Registration Dossier

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The reliability is rated Klimish 1

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Cement, alumina, chemicals
EC Number:
266-045-5
EC Name:
Cement, alumina, chemicals
IUPAC Name:
266-045-5
Details on test material:
- Name of test material (as cited in study report): Ciment Fondu® (and Milled Clincker of Ciment Fondu® for one assay)
- Substance type: mineral
- Physical state: dark grey fine powder
- Purity : 100%
- Quantity Supplied: 50 g
- Composition of test material, percentage of components: Ciment Fondu® is a mixture of more than 99.5% milled clinker of Ciment Fondu® which is the same substance as the registered substance here, and a grinding agent
- Lot/batch No.: 91478 for the first, second, fouth and fifth assay. Batch n° 93539 and batcn N° 93535 for the assay 3.
- Expiration date of the lot/batch: at least up to 20/10/2010
-Storage condition of test material: sealed, at ambient temperature
- Other: Date of sample preparation: 20/04/2010

Method

Target gene:
Histidine operon
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: see below
Metabolic activation:
with and without
Metabolic activation system:
hepatic microsomes from rat livers induced by Aroclor 1254 (incubation period 48 h)
Test concentrations with justification for top dose:
Doses in µg/plate:
0; 50; 150; 500; 1500 and 5000 for assay 1 and ssay 2
0; 150; 500; 1500; 3000 and 5000 for 2 strains in assay 2, and in assay 3
0; 3000; 5000; 10000; 15000 and 20000
0; 500; 1500; 3000 and 5000 in assay 4
and 0; 3000; 5000; 10000; 15000 and 20000 in assay 5
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Sodium azide; 9-amino-acridine; 2-nitro fluorene; mitomycin C; 2-anthramine; benzo[a]pyrene
Remarks:
almost each strain was tested with a different reference products
Details on test system and experimental conditions:
METHOD OF APPLICATION:

Without metabolic activation:
The following technique was performed for each one of the strains used in the test: 0.1 mL of a bacterial suspension from a culture agitated over
night at 37°C and 0.1 mL of the test item at the relevant concentration were successively added to 2 mL of top agar to which 10 % of 0.5 mM biotin histidine solution, maintained in a state of superfusion at 45°C, has been added. The content of each tube was agitated, then spread out in a Petri plate containing 20 mL of minimum agar. Three plates were used per treatment. The plates were then incubated at 37°C for 48 h approximately. Finally, colonies of revertants were counted for each plate.
For controls (without metabolic activation):
• Solvent controls, Positive controls
At the same time, solvent controls (0.1 mL solvent/plate) were performed under the same conditions but using 6 plates per solvent (Gatehouse et al., 1994). Appropriate positive reference controls were also performed .
• Sterility of the media
At each assay, 3 Petri plates containing 20 mL of minimal agar receive 2 mL of top agar only and were incubated under the same conditions of assay for the control of media sterility: no colony must be observed after 48 hours at 37°C.

With metabolic activation:
The method was the same as the one described above except that immediately before spreading in the plates, 0.5 mL of the S9 mix metabolic activation system was added in soft agar.
For controls (with metabolic activation):
Solvent controls, positive controls and assay for the control of media sterility were performed like in the mutagenicity assay without metabolic
activation.

Repeat test
Without metabolic activation
The test was subsequently repeated in an independent assay. The same method was used, but the test dose range may be modified, depending on the results obtained during the first test.

With metabolic activation:
According to the results obtained in the first assay in the presence of metabolic activation, the second assay can either be performed using a
standard plate-incorporation technique, or be extended by use of the pre-incubation modification of the assay.
- where a positive response was observed in the first assay, the same standard plateincorporation technique was used in a repeat assay with the
confirmatory purpose. But the test doses may be modified, depending on the results obtained during the first test.
- where a negative response was observed in the first assay, the method used in a second assay was the pre-incubation test.
The use of the standard plate-incorporation study plan with S9-mix is known to be sub-optimal in the detection of a number of bacterial
promutagens. These include aliphatic N-nitroso compounds (Bartsch et al., 1976), azo-dyes (Bridges et al., 1981) and alkaloids (Yamanaka et al., 1979). The technique with pre-incubation avoids such limitations. The National Toxicology Program (Gatehouse et al., 1990) has specifically adopted
the pre-incubation study plan, which is recommended by OECD for updating the existing guidelines.

On a technical point of view, this method is the same as the one using agar plate incorporation with, however, the following modifications:
The following solutions were added in this order: the bacterial strain to be tested (100 μL), S9-mix (500 μL) and at the end the test item solution (50 μL if an organic solvent was used and 100 μL if aqueous solvent).

The mixture was preincubated with stirring at 37°C for 60 minutes prior to adding soft agar and spreading out in a Petri plate.

DURATION
- Preincubation period: 48h
- Exposure duration:48h


NUMBER OF REPLICATIONS: 1 and assay 2, 4 and 5 was continued for strains TA 98 ans TA 102.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
Evaluation criteria:
After about 48 hours of incubation at 37°C, the prototrophic mutant colonies that have developed in the plates were counted, eventually using a
colony counter. The results are expressed as the mean number of revertants per plate and, for each concentration of the test product,
the following ratio was established:
Mean number of revertants per plate in presence of the test product/ Mean number of revertants per plate without the test product (solvent control)

Given that the procedures followed in order to evaluate mutagenic activity are semi-quantitative, the criteria used to determine a positive may have
been to some extent subjective and based mainly on experience. However, under our experimental conditions, and when the validity criteria are
reached, the following decision criteria may be used.
Strains TA1535, TA1537
A test item causing a positive response proportional to the dose for at least 3 concentrations with, for
the highest increase, a value greater than or equal to 3 times the value for the solvent control, is
considered positive in the assay.
Strains TA98, TA100, TA102
A test item causing a positive response proportional to the dose for at least 3 concentrations with, for
the highest increase, a value greater than or equal to 2 times the value for the solvent control, is
considered positive in the assay.
Statistics:
Data are analysed by means of Dunnett's method (Mahon et al, 1989) allowing the comparison of the mean value for each dose to the mean value for the corresponding solvent control.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA1535; TA 1537; TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
slight to moderate precipitation in all strains both with and without metabolic activation were observed at the 4 highest doses studied: 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
Without preincubation, statistically but not biologically significant increases with no dose-response relationship were observed in 2 independent assays. These increases were not considered as due to a mutagenic effect.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
slight to moderate precipitation in all strains both with and without metabolic activation were observed at the 4 highest doses studied: 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
without
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
In the 3 first assays: mutagenic in the presence of S9-mix. The complementatry assays did not allow to conclude the mutagenic activity.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The examination of the background lawn demonstrated that CIMENT FONDU® induced no toxicity. In return,
slight to moderate precipitates in all strains both with and without metabolic activation were observed at the 4 highest doses studied.
Therefore, the maximum dose retained for the first mutagenicity assay was of 5000 μg/plate in all strains with and without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

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TOXICITY ASSAY

 TOXICITY ASSAY   

 Doses in

µg/plate   

    TA1535

    TA1537

    TA98

    TA100

   TA102 

 T

 P

 T

 P

 T

 P

 T

 P

 T

 P

CIMENT FONDU®

       

Without S9-mix

 0

 -

 -

 -

 -

 -

 -

 -

 -

 -

 -

 50

 -

 -

 -

 -

 -

 -

 -

 -

 -

 -

 150

 -

 +

 -

 +

 -

 +

 -

 +

 -

 +

 500

 -

 +

 -

 +

 -

 +

 -

 +

 -

 +

 1500

 -

 +

 -

 +

 -

 +

 -

 +

 -

 +

 5000

 -

 ++

 -

 ++

 -

 ++

 -

 ++

 -

 ++

 Top Dose in

1st mutagenicity assay

 -

 5000   

    5000

    50000

    5000

    5000

 CIMENT FONDU®

               

with S9 -mix

 0

 -

 -

 -

 -

 -

 -

 -

 -

 -

 -

 50

 -

 -

 -

 -

 -

 -

 -

 -

 -

 -

 150

 -

 +

 -

 +

 -

 +

 -

 +

 -

 +

 500

 -

 +

 -

 +

 -

 +

 -

 +

 -

 +

 1500

 -

 +

 

 +

 -

 +

 -

 +

 -

 +

 5000

 -

 ++

 -

 ++

 -

 ++

 -

 ++

 -

 ++

 Top Dose in

1st mutagenicity assay

 

    5000

    5000

   5000 

    5000

    5000

The test item was placed in solution in the appropriate solvent (sterile distilled water, dimethylsulfoxide, ethanol, acetone etc.). The toxicity assay was carried out in all the test strains to be tested under the same conditions as the mutagenicity test with and without metabolic activation but using only one plate per dose instead of 3 plates per dose. Reference substances were not used in this preliminary assay. The plates were incubated for approximatively 48-72 hours at 37°C, and the revertants were counted. Toxicity was checked by microscopic examination of the background lawn and was noted as follows:

- : non-toxic

+ : slightly toxic

++ : moderately toxic

+++ : strongly toxic

N : no bacterial growth

In this assay, the possible presence of a precipitate was also noted:

- : no precipitate

+ : slight precipitate

++ : moderate precipitate

+++ : heavy precipitate hindering scoring of revertants

The examination of the background lawn demonstrated that CIMENT FONDU® induced no toxicity. In return, slight to moderate precipitates in all strains both with and without metabolic activation were observed at the 4 highest doses studied.

Therefore, the maximum dose retained for the first mutagenicity assay was of 5000 μg/plate in all strains with and without metabolic activation.

MUTAGENICITY ASSAYS

    ASSAY 1           TA 1535     TA 1537     TA 98     TA 100     TA 102      
 Doses in µg/plate  Induction Ratio (a) Doses in µg/plate  Induction Ratio (a) Doses in µg/plate  Induction Ratio (a) Doses in µg/plate  Induction Ratio (a) Doses in µg/plate  Induction Ratio (a)      
 CIMENT FONDU

®

 Without S9 -mix                  
 0  -  0  -  0  -  0  -  0  -      
 50  1.2  50  1.0  50  1.0  50  0.8  50  0.7      
 150  1.4  150  0.9  150  1.0  150  1.0  150  0.8      
 500  1.4  500  0.7  500  0.9  500  0.8  500  1.4      
 1500  1.2  1500  0.8  1500  0.8  1500  1.0  1500  1.3      
 5000  1.5  5000  0.9  5000  0.7  5000 1.0   5000  1.0      
                               
 CIMENT FONDU 

®

 With S9-mix, without preincubation               
 0  -  0  -  0  -  0  -  0  -
 50  1.4  50  1.0  50  1.0  50  1.1  50  0.8
150  1.4  150  1.3  150  1.4  150  1.3  150  0.9
 500  1.2  500  0.8  500  1.5  500  1.4  500  1.1
 1500  1.0  1500  1.0  1500  1.5  1500  1.3  1500  1.5
 50000  0.9  5000  0.6  5000  1.0  5000  1.0  5000  1.0

(a) Induction Ratio = number of revertants in the threated/number of revertants in the control.

   ASSAY 2          TA 1535     TA 1537     TA 98     TA 100     TA 102      
 Doses in µg/plate  Induction Ratio (a) Doses in µg/plate  Induction Ratio (a) Doses in µg/plate  Induction Ratio (a) Doses in µg/plate  Induction Ratio (a) Doses in µg/plate  Induction Ratio (a)      
 CIMENT FONDU 

®

             Without S9 -mix                  
 0  -  0  -  0  -  0  -  0  -      
 50  0.8  50  0.5  50  1.2  50  0.7  50 1.3      
 150  0.8  150  0.8  150  1.2  150  1.1  150  1.3      
 500 0.8  500  0.7  500  0.9  500  0.9  500  1.2      
 1500  0.7  1500  0.4  1500  0.9  1500 0.9  1500  1.6     
 5000  0.9  5000  0.7  5000  1.1  5000 1.2   5000  1.4      
                               
 CIMENT FONDU

®

With S9-mix, without preincubation               
 0  -  0  -  0  -  0  -  0  -
 50  0.6  50  1.2  150  0.9  50  0.8 150  1.1
150  1.2  150  0.8  500  1.1  150 0.8  500  1.6
 500  1.1  500  0.8  1500  0.6  500  0.8  1500  1.4
 1500  1.0  1500  0.8  3000  1.2  1500  0.9  3000  1.7
 5000  1.4  5000  1.2  5000  1.3  5000 0.9  5000 2.3

(a) Induction Ratio = number of revertants in the threated/number of revertants in the control.

   ASSAY 2 -Continued          TA 98     TA 102
 Doses in µg/plate  Induction Ratio (a) Doses in µg/plate  Induction Ratio (a)

 CIMENT FONDU

®

    

         WITH S9 -mix     without pre-incubation             

 0  -  0  -
 150  1.3  150  1.5
500 1.1  500  1.8
 1500 0.8  1500 1.8
3000  0.8  3000  2.2
 5000  0.9  5000  1.9

(a) Induction Ratio = number of revertants in the threated/number of revertants in the control.

FIRST AND SECOND MUTAGENICITY ASSAYS

In strain TA98 in presence of metabolic activation without preincubation, statistically but not biologically significant revertants increases with no dose-response relationship were observed in 2 independent assays. These increases were not considered as significant.

A non-reproducible, non-biologically but statistically significant increase was observed in the second assay in strain TA102 in absence of metabolic activation. This increase in the number of revertants was not considered as due to a mutagenic effect.

In strain TA102 in presence of metabolic activation without preincubation, statistically and/or biologically significant revertants increases with a slight dose-response relationship were observed in 2 independent assays. In the assay performed with preincubation, statistically and biologically significant increases in the number of mutants were also observed. These effects were considered as equivocal to weak.

In order to check if this effect is (or not) specific of the batch 91478, a third assay was implemented on

2 new batches only on TA102 strain with metabolic activation.

Results of the third assay:

      CIMENT FONDU®(batch No 91478)   CIMENT FONDU®  (batch No 93539) CIMENT FONDU®(batch No 93535)  

 ASSAY 3   

    TA 102

    TA 102

    TA 102

 Doses in

µg/plate

 Induction

ratio (a)

  Doses in µg/plate

 Induction ratio (a)  

  Doses in µg/plate

  Induction ratio (a)

 CIMENT FONDU®

With S9 -mix Without preincubation               

 0

 -

 0

 -

 0

 -

 150

 1.0

 150

 1.0

 150

 0.9

 500

 1.1

 500

 1.4

 500

 1.2

 1500

 0.9

 1500

 1.5

 1500

 1.8

 3000

 1.2

 3000

 1.9

 3000

 2.0

 5000

 1.4

 5000

 1.9

 5000

 2.1

 CIMENT FONDU®

 With S9 -mix

With preincubation

 -

 0

 -

 0

 -

 150

 1.0

 150

 1.1

 150

 0.9

 500

 1.3

 500

 1.5

 500

 1.3

 1500

 1.4

 1500

 1.6

 1500

 1.7

 3000

 1.8

 3000

 1.4

 3000

 1.8

 5000

 1.3

 5000

 1.8

 5000

 1.4

(a) Induction Ratio = number of revertants in the threated/number of revertants in the control.

In strain TA102 in presence of metabolic activation with and without preincubation, statistically and/or biologically significant increases in the number of revertants accompanied by a dose-response relationship were observed for the 3 different batches of the test item that confirms the weak mutagenic activity of CIMENT FONDU®. A fourth assay was performed only in strain TA102 and only in the presence of metabolic activation following preincubation method, in order to understand the influence of one or several factors on the mutagenicity activity of the test item CIMENT FONDU® (batch 91478): pH, metabolic activation, i.e. presence of proteins and time of contact with the water.

Results of the fourth assay:

 

    TA 102

  TA 102

       TA 102

    TA 102   

 ASSAY 4

   t = 0

   t = 1 hour

  t = 5 hours     

 t = 24 hours   

 Doses in

µg/plate

 Induction

ratio (a)

  Doses in µg/plate

 Induction ratio (a)  

  Doses in µg/plate

  Induction ratio (a)

  

Doses in

µg/plate

 

 Induction

ratio (a)

 

 TEST ITEM

With S9 -mix

Without preincubation               

 0

 -

 0

 -

 0

 -

 0    -

 500

 1.0

 500

 1.1

 500

 1.3

500 1.1

 1500

 0.9

 1500

 1.0

 1500

 1.5

 1500  1.5

 3000

 1.2

 3000

 1.1

 3000

1.4

 3000  2.0

 5000

 1.2

 5000

 1.0

 5000

 1.6

 5000  2.3
      

 TEST ITEM

 With S9 -mix

With preincubation (inactivated S9)

 -

 0

 -

 0

 -

   0  -

 500

 0.9

 500

 1.3

 500

 0.8

 500  0.9

 1500

 1.1

 1500

 1.3

 1500

 0.7

 1500  1.2

 3000

 0.8

 3000

 1.3

 3000

 0.9

 3000 1.3 

 5000

 1.0

 5000

 1.3

 5000

 1.4

 5000 1.6 

(a) Induction Ratio = number of revertants in the threated/number of revertants in the control.

In the presence of CIMENT FONDU® (batch No.91478), in the assay performed in strain TA102 with preincubation with either standard or heat-inactivated S9, no statistically or biologically significant increase in the number of revertants was observed in the assay performed with the treatment suspensions prepared extemporaneously or 1 hour before the treatment.

In the assay performed 5 hours after preparation of the treatment suspensions, a statistically but not biologically significant increase was observed with active S9. No statistically or biologically significant increase in the number of revertants was observed with heat-inactivated S9.

In the assay performed 24 hours after preparation of the treatment suspensions, statistically and biologically significant increases were observed with active S9. In the presence of heat-inactivated S9, only a statistically significant increase in the number of revertants was observed. The equivocal results previously observed in assays 2 and 3 were not reproduced. Indeed, no mutagenic activity was found when the test item was in contact with water up to 5 hours. Only a weak mutagenic activity was noted when suspensions were used 24 hours after the preparation.

Highly alkaline conditions did not lead to an increase in the number of revertants but lead to toxicity, meaning that the pH did not interfere with the test system.

In order to confirm or not the mutagenic activity of the test item and to definitely conclude, a fifth assay was performed in strain TA102 in the presence of metabolic activation (with active S9) following the preincubation method, with the suspensions prepared extemporaneously, but using a higher range of doses.

 ASSAY 5   

 TA 102   

 Doses in µg/plate  Induction ratio (a)
TEST ITEM               With S9 -mixWith pre-incubation  0  -
 3000  1.1
 5000  1.1
 10000  1.4
 15000  1.7
 20000  1.8

(a) Induction Ratio = number of revertants in the threated/number of revertants in the control.

In the presence of CIMENT FONDU® (batch No.91478), in the assay performed in strain TA102 with preincubation with the treatment suspensions prepared extemporaneously no biologically significant increases in the number of revertants were observed up to 20000 μg/plate. Equivocal results

previously observed were again not reproduced.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The overall conclusion is that the test item can not be considered as mutagenic under these experimental conditions as the non reproducible
equivocal to weak effects observed in few assays were considered as non specific.
Under these experimental conditions a weak mutagenic activity was revealed in strain TA102 in the presence of metabolic activation.
Executive summary:

The search for any mutagenic activity of CIMENT FONDU®, was studied by means of the Ames test (Salmonella his-/microsome system) in compliance with the OECD Guideline 471 using the maximum concentration recommended by OECD Guideline, i.e. 5000 μg/plate for the toxicity assay. Four lower dilutions chosen according to a geometrical (half-log) ratio were also tested. In the main assay, the maximum dose was chosen in accordance with the recommendations of OECD Guideline, i.e. 5000 μg/plate. Additionally to the 2 first main assays, 3 complementary assays were performed exclusively on the strain TA 102.

Preparation of treatment suspension

The CIMENT FONDU® and MILLED CLINKER OF CIMENT FONDU® were not freely soluble in distilled water. In order to suspend the test items, they were firstly grinded using mortar and pestle, and distilled water was added little-by-little. The test items CIMENT FONDU® and MILLED CLINKER OF CIMENT FONDU® were thus suspended in distilled water at the maximal initial concentration of 50 mg/mL in order to obtain the top doses of 5000 μg/plate when added at 100 μL/plate (without and/or

with S9-mix, with and/or without pre-incubation).

In the fifth assay, the test item CIMENT FONDU® was suspended in distilled water at the maximal initial concentration of 200 mg/mL in order to obtain the top dose of 20000 μg/plate when added at 100 μL/plate.

Successive suspensions were also prepared with distilled water and used at 100 μL/plate. During the fourth assay, the highest doses studied of 5000 and 3000 μg/plate hardened 5 hours after the preparation of the treatment formulations. It was necessary to scrape the product off the tubes and a 20-minute time of vortex was needed to save all the hardened test item. Moreover, 24 hours after the preparation of the treatment formulations, all the doses studied were hardened and needed a scraping of the tubes and a time of vortex to save all the hardened test item.

It is to be noted that 24 hours after the preparation of the treatment formulations, and before the scraping and the vortex, the colour of the water above the residue was brown. During bacteriostatic and mutagenicity assays, slight to moderate precipitates, certainly due to the non-solubility of the test items, were noted at the microscopic examination of the back ground lawn both in presence and in absence of metabolic activation at the 4 or 5 highest doses, from 150 to 20000 μg/plate in all strains.

Mutagenicity test

- Without metabolic activation

The following technique was performed for each one of the strains used in the test: 0.1 mL of a bacterial suspension from a culture agitated overnight at 37°C and 0.1 mL of the test item at the relevant concentration were successively added to 2 mL of top agar to which 10 % of 0.5 mM biotin histidine solution, maintained in a state of superfusion at 45°C, has been added. The content of each tube was agitated, then spread out in a Petri plate containing 20 mL of minimum agar. Three plates were used per treatment. The plates were then incubated at 37°C for 48 h approximately. Finally, colonies of revertants were counted for each plate.

