Synthetic wollastonite

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because (i) the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), (ii) it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and (iii) there is no or no significant human exposure

other:

Justification for type of information:

The REACH regulation requires an evaluation of the reproductive and developmental toxicological properties of substances manufactured or imported at >1000 tpa. In the case of Synthetic Wollastonite no reproductive or developmental toxicology data are available from studies performed on the substance itself. However, there are data available from repeat dose toxicity studies carried out on the analogue substances kieselguhr soda ash flux-calcined and silicon dioxide, as well as data from developmental studies carried out on Silicic acid, calcium salt that may be read-across to Synthetic Wollastonite.
In terms of the oral route a reliable 90-day oral toxicity study performed using kieselguhr soda ash flux-calcined is available [Wolfe 1992; IUCLID Section 7.5.1]. In this study rats were dosed with the substance at 1% (average of 698.3 mg/kg bw/day) and 5% (average of 3737.9 mg/kg bw/day). Histopathology revealed no effects on reproductive organs and did not produce any treatment-related effects in urine silica content. In a supporting 90-day oral study [Bertke 1963; IUCLID Section 7.5.1] silica analysis was performed on the liver, kidneys and spleen of rats fed diatomaceous earth at 5%. There was no significant increase in the percentage of residual silica in liver, kidneys and spleen of the test animals compared to control animals which received none of the test material. The data from these studies demonstrate that ingestion of silica does not affect the residual amounts which are found naturally in the body.
UNEP (2004) reports the outcomes of a number of studies on reproduction in the SIAR for synthetic amorphous silica and silicates. An early limited one-generation study on Wistar rats [Degussa 1963] gave no evidence of any adverse effects arising from long-term feeding of Aerosil (500 mg/kg bw/d) to both genders for a premating period of 4.5 months and continued up to 6 months [Degussa 1963]. Five pregnant test females and four pregnant untreated control females delivering 45 and 37 pups, respectively, were included in this test. The study had shortcomings with respect to the low number of pregnant animals used and the mating ratio of 1(m):5(f) which was too low according to current standards.
Within the scope of a comprehensive and valid testing programme, synthetic amorphous silica and silicates (non-amorphous silica and crystalline silica) were examined for embryotoxic and developmental effects during the gestation phase in various animals species, rat, mouse, rabbit and hamster, at oral doses up to 1600 mg/kg bw/d. There were no significant signs of maternal or embryotoxic/developmental toxic effects in any species tested. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the frequencies occurring spontaneously in the sham-treated controls (FDA 1972, FDA 1973a, FDA 1973b). The experimental data on intra-uterine development produced in four animal species across three types of synthetic amorphous silica and silicates suggest that there is no potential for adverse effects on embryonic/foetal development arising from exposure to these substances.
A dominant lethal assay conducted in rats did not produce significant adverse effects on reproductive performance parameters after exposure of male rats to either synthetic amorphous silica or calcium silicate after single or fivefold oral treatment at doses up to 5000 mg/kg bw/treatment [Litton Bionetics 1974].
The one-generation study conducted with a hydrophilic synthetic amorphous silica is not adequate to contribute reliable information for biostatistical reasons. However, numerous subchronic studies as well as a dominant lethal study with a calcium silicate failed to demonstrate any histopathological changes or deleterious effects in the reproductive organs of treated animals. Furthermore, given the inherent physico-chemical properties and ubiquitous nature of this class of compounds, the UNEP (2004) concluded that there is no structural alert to indicate a potential for reproductive and developmental toxicity.
These data support the findings in the 90-day study performed using kieselguhr soda ash flux-calcined and lead to the conclusion that no impairment of fertility/reproductive performance is expected from oral exposure to this substance.
For the inhalation route a 28-day toxicity study using kieselguhr soda ash flux-calcined at exposures up to 700 mg/m3 showed no effects on the reproductive organs of either sex [Schuler 2011; IUCLID Section 7.5.2]. In addition, in this study, the toxicity linked to the respirable crystalline silica was observed locally (i.e. lung) and no systemic changes were noted. It should be noted that this exposure is far in excess of the proposed DNEL of 0.05 mg/m3 for workers. No reproductive studies are available for amorphous or crystalline silica via the inhalation route [ECETOC, 2006]. However, it is considered that the available data for the oral route is also applicable for the inhalation route in terms of the effects of systemically-available silica on reproductive parameters. Further, it would be expected that the lower solubility of crystalline silica compared to amorphous silica would lead to reduced peak exposure, further mitigating the potential for effects. On this basis it is concluded that no impairment of fertility/reproductive performance is expected from inhalation exposure to this substance.
Further weight of evidence exists to demonstrate the lack of potential of crystalline and/or amorphous silica to cause toxicity to reproduction, and is described below.
Silica is essential for normal body function and is ingested orally via the consumption of foods containing silica naturally, as an additive, via dietary supplementation or drinking water. Silica can be found in whole grains (e. g. rice, oats, millet, wheat etc.), root vegetables (e. g. onions, beetroot, potatoes etc.), lettuce, horsetail, nettle, etc. The silica content of foods can vary widely but whole grasses and cereals, for example, may contain 3 – 6% of silica (Watson 2000). The FSA (2003) reports that beer contains 33 – 60 mg silica per kg of beer. Bamboo is said to be the highest of the edible plants in silica (around 1.5 – 2%) and has been eaten both as part of a daily diet and for medicinal purposes (various online sources) for thousands of years, with no evidence of toxicity.
Silicon dioxide (in its amorphous form) is an approved additive, listed as E551 (SCF 1991, JEFCA 2005) and authorized for use according to EC Directive 95/2/EC and its various amendments and adaptations. The maximum levels permitted are 10 or 30 g/kg (depending on the foodstuff), but is also allowed at up to a quantum satis concentration in other foodstuffs (EC Directive 95/2/EC). The physical differences between amorphous and crystalline silica should not affect reproductive toxicity, and therefore similar levels would also be applicable to crystalline silica, if its properties were more suitable to a food additive.
As a nutritional supplement, silica is sold in many commercially available products, with amounts typically up to 500 mg per capsule (FSA 2003). Silica supplements are said to promote the protection the function and structure of connective tissue, enhancement of the immune system, improvement of the elasticity of blood vessels, remineralisation of skeletal system, improvement of skin elasticity, stimulation of cell formation and metabolism (various online sources).
Silica is present in drinking water (De Zuane 1997, Watson 2000). Drinking water surveys in cities of the US reported a silica content of between 0 and 72 mg/L (median of 7.1 mg/L) (De Zuane 1997, p.133). There is no evidence of silica in drinking water constituting a health hazard, and no standards have been set (Watson 2000).
In relation to the ingestion of silicon dioxide from the sources mentioned above, silicon dioxide was assigned an ADI of “not limited” (JECFA 1974, p.16, p. 35), which the JEFCA (1974, p.10) described as an ADI without an explicit indication of the upper limit of intake, which is assigned to substances “…of very low toxicity, especially those that are food constituents or that may be considered as food or normal metabolites in man”. The term “not limited” has since been replaced by “not specified” which the JEFCA describes as applicable to a food substance of very low toxicity which “on the basis of the available data (chemical, biochemical, toxicological, and other), the total dietary intake of the substance arising from its use at the levels necessary to achieve the desired effect and from its acceptable background in food does not, in the opinion of the Committee, represent a hazard to health” (JEFCA 1976, p. 29). Furthermore, a study quoted in JEFCA (1970) (Klösterkotter 1956) which used “quartz water” (water in which silicic acid had been dissolved as a result of contact with quartz powder) given to rats over a prolonged period as the only source of liquid intake did not show any silica storage in tissues.
Therefore, based on the available data it is considered that both reproductive and developmental toxicity studies are scientifically unjustified and may be waived with no risk to either workers or the general population.
References:

De Zuane J (1997) Handbook of Drinking Water Quality. John Wiley and Sons
Degussa AG (1963) Ueber die chronische Toxizität von AEROSIL. Unpublished report, LPT, Degussa AG – US-IT-No. 63-0001-DKT
EC Directive 95/2/EC – European Parliament and Council Directive No. 95/2/EC of 20 February 1995 on Food Additives Other Than Colours and Sweeteners
ECETOC (2006) Synthetic Amorphous Silica (CAS No. 7631-86-9) JACC Report No. 51.
FDA (1972) Teratologic Evaluation of FDA 71-41 (Hydrated calcium silicate). Prep. For: FDA, U. S. Food and Drug Administration; NTIS, National Technical Information Service, U. S. Department of Commerce, USA, PB 221 801, 29 Dec. 1972
FDA (1973a) Teratologic Evaluation of FDA 71-48 (Syloid; silica aerogel). Prep. For: FDA, U. S. Food and Drug Administration; NTIS, National Technical Information Service, U. S. Department of Commerce, USA, PB-223 808
FDA (1973b) Teratologic Evaluation of FDA 71-45 (Sodium Silicoaluminate). Prep. For FDA, U. S. Food and Drug Administration; NTIS, National Technical Information Service, U. S. Department of Commerce, USA, PB 223-810 (01 May 1973)

FSA (2003) Safe Upper Levels for Vitamins and Minerals. Expert Group on Vitamins and Minerals, Food Standards Agency
IARC (1997) IARC Monographs On The Evaluation Of Carcinogenic Risks To Humans: Silica, Some Silicates, Coal Dust And Para-Aramid Fibrils. Vol 68. World Health Organisation, International Agency for Research on Cancer: Lyon, France
JEFCA (1974) Toxicological Evaluation of Certain Food Additives with a Review of General Principles and of Specifications, Seventeenth Report of the Joint FAO/WHO Expert Committee on Food Additives, World Health Organization Technical Report Series No. 539, FAO Nutrition Meetings Report Series 53, WHO: Geneva, Switzerland
JEFCA (1976) Evaluation of Certain Food Additives, Twentieth Report of the Joint FAO/WHO Expert Committee on Food Additives, World Health Organization Technical Report Series No. 599, FAO Nutrition Meetings Report Series No. 1, WHO: Geneva, Switzerland
JEFCA (2005) Endorsement and/or Revision of Maximum Levels for Food Additives and Processing Aids in Codex Standards. Codex Committee on Food Additives and contaminnats, 37th Session: he Hague, The Netherlands
SCF (1991) Reports of the Scientific Committee for Food, Twenty fifth series: First series of food additives of various technological functions. Scientific Committee for Food, commission of the European Communities, Luxemberg
UNEP (2004) Synthetic amorphous silica and silicates - SIDS Initial Assessment Report for SIAM 19. UNEP, Berlin, Germany
Watson RR (2000) Handbook of Nutrition in the Aged. CRC Press
WHO (2000) Concise International Chemical Assessment Document 24 – Crystalline Silica, Quartz, World Health Organization: Geneva, Switzerland

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
- Age at study initiation:
- Weight at study initiation:
- Fasting period before study:
- Housing:
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: water suspension

VEHICLE
- Justification for use and choice of vehicle (if other than water): used as dispersant
- Concentration in vehicle: depending on the dose
- Amount of vehicle (if gavage): 1 mL suspension/kg bw (Tab. 1, p. 15)
[note: not conclusive, is supposed to read 10 mL/kg b.w., comp. corresponding report on NAS]

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: no data
- Length of cohabitation: no data
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

from day 6 to day 15 of gestation

Frequency of treatment:

1x/d

Duration of test:

Gestation day 20: sacrifice of all dams by Caesarian section

Remarks:

Doses / Concentrations:
0, 16, 74, 350 and 1600 mg/(kg*d) (as aqueous suspension)
Basis:

No. of animals per sex per dose:

20 - 22 pregnant female rats

Control animals:

yes, sham-exposed

other: positve control dosed with Aspirin (250 mg/kg bw)

Details on study design:

- Dose selection rationale: no data
- Other: Sham control is said to be dosed with corn oil, see Tab. 1, p. 15
(probably erroneous, contradicting corresponding report on NAS)

Maternal examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: gestation day 0, 6, 11, 15, and 20 (Tab. 4, p. 19)


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes (not documented)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: no data


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: in particular urogenital tract

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (neonatal pup weight) [Appendix II, p. 21]
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 1/3 per litter
- Skeletal examinations: Yes: 2/3 per litter
- Head examinations: yes

Statistics:

no data

Historical control data:

no data

Details on maternal toxic effects:

Maternal toxic effects:no effects

Key result

Dose descriptor:

NOAEL

Remarks:

(highest dose tested)

Effect level:

1 600 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Key result

Dose descriptor:

NOAEL

Remarks:

(highest dose tested)

Effect level:

1 600 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

The administration of up to 1600 mg/kg (body weight) of the test material to pregnant rats for 

10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ
from the number occurring spontaneously in the sham-treated controls.
  

Conclusions:

The administration of up to 1600 mg/kg (body weight) of the test material to pregnant rats for 

10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ
from the number occurring spontaneously in the sham-treated controls.

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation:
- Weight at study initiation: no data
- Fasting period before study: no
- Housing: single
- Diet: ad libitum
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Photoperiod (hrs dark / hrs light): no data

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: water suspension

VEHICLE
- Justification for use and choice of vehicle (if other than water): used as dispersant
- Concentration in vehicle: depending on the dose
- Amount of vehicle (if gavage): 1 mL suspension/kg bw (Tab. 1, p. 2)
[note: not conclusive, is supposed to read 10 mL/kg b.w., comp. corresponding report on NAS]

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: no data
- Length of cohabitation: no data
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

from day 6 to day 15 of gestation

Frequency of treatment:

1x/d

Duration of test:

gestation day 17: sacrifice of all dams by Caesarian section

Remarks:

Doses / Concentrations:
0, 16, 74, 350 and 1600 mg/(kg*d) (as aqueous suspension)
Basis:

No. of animals per sex per dose:

20 - 23 pregnant dams

Control animals:

yes, sham-exposed

other: positive control dosed with Aspirin [150 mg/(kg bw*d)]

Details on study design:

- Other: Sham control is said to be dosed with corn oil, see Tab. 1, p. 2
(probably erroneous, contradicting corresponding report on NAS)

Maternal examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: gestation day 0, 6, 11, 15, and 17 (Tab. 4, p. 6)


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes (not documented)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 17
- Organs examined: in particular urogenital tract

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (neonatal pup weight) [Appendix II, p. 8]
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 1/3 per litter
- Skeletal examinations: Yes: 2/3 per litter
- Head examinations: yes

Statistics:

no data

Historical control data:

no data

Details on maternal toxic effects:

Maternal toxic effects:no effects

Key result

Dose descriptor:

NOAEL

Remarks:

(highest dose tested)

Effect level:

1 600 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Remarks:

(highest dose tested)

Effect level:

1 600 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

The administration of up to 1600 mg/kg (body weight) of the test material to pregnant mice for 

10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ
from the number occurring spontaneously in the sham-treated controls.
   

Conclusions:

The administration of up to 1600 mg/kg (body weight) of the test material to pregnant mice for 

10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ
from the number occurring spontaneously in the sham-treated controls.

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

no

Limit test:

no

Species:

hamster, Syrian

Strain:

other: (outbred)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: no data
- Fasting period before study: no
- Housing: single
- Diet: ad libitum
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Photoperiod (hrs dark / hrs light): no data

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: water suspension

VEHICLE
- Justification for use and choice of vehicle (if other than water): used as dispersant
- Concentration in vehicle: depending on the dose
- Amount of vehicle (if gavage): 1 mL suspension/kg bw (Tab. 1, p. 28)
[note: not conclusive, is supposed to read 10 mL/kg b.w., comp. corresponding report on NAS]

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: no data
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

from day 6 to day 10 of gestation

Frequency of treatment:

1x/d

Duration of test:

day 14: sacrifice of dams by Caesarian section

Remarks:

Doses / Concentrations:
0, 16.0, 74, 345 and 1600 mg/(kg bw*d)
Basis:

No. of animals per sex per dose:

19 - 22 pregnant hamsters

Control animals:

yes, sham-exposed

other: pos. control receiving Aspirin, 250 mg/(kg bw *d)

Details on study design:

- Other: Sham control is said to be dosed with corn oil, see Tab. 1, p. 28
(probably erroneous, contradicting corresponding report on NAS)

Maternal examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: gestation day 0, 6, 8, 10, and 14


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: no data


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 14
- Organs examined: in particular urogenital tract

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: yes (neonatal pup weight) [Appendix II, p. 34]
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 1/3 per litter
- Skeletal examinations: Yes: 2/3 per litter
- Head examinations: No data

Statistics:

no data

Historical control data:

no data

Details on maternal toxic effects:

Maternal toxic effects:no effects

Key result

Dose descriptor:

NOAEL

Remarks:

(highest dose tested)

Effect level:

1 600 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Key result

Dose descriptor:

NOAEL

Remarks:

(highest dose tested)

Effect level:

1 600 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

The administration of up to 1600 mg/kg (body weight) of the test material to pregnant hamsters for 

5 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from
the number occuring spontaneously in the sham-treated controls.

Conclusions:

The administration of up to 1600 mg/kg (body weight) of the test material to pregnant hamsters for 

5 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from
the number occuring spontaneously in the sham-treated controls.

Cross-reference 4

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Exposure: 20 Jul. 1984 - 19 Oct. 1984 / end observation: 15 Oct. 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Published data not containing full information available in the original study report

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

Special modifications as compared with standard study:  Focus upon lung, respiratory  tract, and regional lymph nodes. Post-exposure recovery period up to one year.

Principles of method if other than guideline:

Comparative study including Aerosil 200, Aerosil R 974 (pyrogenic, hydrophobic), Sipernat 22S (precipitated, hydrophilic) as well as quartz (crystalline silica).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Central Institute for Breeding of Laboratory Animals TNO
- Age at study initiation: 6 weeks
- Weight at study initiation: males 107 +/- 1 g - 109 +/- 2 g; females 105 +/- 1 g - 109 +/- 1 g
- Fasting period before study: no
- Housing: single in stainless steel wire cages during exposure. 5 males/5 females/ stainless steel cage after the exposure period
- Diet: no access during exposure
- Water:unflouridated tap water ad libitum (no access during exposure)
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-1
- Humidity (%):65 - 75
- Air changes (per hr): 12x/h (airflow approximately 40 m3/h)
- Photoperiod (hrs dark / hrs light): no data

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Remarks on MMAD:

MMAD / GSD: no monitoring data due to technical difficulties (see above "Details on inhalation exposure")

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Hazleton H 1000 inhalation chambers
- Exposure chamber volume: 2.3 m3
- Method of holding animals in test chamber: single
- Source and rate of air: Aerosol entrance at top of the chamber
- Method of conditioning air: no data
- System of generating particulates/aerosols: Institute´s dust generator composed of a dust feed mechanism and an atomizer operated by compressed air
- Temperature, humidity, pressure in air chamber: av. 21 - 23 °C/65 - 75 % rel. humidity
- Air flow rate: approx. 40 m3/h
- Air change rate: 40 / 2.3 = ~17/h
- Method of particle size determination: Primary particle size calculated using arithmetic mean of transmission electron micrograph magnifications. These particles form agglomerates and aggregates for which it was not possible to determine the aerodynamic size distribution in the test atmosphere due to the weakness of the bonds and the electrostatic charge fo the particles.
- Treatment of exhaust air: filtered before release


TEST ATMOSPHERE
- Brief description of analytical method used: gravimetry - Air samples are drawn through glass fiber filters (Sartorius SM 13430) and weighed (3 - 4 times per day)
- Samples taken from breathing zone: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the test material in the test atmospheres were determined by gravimetry. Samples of the test atmospheres were drawn through glass fibre filters (Sartorius SM 13430). The filters were weighed just before and after sampling. No further details are available in the literature paper.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
1.3, 5.9 or 31 mg/m3 (mean analytical values)
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
1, 6 and 30 mg/m3 (target concentrations)
Basis:
nominal conc.

No. of animals per sex per dose:

70 animals/sex/dose
Aerosil 200: assigned dose groups B, C, and D, each sub-divided in 7 sub-groups a, b, c , d, e, f, and g
10 each (sacrificed after 13 wks),
50 each kept for a recovery period of at most 52 wks (13, 26, 39, and 52 wks). 

Control animals:

yes

Details on study design:

- Dose selection rationale: based on range findings (14 d)
- Rationale for selecting satellite groups: post-exposure recovery period for examination of reversibility of effects
- Post-exposure recovery period in satellite groups: 13, 26, 39, and 52 wks

Positive control:

Quartz (crystalline silica, 58 mg/m3) included (assigned Group G)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes
- Time schedule: 2x/day, 1x/d (weekends)
- Cage side observations (mortalities) were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: see body weight


BODY WEIGHT: Yes
- Time schedule for examinations: start, weekly during exposure, 1x/wk during recovery


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION: No data
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13, 26, 39, 52, 65 (i.e. including recovery period)
- Anaesthetic used for blood collection: No (data)
- Animals fasted: No data
- How many animals: 10 males, 10 females



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 14, 27, 40, 53, and 66
- Animals fasted: Yes overnight
- How many animals: 10 males, 10 females



URINALYSIS: Yes
- Time schedule for collection of urine: week 13, 26, 40/41, 52, and 65
- Animals fasted: Yes



NEUROBEHAVIOURAL EXAMINATION: No


OTHER: ---

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Relative organ weights
HISTOPATHOLOGY: Yes, in particular lung and lymph nodes
in addition:
Si contents of lung and lymph nodes
Collagen content in lung

Other examinations:

Relative organ weights

Statistics:

Body weights: analysis of co-variance followed by Dunnett´s test
Histopathological changes and mortality: Fisher´s exact probability test
Organ weights, blood parameter: analysis of variance and Dunnett´s test

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
Respiration rate: concentration-related increase
No mortality

BODY WEIGHT AND WEIGHT GAIN
No effect in females at all dose levels
Depressive effect in males:
1 mg/m3: slightly at day 14 of exposure only (~ -5%)
6 mg/m3: slightly from day 49 to day 77 of exposure (~ - 6 to <5 %)
no more significant by end of exposure (day 91)
30 mg/m3: significantly throughout exposure: ~ -7 - -10 %, day 91: -7 %
Recovery: no difference from control at day 455 (52 weeks post-exposure)


HAEMATOLOGY
1 mg/m3: no effects
6 mg/m3: White blood cell count elevated in both males and females due to increases in the numbers of neutrophilic leukocytes,
but concentration-response relationship was poor.
After 3 months recovery, these blood parameters normalized in males and females.
30 mg/m3: Red blood cell count and hemoglobin were statistically higher in males, but not in females.
White blood cell count elevated in both males and females due to increases in the numbers of neutrophilic leukocytes,
at 3 months of recovery (days 176/177), but concentration-response relationship was poor.
In females, a slight increase above the control group apparently still existed after 6 months of recovery (day 275).


CLINICAL CHEMISTRY
no significant effects

URINALYSIS
no significant effects

ORGAN WEIGHTS
No changes in heart, thyroid, thymus, adrenals, testes, brain, spleen, kidney
Treatment-related degrees of severity: swollen lungs and enlarged mediastinal lypmph nodes at the end of exposure
LUNG
1 mg/m3: no significant increase
6 mg/m3: mean increase in relative weight 1.7x (males), 1.4x (females)
30 mg/m3: mean increase in relative weight 2.3x (males), 2.0x (females)
LYMPH NODE: no weight data


PATHOLOGY
Swollen and spotted lungs and enlarged mediastinal lymph nodes, the degree of severity being treatment-related.
At 6 and 30 mg/m3, collagen content in the lungs was clearly increased, most pronounced in males.

The above-mentioned effects gradually subsided after the exposure period,
but in males exposed to 6 and 30 mg/m3 the collagen content was still above control values at the end of the study.


HISTOPATHOLOGY: NON-NEOPLASTIC
Accumulation of alveolar macrophages and granular material, cellular debris, polymorphonuclear leucocytes, increased septal cellularity.
Alveolar bronchialisation, focal interstitial fibrosis, cholesterol clefts and granuloma-like lesions in the lung.
The granuloma-like lesions were seen in a few animals at the end of exposure period and after 13 weeks of recovery. They did not show fibroblastic activity and hyalinization and regressed during recovery [not progressive, i.e. no silicogenic nodules formed (no silicosis)].

Accumulation of macrophages were seen in the mediastinal lymph nodes (disappeared after wk 39 post-exposure).
Treatment-related microscopic changes in the nasal region were occasionally found at the end of exposure period,
such as focal necrosis and slight atrophy of the olfactory epithelium.

Interstitial fibrosis was not noted directly after the exposure period, but appeared with a delay,
for the first time observed after 13 wks post-exposure: increasing incidence especially in 30-mg rats, and a few in the 6-mg group,
but decreased in severity and frequency until the end of the study (Report p. 51).

All types of pulmonary lesions were more marked in males than in females.


The level of 1.3 mg/m3 induced only slight changes after 13-wk exposure, which generally recovered quickly (no differences from control after 13-wk post-exposure).

Morphological changes after 13-wk exposure, that were considered statistically significant at 1.3 mg/m3:

males females ! males females
treated ! untreated controls
------------------------------------------------------------------------------------------------------------------------------
Accumulation of alveolar macrophages: slight in 10/10 (very) slight in 10/10 ! (very) slight 4/10 slight in 1/10
Intra-alveolar polymorphonuclear !
leukocytes: (very) slight in 6/10 (very) slight in 8/10 ! 0/10 0/10
Increased septal cellularity: ( very) slight in 10/10 (very) slight in 9/10 ! very slight 1/10 very slight 1/10
Olfactory epithelial atrophy: (very) slight in 5/10 (very) slight in 8/10 ! 0/10 0/10
Intracytoplasmic proteinaceous droplets !
-respiratory epithelium: in 8/10 in 9/10 ! 1/10 0/10
Mediastinal lymph node !
-macrophage accumulation: (very) slight in 8/10 (very) slight in 8/10 ! 0/10 0/10
------------------------------------------------------------------------------------------------------------------------------



HISTOPATHOLOGY: NEOPLASTIC
No particular findings


HISTORICAL CONTROL DATA (if applicable): no data


OTHER FINDINGS - SILICA DEPOSITION
Silica could be detected in lungs only in relatively small amounts at the end of the exposure period:
on the average 0.1 - 0.2 mg per lung of male animal groups (not dose-related), 0.05 - 0.21 mg per lung of female groups (dose-related).
Only one male exposed to 30 mg/m3 showed a small amount of silica in the regional lymph node.
90 days after termination of exposure (day 188), no silica could be recovered from any animal.

Key result

Dose descriptor:

NOAEC

Effect level:

1.3 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: Histopathology: based on the slight and fully reversible pulmonary response noted at this exposure level (inflammation reaction) (see details below "Details on results")

Key result

Dose descriptor:

LOAEC

Effect level:

5.9 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: (haematology); organ weights (hypertrophy lung); histopathology (collagen increase, sporadic focal fibrosis)

Key result

Dose descriptor:

NOEC

Effect level:

< 1.3 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: Histopathology: based on the pulmonary response (inflammation reaction) (see details below "Details on results")

Critical effects observed:

not specified

Please refer to attached document 'Tables and figures' for more information on the results from this study

Findings for quartz-exposed rats:

Clinical signs:None

Body weight:The body weight of quartz-exposed rats was not affected during the exposure period. The rats showed a slightly progressive reduction in weight gain throughout the post exposure period.

Haematology:There was a statistically significant increase in neutrophilic leucocyte counts during the exposure period. The counts increased during the first 13 weeks after the end of the exposures and remained high during the whole post exposure period. Red blood cell counts, haemoglobin content and packed cell volumes had slightly increased in males exposed to quartz by the end of the exposure period. The males continued to show high red blood cell values throughout the observation period. The remaining haematological parameters that were examined did not show differences that could be related to treatment.

Clinical chemistry:From 13 weeks after exposure alanine aminotransferase activities increased in quartz-exposed rats. In males the increase was approximately 50-90% (p<0.01) in comparison with controls. Alkaline phosphatase activity had increased in rats exposed to quartz 52 weeks after the exposure period. The remaining biochemical parameters that were examined did not show differences that could be related to treatment.

Organ weights:At the end of the exposure period both absolute and relative lung weight were statistically significantly increased compared to controls. The increase was greater in males than in females. Lung weight increased progressively during the post-exposure period to levels three or more times higher than those of control animals. The only other organ that showed increased weight was the thymus. Both the absolute and relative thymus weights were significantly increased (by about 140% of control weight in males at day 188). This increase was most pronounced in males and had disappeared 39 weeks after the end of exposure.

Lung collagen contents:At the end of the exposure period, the lung collagen content of quartz exposed rats was slightly higher than in controls, but in the course of the post-exposure period it had increased markedly.

Silicon in the lungs and associated lymph nodes:Silicon levels in the lungs of males were higher 26 weeks after the end of exposure than at 13 weeks after the end of exposure. Males rats exposed to quartz exhibited even higher values at week 39 than week 26 after exposure. This finding was not observed in females, but this could not be explained by the authors.

Pathology:Most of the rats exposed to quartz and killed at the end of the exposure period had swollen and spotted lungs with a spongy consistency and/or irregular surface and enlarged lung-associated lymph nodes. These changes were more pronounced than in the amorphous silica-exposed rats. The gross lesions in the lungs and lung-associated lymph nodes remained present during the whole post-exposure period. Microscopic changes were mainly observed in the lungs. Changes in rats killed at the end of the exposure period comprised slight to severe accumulation of alveolar macrophages, intra-alveolar granular material, cellular debris and polymorphonuclear leucocytes in the alveolar spaces and increased septal cellularity, seen as an increase in the number of type II pneumocytes and macrophages within the alveolar walls. During the post-exposure period no recovery from lung lesions was observed in quartz-exposed rats. Accumulation of intra-alveolar granular material, cellular debris and polymorphonuclear leucocytes were found in all quartz-exposed rats during the post-exposure period. Alveolar broncholization persisted mainly in quartz-exposed animals. Focal interstitial fibrosis, seen as amorphous eosinophilic, collagen-containing thickenings of the septa, was first observed 13 weeks after exposure and became more severe during the post exposure period. Alveolar cholesterol clefts were observed for the first time after 26 weeks of non-exposure. During the remaining recovery period this lesion became more pronounced. Granulomas, seen as aggregates of macrophage-like cells were observed in nearly all rats exposed to quartz at the end of the exposure period. Slight fibrosis was demonstrated in the granulomas in animals of the quartz group. One year after the end of the exposure period, one male rat had a focus of squamous metaplasia in the periphery of the lung. In addition, in one female a small but unequivocal squamous cell carcinoma was found in the lung parenchyma.

