2020
- Stability under test conditions: stable
- Storage condition of test material: room temperature, sealed container

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 to 12 weeks old
- Weight at study initiation: Males: 271.2 - 310.4g; Females: 172.1 - 201.7g;
- Fasting period before study: N/A
- Housing: Cages conforming to 'Code of practice for the housing and care of animals used in scientific procedures' (Home Office, London, 1989)
- Diet: ad libitum
- 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: 26th January 2010 To: 6th April 2010

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: MC (methyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: daily

VEHICLE
- Justification for use and choice of vehicle (if other than water): to ensure homogenous suspension of the substance for dosing as it is insoluble in water
- Amount of vehicle (if gavage): 10ml/kg

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 15 days
- Proof of pregnancy: vaginal plug / sperm in vaginal washing referred to as day 0 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:

Verification of composition at start and end of sampling carried out by XRF analysis, concentrations verified by gravimetric analysis of start and end samples.

Duration of treatment / exposure:

40 days to 55 days

Frequency of treatment:

daily dosing

Doses / concentrations

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Dose selection rationale: The dose level for both test articles was chosen after consultation with the Sponsor and 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.

Positive control:

none used

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily start and end (nominal) of the working day 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.
- Cage side observations checked; ill health or overt toxicity. Any abnormalities of appearance or behaviour or other signs of reaction to treatment or ill health were recorded

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly, at time of body weight examination Clinical signs for females during the gestation and lactation periods, were recorded more frequently that weekly due to days of body weight recording.

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
The food consumed by each cage of animals was determined weekly during the pre pairing periods.
Individual food intake of mated females was recorded on Days 0 to 6, 7 to 13 and 14 to 19 of gestation, and on Days 1 to 3 of lactation.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other:]

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after assessment of females and offspring
- Maternal animals: All surviving animals 5 days post-partum

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues listed below were prepared for microscopic examination and weighed, respectively.
ovaries (with oviducts) testes
uterus epididymides
cervix seminal vesicles
vagina prostate
pituitary coagulating gland
gross lesions animal identification

Postmortem examinations (offspring):

SACRIFICE
- The offspring were sacrificed at 5 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

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

Statistics:

Statistical analysis used SAS

Reproductive indices:

Mating Index
Female fecundity index
Male fecundity index
Female fertility index
Male fertility index
Median pre-coital time

Offspring viability indices:

Post implantation survival index
Viability index 1
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

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no deaths during the study, all animals survived until scheduled necropsy. There were no clinical observations recorded that were considered to be related to treatment with AES Covance

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There was no adverse effect of treatment on mean body weight or body weight gain in males or females. In males and females, there was no effect of treatment on mean food intake.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
the majority of females mated within one oestrous cycle.
There was no effect of treatment on fertility or fecundity indices

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Qualitative testis staging did not indicate any abnormalities in the integrity of the various cell types present within the different stages of the spermatogenic cycle.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There was no effect of treatment on mating, the majority of females mated within one oestrous cycle.
There was no effect of treatment on fertility or fecundity indices.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There was no adverse effect of treatment on adult male or female organ weights.

GROSS PATHOLOGY (PARENTAL ANIMALS)
there was no effect of treatment on adult male or females

HISTOPATHOLOGY (PARENTAL ANIMALS)
Macroscopic findings
Most tissues were macroscopically unremarkable and the findings seen were generally consistent with the usual pattern of findings in rats of this strain and age. There were no macroscopic findings suggestive of effects of the test articles.
Microscopic findings
Microscopic findings were generally infrequent, of a minor nature and consistent with the usual pattern of findings in rats of this strain and age. There were no microscopic findings in treated animals due to effects of the test articles.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

> 250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

There was no effect of treatment on gestational length, the number of implantation sites, pup numbers, pup survival or body weights to Day 4 post partum and no gross external malformations of the pups.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Group mating data

Test article	Control	AES Covance	RCF Covance (not applicable)
Group	1	2	3
Level (mg/kg/day)	0	250	250

 

5.1 Mating Performance
		Group 1	Group 2	Group 3 (not applicable)
Group size at pairing:
Male		10	10	10
Female		10	10	10
Number mating on Day of pairing	1	2		2
	2	3	6	4
	3	3	1	2
	4	2	3
	5
	6
	7			2
	8
	9
	10
	11
	12
	13
	14
	15
	Total:	10	10	10
Median pre-coital time (days)		2.5	2	2

5.2 Summary of mating record
	Group 1	Group 2	Group 3 (not applicable)
Number of females paired	10	10	10
Pregnant:
Mating from vaginal plugin situ		2	1
Mating from positive smear:
Sperm/oestrus/pro-oestrus	1		1
Sperm/metoestrus/dioestrus
Sperm/stage of oestrus not recorded	8	8	7
Absence of positive smear
Pregnancy rate (%)	9 (90.0)	10 (100.0)	9 (90.0)
Non-pregnant:
Vaginal plugin situ
Positive smear	1		1
Absence of positive smear
Total showing vaginal plugin situor positive smear	10	10	10

5.3 Mating indices
	Group 1	Group 2	Group 3 (not applicable)
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

Group mean litter data

 

6.1 Pup numbers – females with live pups at Day 4post-partum
	Group 1	Group 2	Group 3 (not applicable)
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

6.2 Neonatal survival indices – females with live pups at Day 21post-partum
		Group 1	Group 2	Group 3 (not applicable)
	Post-implantation survival index %	91.9	97.4	90.3
	Live birth index %	100.0	100.0	100.0
	Viability index 1 %	97.0	99.1	100.0

6.4 Summary necropsy data – pups surviving to Day 5 necropsy
	Group 1	Group 2	Group 3 (not applicable)
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:

Administration of AES Covance by oral gavage to male rats for at least 55 days and to female rats for at least 41 days at 250 mg/kg/day elicited no signs of adverse toxicity.
The no observed adverse effect level (NOAEL) for reproductive and developmental toxicity was considered to be at least 250 mg/kg/day for both AES Covance

Executive summary:

The objective of the study was to provide a preliminary evaluation of the effects of the test articles, AES Covance, on the reproductive/developmental toxicity in the rat.

Groups of 10 male and 10 female rats were given 250 mg/kg/day of either AES Covance orally, by gavage, for two weeks prior to pairing, during the pairing period and until Day 4post-partumfor the females and until the day before necropsy in Week 8 for the males. A similar group of 10 males and 10 females were given 1% (w/v) methylcellulose over the same period to act as controls.

There were no deaths during the study, and no treatment-related clinical observations.

Mean body weight gains and food intake were unaffected by treatment.

Mating data were unaffected by treatment. There was no effect of treatment on mean uterine/implantation data or mean litter data.

Mean organ weights were unaffected by treatment. At necropsy, macroscopic findings were unremarkable.

Microscopic examination revealed no treatment-related findings and there were no findings at testis staging due to effects of the test articles.

In conclusion, administration of AES Covance by oral gavage to male rats for at least 55 days and to female rats for at least 41 days at 250 mg/kg/day elicited no signs of adverse toxicity.

The no‑observed‑adverse‑effect‑level (NOAEL) for reproductive and developmental toxicity was considered to be at least 250 mg/kg/day for both AES Covance.

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