Controls:

• Solvent controls, Positive controls

At the same time, solvent controls (0.1 mL solvent/plate) were performed under the same conditions

but using 6 plates per solvent (Gatehouse et al., 1994). Appropriate positive reference controls were

also performed.

• Sterility of the media

At each assay, 3 Petri plates containing 20 mL of minimal agar receive 2 mL of top agar only and were incubated under the same conditions of assay for the control of media sterility: no colony must be observed after 48 hours at 37°C.

- With metabolic activation

The method was the same as the one described above (§ 4.5.1.) except that immediately before spreading in the plates, 0.5 mL of the S9 mix metabolic activation system (§ 4.8.) was added in soft agar.

Controls:

Solvent controls, positive controls and assay for the control of media sterility were performed like in the mutagenicity assay without metabolic activation.

Repeat test:

- Without metabolic activation

The test was subsequently repeated in an independent assay. The same method was used, but the test dose range may be modified, depending on the results obtained during the first test.

- With metabolic activation

According to the results obtained in the first assay in the presence of metabolic activation, the second

assay can either be performed using a standard plate-incorporation technique, or be extended by use

of the pre-incubation modification of the assay.

The mixture was preincubated with stirring at 37°C for 60 minutes prior to adding soft agar and spreading out in a Petri plate.

Toxic activity

- Toxicity assay: When assessing its toxic activity by microscopic examination of the background lawn, the test item CIMENT FONDU® induced no toxicity whatever the dose and the strains tested, both with and without metabolic activation and with or without pre-incubation.

- Toxicity in mutagenicity assays: In both assays, no toxicity was noted at the microscopic examination of the background lawn, either with or without metabolic activation.

Mutagenicity assays

The mutagenic activity of the test item was assessed by means of the Ames’s test in the five Salmonella typhimurium strains TA1535,

TA1537, TA98, TA100 and TA102 tested either in presence or in absence of metabolic activation, in two to four independent assays. Indeed, 3 complementary assays were carried out in strain TA102 only in presence of S9-mix. Under these experimental conditions an equivocal to weak mutagenic activity was revealed exclusively in strain TA102 only in the presence of metabolic activation. In return, no mutagenic activity was found either with and without metabolic activation in

strains TA1535, TA1537, TA98 and TA100.

The first complementary assay (third assay), performed in order to check if the previous observed effect was specific to this batch, allowed to show that the weak mutagenic activity noted with the test item CIMENT FONDU® was also observed with 2 other batches, i.e. CIMENT FONDU® and MILLED CLINKER OF CIMENT FONDU®.

During the complementary assay, results obtained with the 3 different batches of CIMENT FONDU® and MILLED CLINKER OF CIMENT FONDU® firstly tend to confirm a weak mutagenic activity with metabolic activation in strain TA102 not

batch specific. The test item CIMENT FONDU® was mutagenic in the presence of an exogenous metabolic activation system in strain TA102.

On one hand, it was considered as surprising that the test item CIMENT FONDU® i.e. a mineral substance, needs metabolic activation. On the other hand, it is known that CIMENT FONDU® lead to an exothermic and alkaline reaction leading to local strong increase in the pH (up to near 13). Nevertheless, in order to understand the influence of one or several factors (pH, presence of heat-inactivated S9, time of contact with the water) on the mutagenicity activity of the test item CIMENT FONDU® , a fourth assay was performed only in strain TA102 and only in the presence of metabolic activation following preincubation method. No mutagenic activity was found when the test item was in contact with water up to 5 hours. Only a weak mutagenic activity was noted when suspensions were used 24 hours after the preparation Previous equivocal to weak effect was not reproduced.

A fifth assay was performed only in strain TA102 and only in the presence active S9 following preincubation method, with the suspensions prepared extemporaneously but using a higher range of doses. The increase in doses did not allow to confirm the mutagenic activity of the test item and to definitely conclude. Previous equivocal to weak effect was not reproduced.

Conclusion:

The overall conclusion is that the test item can not be considered as mutagenic under these experimental conditions as the non reproducible equivocal to weak effects observed in few assays were considered as non specific.

Otherwise, it can not be excluded that the test system is not actually appropriate for this kind of test item, i.e. particles.