Kaolin, calcined

Genetic toxicity: in vitro

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

Study period:

18 Nov - 27 Nov 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP -Guideline study with acceptable deviations (methodic deficiencies in cytotoxicity testing (determination of titre); however, as no cytotoxic effects were observed in the main experiment (no dose-dependent reduction in the number of revertants per plate), this was considered uncritically regarding the overall result of the study.)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

; methodic deficiencies in cytotoxicity testing

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Strasse 7, D-55116 Mainz

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Silicic acid, aluminum salt

EC Number:

215-628-2

EC Name:

Silicic acid, aluminum salt

Cas Number:

1335-30-4

Details on test material:

- Name of test material (as cited in study report): Silicic acid, aluminium salt
- Physical state / appearance: white powder
- Analytical purity: >99%
- Lot/batch No.: 1000168802
- Expiration date of the lot/batch: 04/2011
- Stability: stable
- Solubility: 7 mg/L in H20
- Storage condition of test material: room temperature, 20 +/- 5°C, dry, closed vessel protected from humidity

Method

Target gene:

TA97a: hisD6610
TA98: hisD3052
TA100 and TA 1535: hisG46
TA102: hisG428

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 97a, TA98, TA 100 and TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (obtained from Trinova Biochem, Gießen, Germany; Batch No. 2334; it was produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg bw i.p.)

Test concentrations with justification for top dose:

First experiment: 50, 151, 502, 1508 and 5017 µg/plate
Second experiment: 311, 627, 1249, 2510 and 5006 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
water (deionised) was used for the test substance and for the positive control sodium azide
DMSO was used for the positive control substances 4-Nitro-1,2-phenylene diamine, 2-Amino-anthracene and Benzo-a-pyrene

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with (2-Amino-anthracene, Benzo-a-pyrene) and without metabolic activation (4-Nitro-1,2-phenylene diamine and Sodium azide)

Positive control substance:

other: 4-Nitro-1,2-phenylene diamine (20µg/plate); Sodium azide (1 µg/plate); 2-Amino-anthracene (1 µg/plate); Benzo-a-pyrene (20µg/plate)

Remarks:

4-Nitro-1,2-phenylene diamine for strains TA 97a, TA 98 and TA 102 and Sodium azide for TA 100 and TA 1535 without S9; 2-Aminoanthracene for TA 97a, TA 100, TA 102 and TA 1535 and Benzo-a-pyrene for TA 98 with S9

Details on test system and experimental conditions:

Culture of Bacteria
10 hours before the start of each experiment, one pellet lyophilisate of each strain was placed into a culture flask containing 70 mL nutrient broth. For the incubation of strains TA97a, TA98, TA100, TA102 ampicilline was added to the nutrient broth (25 mg/L), for the incubation of strain TA102, tetracycline was added (20 mg/L) in addition to ampicilline. TA1535 was incubated without the addition of antibiotica. The flasks were incubated at 37°C for 10 hours.

Preparations
In the days before each test, the media and solutions were prepared. Two days before the test, the plates were sterilized and the first batches poured. On the day before the test, the remaining plates were poured.
On the day of the test, the overnight cultures were checked for growth. The incubation chambers were heated to 37 °C. The water bath was turned to 43 °C. The table surface was disinfected.
The S9 mix was freshly prepared and stored at 0 °C.

METHOD OF APPLICATION: in agar (plate incorporation) and pre-incubation method

Plate incorporation method
Per strain and dose, four plates with and four plates without S9 mix were used. The upper three concentrations of the test item were weighed directly into sterile test tubes and auto-claved; then, the tubes were filled up with sterile water. The other two concentrations were diluted from these suspensions. All suspensions were constantly stirred during the test. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine-biotin-solution 0.5 mMol per 100 mL basis was added and the bottle was placed in the water bath at 45 °C.
0.1 mL of the appropriate suspension of the test item was given into a sterile tube. After mixing with 0.1 mL overnight culture of the respective strain and 0.5 mL phosphate buffer (only for treatments without S9) or 0.5 mL S9 mix, 2 mL Top-Agar were added. The mix-ture was gently vortexed, then poured on a minimal glucose plate and distributed evenly, the plate was shaked slowly. The plates were closed and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 °C.

