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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15-03-2010 to 06-08-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, according to the OECD 422 technical guideline
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Flue dust, portland cement
EC Number:
270-659-9
EC Name:
Flue dust, portland cement
Cas Number:
68475-76-3
Molecular formula:
It is a UVCB.
IUPAC Name:
tricalcium dipotassium silanedione carbonate sulfate silicate
Details on test material:
The test substance was supplied by the sponsor. Two containers each containing 800 g Flue dust T (REACH) were received in good condition on 4 March 2010. Gross weights were 870.95 g and 875.67 g, respectively. This batch was given TNO Dispense Reference No. 10005B.

The following characteristics were provided by the sponsor:

Name : Flue dust T (REACH)
Other names : Flue dust, Portland cement (EC number 270-659-9); Cement kiln dust
Colour / appearance : Beige/grey powder
CAS reg. number : 68475-76-3
Purity : UVC substance
Storage conditions : Ambient temperature
Batch number : 12-2009
Expiry date : 1 December 2010

The certificate of analysis pertaining to the batch of test substance used during this study was
provided by the sponsor.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Both the dose-range finding study and the main study were conducted with Wistar rats. The rat was chosen because this species is considered one of the most suitable species for this type of study, and is usually required for regulatory agencies. Wistar outbred rats, 8-9 weeks, were obtained from a colony maintained under SPF conditions from Charles River Wiga GmbH, Sulzfeld, Germany.

Upon arrival, the rats were quarantined (animal room 05.1.11, DRF and main study) and checked for overt signs of ill health and abnormalities. During the quarantine period serological examinations of the microbiological status of the rats were conducted in a random sample. After 5 days (DRF) and 2 days (main study) the results of serology appeared to be satisfactory and the animal room was cleared for use as experimental room. The animals were acclimatized to the laboratory conditions for 6 days. The day before the first exposure (day -1 of the study), on 15 March 2010 (DRF) and 5 April 2010 (main study), the animals (males and females separately) were allocated to the various groups by computer randomization proportionately to body weight (DRF: Appendix 1; main study: Appendix 6). Surplus animals were kept in the animal room for monitoring during the study. They were sacrificed at the end of the in-life segment of the study.

The study was identified as TNO study number 8899DRF (dose-range finding study) and 8899 (main study). During the acclimatization period animals were identified by a temporary tail mark. After allocation to the groups the individual rats were identified by a unique even (male) or odd (female) animal identification number, one digit was tattooed in each ear and (if necessary), the ear were clipped for hundreds.
During the study each group of rats was coded by a group number and a colour. Each cage was provided with a card showing the colour code, the animal identification number(s), the group number and the study number.


Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: rodent diet
Details on oral exposure:
Feed and drinking water were provided ad libitum from the arrival of the rats until the end of the study.
The rats received a cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Witham, England). Each batch of RM3 diet was analysed by the supplier for nutrients and contaminants. The certificates of analysis pertaining to the batch (no. 7388) used in this study are included in Annex 3 of this report. The feed was provided as a powder, in stainless steel cans, covered by a perforated stainless steel plate that served to prevent spillage. The feed in the feeders was refreshed twice per week during the dose-range finding study and once per week during the main study.
Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chemical and microbiological examination of drinking water as conducted by the supplier were made available to the test facility. In addition, the supplier periodically (twice
per year) analyses water samples taken at the premises of TNO in Zeist for a limited number of physical, chemical and microbiological variables.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine the homogeneity and content of aluminium of the test substance in the diets of the main study wase conducted using Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES), after melting of the diet with a mixture of sodium carbonate and sodium borate.
Before analysis of study samples, the method was validated for the matrix under examination (viz. the rodent diet RM3) to conform to the following criteria: - Linearity: the correlation coefficient of the calibration curves should be greater than or equal to
0.996;
- Recovery: the recovery of the test substance from test diet should be between 80% and 110% at each of the concentrations tested;
- Repeatability: the relative standard deviation in the percentage recovery of three spiked diet samples per concentration level should be less than 10%.
Samples were taken from the batch of experimental diets prepared in the dose-range finding study as backup samples for possible analysis in the analytical validation process. These samples were not used and they will be discarded at finalization of the report.
The homogeneity and content (achieved concentration) of the test substance in the experimental diets was demonstrated in the batch of diets used in the main study, by analysing five samples (taken at different locations in the feed container) of each test diet; one sample of the control diet was analysed in the same series.
Analysis for stability of the test substances in the diet were not conducted, since it is not possible to measure stability in a complex mixture. In addition, aluminium is known to be stable in the diet.
Duration of treatment / exposure:
male rats 28 days
female rats 6-7 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2000 mg/kg diet
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
6000 mg/kg diet
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
16000 mg/kg diet
Basis:
nominal in diet
No. of animals per sex per dose:
3
Control animals:
yes, plain diet
Details on study design:
Preceding the main study, Flue dust T (REACH) was studied in a 1-week dose-range finding study at dose levels of 0, 2,500, 5,000, 10,0000 or 20,000 mg Flue dust T (REACH)/kg diet. Body weight change of the female animals of the top-dose group (20,000 mg/kg diet) was statistically significantly decreased between day 3-7 of the study. No other treatment-related effects were observed.
Considering the duration of the main study, at least 28 days for males and approx. 6-7 weeks for females, the following dose levels were chosen for the main study: 0 (control), 2,000 (low-dose), 6,000 (mid-dose) and 16,000 (high-dose) mg Flue dust T (REACH)/kg diet. It was intended that the mean substance intake in the high-dose group was comparable to approx. 1 g Flue dust T (REACH)/kg body weight/day.

The objective of this main study was to provide data on the possible effects of Flue dust T (REACH) on the reproductive performance of male and female Wistar Crl:Wi(WU) rats and on the growth and development of the offspring after oral exposure via the diet.
Groups of 12 animals/sex were administered via the diet with 0, 2,000, 6,000 or 16,000 mg Flue dust T (REACH)/ kg diet for 30 days (males) or during 2 weeks premating, mating gestation and up to day 4 of lactation or shortly thereafter (females).
Clinical signs were determined at least daily. Detailed clinical observations outside the home cage were performed prior to the first exposure and then once weekly. Functional Observational Battery (FOB) tests, including assessment of grip strength and sensory reactivity to stimuli of different types, and spontaneous motor activity measurements were performed in 5 males/group prior to the end of dosing and 5 females/group prior to day 4 of lactation.
Body weight and food consumption were recorded once weekly. Prior to the end of the premating period, 5 rats/sex/group were fasted overnight and blood was taken for haematology and clinical chemistry. Male and female animals were mated within the groups. Female animals were allowed to litter. The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups were evaluated on
days 1 and 4 of lactation. The pups were individually weighed on days 1 and 4 of lactation. Females and their pups were sacrificed at or shortly after day 4 of lactation. Male animals were sacrificed after 30 days of administration of the test substance. Reproductive organs of 12 animals/sex/group were preserved and thereafter microscopically examined. The testes and epididymides of these animals were weighed. In addition, for 5 animals/sex/group extra organs were weighed and preserved and thereafter microscopically examined.

Examinations

Observations and examinations performed and frequency:
Each animal was observed daily in the morning hours by cage-side observation and, if necessary, handled to detect signs of toxicity starting from the beginning of the study. On working days, all cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. On Saturdays, Sundays and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.

Detailed clinical observations were conducted in all animals. Detailed clinical observations outside the home cage were performed prior to the first exposure and then once weekly. Functional Observational Battery (FOB) tests, including assessment of grip strength and sensory reactivity to stimuli of different types, and spontaneous motor activity measurements were performed in 5 males/group prior to the end of dosing and 5 females/group prior to day 4 of lactation.



Sacrifice and pathology:
Male animals were sacrificed after the mating period. Female animals were sacrificed at or shortly after day 4 of lactation.
Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which was preserved in Bouin's fixative:
- ovaries
- uterus (after counting of the implantation sites )
- testes
- epididymides
- seminal vesicles
- prostate
- all gross lesions
In addition of 5 animals/sex from each group (see Annex 5), the following organs were preserved:
adrenals
bone marrow (femur)
brain (including sections of cerebrum, cerebellum, medulla/pons)
heart
small and large intestines (including Peyer’s patches)
kidneys
liver
lungs
lymph nodes
peripheral nerve (sciatic or tibial)
spinal cord (cervical, mid-thoracic, and lumbar)
spleen
stomach
thymus
thyroid
trachea
urinary bladder
The underlined organs were weighed (paired organs together) as soon as possible after dissection to avoid drying.
Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 μm, and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS haematoxylin. Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high-dose group. As no treatment-related changes were observed in the high-dose groups, the evaluation of these tissues/organs was not extended to the intermediatedose groups (2 and 3).
Statistics:
The resulting data were analyzed using the methods mentioned below. P < 0.05 was considered as a level of significance.
- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, organ weights and food consumption data were subjected to one-way analysis of variance (ANOVA)
- Fisher's exact probability test was be used to evaluate the number of mated and pregnant females and females with live pups.
- Number of implantation sites, live and dead fetuses or pups were evaluated by Kruskal-Wallis nonparametric analysis of variance
- Mortality data and data of the pathology of parent animals were evaluated by the Fisher’s exact probability test.
- Functional observational battery: one way analysis of variance (Anova) followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric Anova followed by multiple comparison tests (rank order data) or Pearson chi-square test followed by multiple comparison tests (categorical data).
- Motor activity assessment: one-way analysis of variance followed by Dunnett’s multiple comparison tests; effects of treatment on habituation: repeated measures analysis of variance on time blocks (each session consisted of 5 time blocks of 6 minutes each).

Results and discussion

Results of examinations

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:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Increased number of thrombocytes in males of the high-dose group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males of the high-dose group, a statistically significant increase was observed in the mean amount of plasma urea. Urea also tended to be increased in high-dose females. In addition, PO4 was statistically significantly increased in high-dose males.
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Effect levels

Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

No NOAEL could be identified.

Applicant's summary and conclusion

Conclusions:
Preceding the main study, Flue dust T (REACH) was studied in a 1-week dose-range finding study at dose levels of 0, 2,500, 5,000, 10,0000 or 20,000 mg Flue dust T (REACH)/kg diet.
Body weight change of the female animals of the top-dose group (20,000 mg/kg diet) was statistically significantly decreased between day 3-7 of the study.
Considering the duration of the main study, at least 28 days for males and approx. 6-7 weeks for females, the following dose levels were chosen for the main study: 0 (control), 2,000 (low-dose), 6,000 (mid-dose) and 16,000 (high-dose) mg Flue dust T (REACH)/kg diet. It was intended that the mean substance intake in the high-dose group was comparable to approx. 1 g Flue dust T (REACH)/kg body weight/day.
The objective of this main study was to provide data on the possible effects of Flue dust T (REACH) on the reproductive performance of male and female Wistar Crl:Wi(WU) rats and on the growth and development of the offspring after oral exposure via the diet.
Groups of 12 animals/sex were administered via the diet with 0, 2,000, 6,000 or 16,000 mg Flue dust T (REACH)/kg diet for 30 days (males) or during 2 weeks premating, mating gestation and up to day 4 of lactation or shortly thereafter (females).
Clinical signs were determined at least daily. Detailed clinical observations outside the home cage were performed prior to the first exposure and then once weekly. Functional Observational Battery (FOB) tests, including assessment of grip strength and sensory reactivity to stimuli of different types, and spontaneous motor activity measurements were performed in 5 males/group prior to the end of dosing and 5 females/group prior to day 4 of lactation.
Body weight and food consumption were recorded once weekly. Prior to the end of the premating period, 5 rats/sex/group were fasted overnight and blood was taken for haematology and clinical chemistry. Male and female animals were mated within the groups. Female animals were allowed to litter. The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups were evaluated on days 1 and 4 of lactation. The pups were individually weighed on days 1 and 4 of lactation. Females and their pups were sacrificed at or shortly after day 4 of lactation.
Male animals were sacrificed after 30 days of administration of the test substance. Reproductive organs of 12 animals/sex/group were preserved and thereafter microscopically examined. The testes and epididymides of these animals were weighed. In addition, for 5 animals/sex/group extra organs were weighed and preserved and thereafter microscopically examined.

The content of Flue dust T (REACH) was considered to be close to the intended concentration for the mid-dose and high-dose level. At the low-dose level the determined content of Flue dust T (REACH) was higher than intended, but this was attributed to analytical variation resulting from the high level of aluminium in the blank diet.

No mortalities or treatment-related clinical observations were observed.

Weekly detailed clinical observation and neurobehavioural testing did not indicate a treatmentrelated effect.

Body weight change of the female animals of the high-dose group (16,000 mg/kg diet) was statistically significantly decreased during the lactation period.. No other treatment-related effects were observed on body weight and body weight change in the male and female animals.

No exposure-related differences were observed on food consumption.

No effect was observed on the fertility and reproduction parameters.

No statistically significant or exposure-related differences were observed in the litter data between the control and the Flue dust T (REACH)-dosed groups.

Haemoglobin and mean corpuscular volume were statistically significantly decreased in high-dose females. Haemoglobin also tended to be decreased in high-dose males. In males of the high-dose group, a statistically significant increase was observed in the mean amount of plasma urea. Urea also tended to be increased in high-dose females. No other relevant differences were observed on haematology and clinical chemistry between the control and the groups of animals dosed with Flue Dust T (REACH).

No treatment-related differences were observed in the reproductive organ weights of all male animals. The organ weights of the control and Flue dust T (REACH)-dosed groups recorded in 5 animals/sex/group: adrenals, brain, heart, kidneys, liver, lung, spleen, stomach and thymus did not show treatment-related differences.
Microscopic examination of testes, epidymides, seminal vesicle, prostate, uterus and ovaries of 12 animals/sex of the control and high-dose groups, did not reveal any treatment-related effects. Microscopic examination of the adrenals, brain, caecum, colon, femur, heart, kidneys, larynx, liver, lung, nasal cavity, rectum, small intestines, spinal cord, spleen, stomach, thymus, thyroid, trachea/bronchi, and trachea/bronchial lymph nodes in 5 animals/sex of the control and high-dose groups did not reveal treatment-related histopathological changes in any of the sampled organs and tissues.

Based on the results found in this study (decreased body weight change during lactation, decreased haemoglobin in females and increased plasma urea in males) the No Observed Adverse Effect Level for parental toxicity was 6,000 mg Flue dust T (REACH)/kg diet (374 and 483 mg Flue dust T (REACH)/kg body weight, respectively for male and female animals). The No Observed Adverse Effect Level for fertility and developmental toxicity was 16,000 mg Flue dust T (REACH)/kg diet as no effects were observed at the highest dose-level used in this study (1010 and 1216 mg Flue dust T (REACH)/kg body weight, respectively for male and female animals).


Executive summary:

1. Preceding the main study, Flue dust T (REACH) was studied in a 1-week dose-range finding study at dose levels of 0, 2,500, 5,000, 10,0000 or 20,000 mg Flue dust T (REACH)/kg diet. Body weight change of the female animals of the top-dose group (20,000 mg/kg diet) was statistically significantly decreased between day 3-7 of the study. No other treatment-related effects were observed. Considering the duration of the main study, at least 28 days for males and approx. 6-7 weeks for females, the following dose levels were chosen for the main study: 0 (control), 2,000 (low-dose), 6,000 (mid-dose) and 16,000 (high-dose) mg Flue dust T (REACH)/kg diet. It was intended that the mean substance intake in the high-dose group was comparable to approx. 1 g Flue dust T (REACH)/kg body weight/day.

2. The objective of this main study was to provide data on the possible effects of Flue dust T (REACH) on the reproductive performance of male and female Wistar Crl:Wi(WU) rats and on the growth and development of the offspring after oral exposure via the diet. Groups of 12 animals/sex were administered via the diet with 0, 2,000, 6,000 or 16,000 mg Flue dust T (REACH)/ kg diet for 30 days (males) or during 2 weeks premating, mating gestation and up to day 4 of lactation or shortly thereafter (females). Clinical signs were determined at least daily. Detailed clinical observations outside the home cage were performed prior to the first exposure and then once weekly. Functional Observational Battery (FOB) tests, including assessment of grip strength and sensory reactivity to stimuli of different types, and spontaneous motor activity measurements were performed in 5 males/group prior to the end of dosing and 5 females/group prior to day 4 of lactation. Body weight and food consumption were recorded once weekly. Prior to the end of the premating period, 5 rats/sex/group were fasted overnight and blood was taken for haematology and clinical chemistry. Male and female animals were mated within the groups. Female animals were allowed to litter. The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups were evaluated on days 1 and 4 of lactation. The pups were individually weighed on days 1 and 4 of lactation. Females and their pups were sacrificed at or shortly after day 4 of lactation. Male animals were sacrificed after 30 days of administration of the test substance. Reproductive organs of 12 animals/sex/group were preserved and thereafter microscopically examined. The testes and epididymides of these animals were weighed. In addition, for 5 animals/sex/group extra organs were weighed and preserved and thereafter microscopically examined.

3. The content of Flue dust T (REACH) was considered to be close to the intended concentration for the mid-dose and high-dose level. At the low-dose level the determined content of Flue dust T (REACH) was higher than intended, but this was attributed to analytical variation resulting from the high level of aluminium in the blank diet.

4. Overall test substance intake (mg Flue dust T (REACH)/kg body weight/day)

Low-dose 2,000 mg/kg diet*           Mid-dose 6,000 mg/kg diet*       High-dose 16,000 mg/kg diet*

Males:       119                                                  374                                                 1010

Females:   164                                                  483                                                 1216

Overall intake during the entire study *concentration in the diet

5. No mortalities or treatment-related clinical observations were observed.

6. Weekly detailed clinical observation and neurobehavioural testing did not indicate a treatment-related effect.

7. Body weight change of the female animals of the high-dose group (16,000 mg/kg diet) was statistically significantly decreased during the lactation period. No other treatment-related effects were observed on body weight and body weight change in the male and female animals.

8. No exposure-related differences were observed on food consumption.

9. No effect was observed on the fertility and reproduction parameters.

10. No statistically significant or exposure-related differences were observed in the litter data between the control and the Flue dust T (REACH)-dosed groups.

11. Haemoglobin and mean corpuscular volume were statistically significantly decreased in highdose females. Haemoglobin also tended to be decreased in high-dose males. In males of the high-dose group, a statistically significant increase was observed in the mean amount of plasma urea. Urea also tended to be increased in high-dose females. No other relevant differences were observed on haematology and clinical chemistry between the control and the groups of animals dosed with Flue Dust T (REACH).

12. No treatment-related differences were observed in the reproductive organ weights of all male animals. The organ weights of the control and Flue dust T (REACH)-dosed groups recorded in 5 animals/sex/group: adrenals, brain, heart, kidneys, liver, lung, spleen, stomach and thymus did not show treatment-related differences.

13. Microscopic examination of testes, epidymides, seminal vesicle, prostate, uterus and ovaries of 12 animals/sex of the control and high-dose groups, did not reveal any treatment-related effects.

14. Microscopic examination of the adrenals, brain, caecum, colon, femur, heart, kidneys, larynx, liver, lung, nasal cavity, rectum, small intestines, spinal cord, spleen, stomach, thymus, thyroid, trachea/bronchi, and trachea/bronchial lymph nodes in 5 animals/sex of the control and high-dose groups did not reveal treatment-related histopathological changes in any of the sampled organs and tissues.

15. Based on the results found in this study (decreased body weight change during lactation, decreased haemoglobin in females and increased plasma urea in males) the No Observed Adverse Effect Level for parental toxicity was 6,000 mg Flue dust T (REACH)/kg diet (374 and 483 mg Flue dust T (REACH)/kg body weight, respectively for male and female animals). The No Observed Adverse Effect Level for fertility and developmental toxicity was 16,000 mg Flue dust T (REACH)/kg diet as no effects were observed at the highest dose-level used in this study (1010 and 1216 mg Flue dust T (REACH)/kg body weight, respectively for male and female animals).