Cross-reference 5

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Exposure: 20 Jul. 1984 - 19 Oct. 1984 / end observation: 1

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Remarks:

Comparable to guideline study with acceptable restrictions in synopsis of the whole testing programme (see Reusal et al (1991) Aero200)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

Special modifications as compared with standard study:  Focus upon lung, respiratory  tract, and lymph nodes. Post-exposure recovery period up to one year. One high exposure level only within a combined study (in contrast to Aerosil 200, see other entry).

Principles of method if other than guideline:

Comparative study including Aerosil 200, Aerosil R 974 (pyrogenic, hydrophobic), Sipernat 22S (precipitated, hydrophilic) as well as quartz (crystalline).

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Central Institute for Breeding of Laboratory Animals TNO, Zeist/NL
- Age at study initiation: 4 weeks
- Weight at study initiation: 50 - 70 g
- Fasting period before study: no
- Housing: single during exposure
- Diet: no access during exposure
- Water: no access during exposure
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +-1
- Humidity (%): 50 - 70
- Air changes (per hr): 12x/h
- Photoperiod (hrs dark / hrs light): no data

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Remarks on MMAD:

MMAD / GSD: no monitoring data due to technical difficulties (see above "Details on inhalation exposure")

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel exposure chamber, multitiered (manufactered by Hazelton)
- Exposure chamber volume: 2.3 m3
- Method of holding animals in test chamber: single
- Source and rate of air: Aerosol entrance at top of the chamber
- Method of conditioning air: no data
- System of generating particulates/aerosols: Institute´s dust generator with compressed air operating atomizer
- Temperature, humidity, pressure in air chamber: av. 21 - 23 °C, minimum 19.1, max. 25.4 °C /
65 - 75 % rel. humidity, during extreme weather occasionally up to 95.5 % or down to 48 %.
- Air flow rate: approx. 40 m3/h
- Air change rate: 40 / 2.3 = ~17/h
- Method of particle size determination: due to electrostatic charge of the particles not measured:
technical failure of the 10-stage Mercer cascade impactor and the QCM cascade
- Treatment of exhaust air: filtered before release


TEST ATMOSPHERE
- Brief description of analytical method used: gravimetrically - Air samples are drawn through glass fiber filters (Sartorius)
and weighed (3 - 4 time per day)
- Samples taken from breathing zone: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

see Report Tables (Part 2), Table 2: Daily mean concentrations are documented:
based on 254 measurements: 34.91 (SEM 0.49) mg/m3

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
35 mg/m3 (mean analytical values)
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
30 mg/m3 (target concentration)
Basis:
nominal conc.

No. of animals per sex per dose:

70
Sipernat 22S: assigned dose groups F, sub-divided in 7 sub-groups a, b, c , d, e, f, and g
10 each (sacrificed after 13 wks),
50 each kept for a recovery period of at most 52 wks (13, 26, 39, and 52 wks). 

Control animals:

yes

Details on study design:

- Dose selection rationale: based on range findings (14 d)
- Rationale for selecting satellite groups: post-exposure recovery period for examination of reversibility of effects
- Post-exposure recovery period in satellite groups: 13, 26, 39, and 52 wks

Positive control:

Quartz (crystalline silica) included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes
- Time schedule: 2x/day, 1x/d (weekends)
- Cage side observations (mortalities) were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: see body weight


BODY WEIGHT: Yes
- Time schedule for examinations: start, weekly during exposure, 1x/wk during recovery

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION: No data
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13, 26, 39, 52, 65 (i.e. including recovery period)
- Anaesthetic used for blood collection: No (data)
- Animals fasted: No data
- How many animals: 10 males, 10 females


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 14, 27, 40, 53, and 66
- Animals fasted: Yes overnight
- How many animals: 10 males, 10 females


URINALYSIS: Yes
- Time schedule for collection of urine: week 13, 26, 40/41, 52, and 65
- Animals fasted: Yes


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Relative organ weights
HISTOPATHOLOGY: Yes, in particular lung and lymph nodes
in addition:
Si contents of lung and lymph nodes
Collagen content in lung

Other examinations:

Relative organ weights

Statistics:

Body weights: analysis of co-variance followed by Dunnett´s test
Histopathological changes and mortality: Fisher´s exact probability test
Organ weights, blood parameter: analysis of variance and Dunnett´s test

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
No particular observations
No mortality

BODY WEIGHT AND WEIGHT GAIN
Slightly decreased body weight, ~ -5 % by 13 wks exposure
Recovery: no significant difference from control at day 455, still ~ -4 % (52 weeks post-exposure)


HAEMATOLOGY
No significant effects, but white blood cell count elevated in both males and females at the end of exposure period ,
but not clearly attributable to increases in the numbers of neutrophilic leukocytes.
After 13 weeks of recovery (day 176/177), neutrophil count still tended to be higher than the control in males and females,
and normalized by 26 weeks of recovery (day 274/275).

CLINICAL CHEMISTRY
no significant effects

URINALYSIS
no significant effects

ORGAN WEIGHTS
No changes in heart, thyroid, adrenals, testes, brain, spleen, kidney
Increased organ weights of lung and thymus at the end of exposure.
Swollen lungs and enlarged mediastinal lypmph nodes
LUNG
Slight mean increase in relative weight: 1.3x (males, females) as compared to control
LYMPH NODE: no weight data


PATHOLOGY
Swollen and spotted lungs and enlarged mediastinal lymph nodes.
The effects gradually subsided after the exposure period:
Lung weight normalised after 13 weeks post-exposure in males and females.


HISTOPATHOLOGY: NON-NEOPLASTIC
In the lung: Accumulation of alveolar macrophages, intra-alveolar polymorphonuclear leukocytes,
and increased septal cellularityin malesand females.
Treatment-related microscopic changes in the nasal region were occasionally found at the end of exposure period,
such as very slight focal necrosis and slight atrophy of the olfactory epithelium, intracytoplasmic proteinaceous droplets.

Accumulation of macrophages were seen in the mediastinal lymph nodes (disappeared after wk 39 post-exposure).

Collagen content in the lungs was slightly increased at the end of exposure.
During the recovery period all changes disappeared mostly within 13 to 26 week.


HISTOPATHOLOGY: NEOPLASTIC
No particular findings


HISTORICAL CONTROL DATA (if applicable) no data


OTHER FINDINGS - SILICA DEPOSITION
Silica could be detected in lungs only in relatively small amounts at the end of the exposure period (Tables 59):
on the average 0.5 mg per lung of male animal groups, 0.35 mg per lung of female groups, decreasing over time and
no longer measurable after 39 weeks post exposure (day 370).

Dose descriptor:

NOAEC

Basis for effect level:

other: The study was performed using a single dose level and adverse effects were noted at this level

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Critical effects observed:

not specified

Please refer to attached document 'Tables and figures' in Read Across SAS 01_Degussa 87-0004-DGT_ Aerosil 200_inhal, 13 wk, rat for more information on the results from this study.

Cross-reference 6

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Septmber 24 1991 - December 30 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: 6 weeks
- Weight at study initiation: 163 - 220 g for males and 136 - 171 g for females
- Housing: Stainless steel, hanging wire-mesh cages
- Diet: Pelleted Purina Certified Rodent Chow #5002 available ad libitum during acclimation period
- Water: Tapwater via an automatic watering system was available ad libitum during both acclimation and study periods
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 6°F
- Humidity: 50 ± 20%
- Photoperiod: 12hrs dark / 12 hrs light:

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The test materials were formulated into pellets with a single lot of Purina Certified Rodent Chow #5002. Purina Certified Rodent Chow #5002 was also formulated into pellets to serve as the control material

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The total silica and crystalline silica (quartz and cristobalite) were determined in the feed mixtures priorto the study and in the pellets at weeks 1, 7 and 13. The percent total silica in the 5% feed mixtures and pellets of Natural Diatomaceous Earth was 4.8% with 0.44% quartz and no cristobalite. The percent total silica in the 1% feed mixtures and pellets of Flux Calcined Diatomaceous Earth was 1.2% with 0.24% quartz and 0.26& cristobalite. The percent total silica in the 5% feed mixtures and pellets of Flux Calcined Diatomaceous Earth was 5.1% with 0.43% quartz and 1.70% cristobalite.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
Natural Diatomaceous Earth 5% (average of 3765.5 mg/kg/day)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
Flux Calcined Diatomaceous Earth 1% ( average of 698.3 mg/kg/day)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
Flux Calcined Diatomaceous Earth 5% ( average of 3737.9 mg/kg/day)
Basis:
actual ingested

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Time schedule: Twice daily for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: Once daily


BODY WEIGHT: Yes
Time schedule for examinations: Measured and recorded at randomization, prior to treatment and weekly, thereafter

WATER CONSUMPTION: No


FOOD CONSUMPTION AND COMPOUND INTAKE (: Yes
Time schedule for examinations: Measured and recorded weekly for weeks 1-13

HAEMATOLOGY: Yes
Time schedule for collection of blood: During week 13 of the study
Animals fasted: Yes
How many animals: first ten animals/sex/group
Parameters checked: cell morphology, corrected leukocyte count (COR WBC), erythrocyte count (RBC), haematocrit (HCT), haemoglobin(HGB), leukocyte count (WBC), leukocyte differential, platelet count

CLINICAL CHEMISTRY: Yes
Time schedule for collection of blood: During week 13 of the study
Animals fasted: Yes
How many animals: first ten animals/sex/group
Parameters checked: alanine aminotransferase (ALT), albumin, aspartate aminotransferase (AST), blood urea nitrogen (BUN), calcium, chloride, creatine , gamma glutamyltransferase (GGT), glucose, inorganic phosphorous (IN PHOS), potassium, sodium, total bilirubin (T BILI), total protein (T PROT), creatinine, total protein (T PROT),

URINALYSIS: Yes
Time schedule for collection of blood: During week 13 of the study
Animals fasted: Yes
How many animals: last five animals/sex/group
Parameters checked: Urine samples were analyzed for total silica content

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: An indirect opthalmoscopic examination was performed on each animal prior to treatment and on all group 1 and 4 animals during week 13

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY:
Performed on all animals. The necropsy included: adrenals, aorta, brain, cervical spinal cord, colon, cecum, rectum, duodenum, jejunum,ileum, esophagus, eyes, heart, kidneys, lesions, liver, lumbar spinal cord, lung, mammary gland, mesenteric lymph node, mid-thoracic spinal cord, ovaries, pancreas, pituitary, prostat, sciatic nerve, spleen, salivary glands, sternum with bone marrow, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus with vagina and cervix.

HISTOPATHOLOGY:
Histopathology performed on all preserved tissue from groups 1, 2 and 4

ORAN WEIGHTS:
Adrenals, kidneys, liver, testes with epididymides

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived to study termination. Alopecia and sores were the clinical signs most frequenly observed but occurred sporadically at a low frequency and were not considered related to treatment.


BODY WEIGHT AND WEIGHT GAIN
During the study there were no statistically significant differences noted in the mean body weight or mean body weight change data between the treated and control groups of either sex

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
mean food consumption for the group 3 males was significantly less than the respective control value for weeks 1-4, 1-8, and 1-13. No other statistically significant differences were noted in food consumption data for either sex at the intervals analyzed. Mean overall compound consumption (mg/kg/day) for weeks 1-13 was 3533.3, 655.3 and 3543.4 for the group 2, 3 and 4 males, respectively, and 3997.8, 740.7, and 3932.3 for the group 2, 3 and 4 females, respectively.

FOOD EFFICIENCY
In the males, mean overall food efficiency (%) for weeks 1-13 was 14.3, 14.6, 15.5 and 14.8 for groups 1 through 4, respectively, and in females was 9.1, 8.8, 9.3 and 9.0 forgroups 1 through 4 respectively.


OPHTHALMOSCOPIC EXAMINATION
During the 13 week ophthalmic examinations, a group 4 female exhibited chromodacryorrhea of the right eye. This finding was not considered treatment related. No other ophthalmic lesions were observed at the week 13 examinations.

HAEMATOLOGY
There were no significant findings in the hematology data

CLINICAL CHEMISTRY
There were no significant findings in the serum chemistry data

URINALYSIS
Refer to table 2 for data

ORGAN WEIGHTS
Statistical analysis of mean organ weight data revealed no significant differences between treated and control groups.


GROSS PATHOLOGY
There were few gross pathology findings at terminal scarifice and the findings were of the type routinely observed in rats. None were considered treatment related.

HISTOPATHOLOGY: NON-NEOPLASTIC
No compound related histomorphological findings were observed after 13 weeks of treatment. Similar frequency and severity of commonly seen spontaneous disease lesions and incidental findings were noted in control and experimental rats of both sexes.

Dose descriptor:

NOAEL

Remarks:

Flux Calcined DE

Effect level:

3 737.9 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: No treatment related effects

Critical effects observed:

not specified

Table 2: Mean total silica content in urine ( µg/g)

Group	Week 1		Week 7		Week 13
	Male	Female	Male	Female	Male	Female
1	31	28	29	34	28	58
2	36	40	48	52	42	91
3	34	24	23	30	37	43
4	35	45	30	46	32	49

Results were obtained from 5 animals/group/sex

Within one standard deviation the silica content in the urine of both males and females fed the control diet, the 5% natural DE pellets and the 1% flux calcined pellets or the 5% flux calcined pellets did not changed between 1 and 13 weeks during the study. Within one standard deviation, the silica content in the urine of the males and females ded the 5% natural DE pellets, the 1% flux calcined pellets or the 5% flux calcined pellets did not differ from the silica content in males and females fed the control diet.

Conclusions:

In conclusion, administration of Natural DE at a dose level of 5% (average of 3765.6 mg/kg/day) or Flux Calcined DE at dose levels of 1% (average of 698.3 mg/kg/day) and 5% (average of 3737.9 mg/kg/day) did not produce any treatment related effects.

Cross-reference 7

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Study not performed to GLP or guideline . The study does not fulfill the requirements of a 90 day guideline study as only histopathologic changes and weight gains were examined. The study is provided as supporting information.

Principles of method if other than guideline:

The purpose of the toxicity test was to determine the possible damage and to ascertain the maximum concentration of diatomaceous earth (DE) that would not produce tissue damage.

GLP compliance:

no

Remarks:

Predates GLP

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: Average weight 50 g
- Diet: ad libitum
- Water: ad libitum

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

To ensure a constant concentration the DE was mixed with water to obtain a thin paste and added, a small quantity at a time, to pre-weighed feed in a tumbling apparatus. It was possible to obtain uniform distribution of the test substances on the pellets. The pellets were dried prior to use.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
5% DE
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
3% DE
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
1% DE
Basis:
nominal in diet

No. of animals per sex per dose:

15

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

Weekly weight changes were recorded for each rat. Organ weight: body weight ratios were calculated for each animal. Parts of these same organs were analyzed for residual silica.

Sacrifice and pathology:

Histopathology sections were made on the following organs: stomach, small and large intestines, liver, kidneys, spleen, lung, urinary bladder, adrenal glands, mesenteric lymph nodes, and testes or ovaries. Organ weights were recorded for the livers, kidneys and spleens (table 2)

Other examinations:

Organ analysis for silica in the liver, kidney and spleen of the 5% level animals is given in table 1.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

effects observed, treatment-related

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

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

It was noted that after the first week, the animals fed 5% DE had a higher weight gain than the control animals. The maximum weight gain differential was reached during the sixth week. The following weeks, although the test animals continued to gain more weight than the controls, the weight gain differential became progressively smaller. At the end of the test the test animals were still gaining more than than the controls. Projection beyond the 90 day period indicated that the weight gains would possibly be equal sometime between the fourteenth and fifteenth week.No accurate averages were made on the lower two levels; however it suffices to estimate that those animals on the 3% diet had a weight gain similar to that of animals at the higher level, while those animals on the 1% level had weight gain similar to that of controls.

Complete histolgic studies were made on the 5% group; in addition six animals from both the 3% and the 1% groups were examined for comparative purposes. No differences were observed in sections from the various organs of the 5% group and comparable sections of animals fed no DE. In both the 5% and control groups there were observed an equal number of animals which had a form of lobar pneumonia. In addition, inner zonal fatty degeneration was noted in the livers of five control animals and six animals on the 5% diet. Of the animals fed the lower two concentrations, the sections of the various organs showed no histologic changes when compared with similar areas from the controls.

Critical effects observed:

not specified

Table 1: The analysis of residual silica from test animals as compared to controls:

Animal	% Silica
Liver
Control males	0.002
5% males	0.002
Control females	0.002
5% females	0.002
Kidney
Control males	0.002
5% males	0.002
Control females	0.002
5% females	0.001
Spleen
Control males	0.002
5% males	0.003
Control females	0.003
5% females	0.003

Table 2: Comparison of average organ weights in grams of control animals to the 5% test group (15 animals per group)

Organ	Control animals		5% test animals
	Male	Female	Male	Female
Liver	15.46	9.68	14.60	11.05
Kidney	1.58	1.13	1.43	1.20
Spleen	1.46	1.10	1.42	1.01

Table 3: Ratio of organ weight to body weight of the 5% test animals as compared to the control analysis:

Animal group	Organ weight: body weight
Control males
Liver	0.0480
Kidney	0.00544
Spleen	0.00520
5% males
Liver	0.0481
Kidney	0.00532
Spleen	0.00520
Control females
Liver	0.0521
Kidney	0.00531
Spleen	0.00542
5% females
Liver	0.00320
Kidney	0.00538
Spleen	0.00420

Conclusions:

The effects of 5% DE in the diet fed to white rats over a 90 day period indicated no mechanical or chemical changes to tissues. There was no significant increase in the % residual silica in liver, kidneys and spleens of the test animals. Comparison of weight changes in the 5% group with the control showed a greater weight gain in the 5% test group. Histologic sections of animals fed 3% and 1% DE also showed no damage.