Pre-incubation method
Per strain and dose, four plates with and four plates without S9 mix were used. All five concentrations of the test item were weighed directly into sterile test tubes and autoclaved; then, the tubes were filled up with sterile water. All suspensions were constantly stirred during the test. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine-biotin-solution 0.5 mMol per 100 mL basis was added and the bottle was placed in the water bath at 45 °C.
0.1 mL of the appropriate suspension of the test item were given into a sterile tube. After mixing with 0.1 mL overnight culture of the respective strain, 0.5 mL phosphate buffer (only for treatments without S9) or 0.5 mL S9 mix were added. The mixture was incubated in an incubation chamber at 37°C for 20 minutes. During this time the vessels were aerated through careful shaking. Then 2 mL top agar was added. The mixture was vortexed gently, then poured on a minimal glucose plate and distributed evenly, the plate was shaked slowly. The plates were closed and left to harden for a few minutes, then inverted and placed in the dark incubator at 37°C.

The colonies were counted visually, the numbers were recorded.

Evaluation criteria:

A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor >= 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Statistics:

A spreadsheet software (Microsoft Excel(R)) was used to calculate mean values and standard deviations as well as the increase factor of revertant induction.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item did not show mutagenic effects in both experiments either with or without the addition of a metabolic activation system (S9 mix). The number of revertant colonies was not increased in comparison with the spontaneous revertants (solvent only).
Cytotoxicity of the test item was not detected. The background lawn was visible and the number of revertants was not significantly decreased.
Therefore it can be stated, that under the test conditions, the test item silicic acid, aluminium salt is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

1st experiment	mean number of revertants per plate ± SD of vehicle control (water)	mean number of revertants per plate ± SD of positive control	mean number of revertants per plate ± SD of test material [test concentration (µg/plate) at which max. number of revertants occured]
without S9-mix
TA 97a	121 ± 10.6	381 ± 18	119 ± 9 [50]
TA 98	11 ± 3.7	96 ± 19	15 ± 2 [1508]
TA 100	114 ± 12.5	331 ± 20	116 ± 16 [502]
TA 102	178 ± 18.4	700 ± 251	243 ± 23 [502]
TA 1535	8 ± 2.5	113 ± 13	12 ± 3 [1508]
with S9-mix
TA 1535	108 ± 2.5	360 ± 59	123 ± 5 [5017]
TA 100	9 ± 4.2	103 ± 10	12 ± 4 [502]
TA 1537	114 ± 10.7	421 ± 39	112 ± 17 [1508]
TA 1538	146 ± 41.2	1003 ± 43	254 ± 14 [5017]
TA 98	7 ± 1.7	101 ± 18	11 ± 3 [50]
2nd experiment	mean number of revertants per plate ± SD of vehicle control (water)	mean number of revertants per plate ± SD of positive control	mean number of revertants per plate ± SD of test material [test concentration (µg/plate) at which max. number of revertants occured]
without S9-mix
TA 97a	111 ± 5.4	327 ± 19	111 ± 4 [311]
TA 98	15 ± 1.7	103 ± 12	13 ± 2 [2510]
TA 100	111 ± 13.7	396 ± 21	123 ± 24 [627]
TA 102	237 ± 11.5	711 ± 121	208 ± 21 [627]
TA 1535	10 ± 3.5	123 ± 9	16 ± 5 [5006]
with S9-mix
TA 97a	116 ± 15.1	330 ± 41	123 ± 6 [627]
TA 98	12 ± 1.8	101 ± 15	14 ± 3 [5006]
TA 100	107 ± 15.4	449 ± 33	109 ± 9 [311]
TA 102	191 ± 16.1	709 ± 76	235 ± 14 [5006]
TA 1535	8 ± 2.6	120 ± 13	12 ± 4 [1249]

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative