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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:
experimental study
Adequacy of study:
key study
Study period:
From 2008-02-12 to 2008-04-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to the guidelines and in compliance with the GLP. Moreover, the data are available on the test substance.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Curb Crop Spray Powder, Aluminium ammonium sulfate

Method

Target gene:
The histidine gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: no data
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
other: histidine deficient
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
- Type and identity of media: no data
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
other: histidine deficient
Metabolic activation:
with and without
Metabolic activation system:
S9 mix: 5% and 10% v/v consisted of an S9 fraction (Aroclor 1254 induced rat liver homogenate) supplemented with cofactors
Test concentrations with justification for top dose:
See table 7.6.1/1
Cytotoxicity (TA100): from 0.00 9to 5.0 mg/plate (+/- 5% v/v S9 mix).
Trial 1: 0.009, 0.019, 0.039, 0.078, 0.156, 0.312, 0.625, 1.25, 2.5 and 5.0 mg /plate (+/- 5% v/v S9 mix)
Trial 2: 0.051, 0.128, 0.32, 0.8 and 5.0 mg/plate (+ 10% v/v S9 mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: the test substance formed solution with distilled water.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: different positive control substances according to the strain and the presence or absence of metabolic activation
Remarks:
no remarks
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: no data
- Exposure duration: 48h
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): histidine
SPINDLE INHIBITOR (cytogenetic assays): no data
STAIN (for cytogenetic assays): no data

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: cytotoxicity is characterised by inhibition of the background bacterial lawn and reduction in the number of colonies.

OTHER EXAMINATIONS:
- Determination of polyploidy: no data
- Determination of endoreplication: no data
- Other: no additional data

OTHER: no data
Evaluation criteria:
A positive result is defined as a dose-dependent increase or a reproducible increase at one or more concentrations in the number of histidine independant revertant colonies per plate in at least one strain (in the presence and absence of metabolic activation).
A test substance for which the results do not meet the above criteria is considered non mutagenic in this test. If the test substance does not induce a statistically significant, dose-dependent increase in revertant frequency but does induce an increase in revertant frequency at one dose level that is at least two-fold the spontaneous control value, the result is considered equivocal.
Negative and equivocal results in the first trial are confirmed by a second trial with an alteration in concentration spacing and/or metabolic activation.
Statistics:
Simple linear regression analysis was performed for TA1537, TA1535, TA98, TA100 and TA102, separately, to assess the dose dependent nature of any increase in revertant colonies.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: yes
- Precipitation: not observed at the maximum concentration of 5mg/plate.
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: 5.0 mg of Aluminium ammonium sulfate spray powder / plate was selected as the highest concentration to be tested in the mutagenicity test, for all the tester strains.

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxicity is characterised by inhibition of the background bacterial lawn and reduction in the number of colonies. Partial background bacterial lawn inhibition was observed at the concentration of 2.5 mg/plate in the presence (5% v/v S9 mix) of metabolic activation. Reduction in the number of colonies was not observed both in the absence and the presence (5% v/v S9 mix) of the metabolic activation system.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/2: Trial 1: Mean count of His+ revertant colonies in negative and positive controls and treatment plates in the absence of metabolic activation

Concentration of Aluminium ammonium sulfate (mg/plate)

His+Revertant Colonies/Plate (Absence of Metabolic Activation)

TA1537

TA1535

TA98

TA100

TA102

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (DW)

6.33

0.58

15.0

1.0

22.0

1.0

161.67

5.86

232.0

3.61

0.156

5.0

1.0

14.33

1.15

22.0

1.0

160.33

3.79

236.33

4.04

0.312

5.33

0.58

14.33

0.58

21.67

0.58

160.67

3.79

230.0

8.54

0.625

5.0

1.0

15

1.0

22.33

0.58

160

3.61

230.0

6.24

1.25

6.0

1.0

14.33

0.58

21.33

0.58

159.67

5.03

231.0

5.0

2.5

5.33

0.58

15.67

1.15

21.33

0.58

160.67

2.52

238.33

4.04

5.0

3.67

0.58

14.0

1.0

21.67

1.53

157.33

3.21

227.67

5.13

PC

416.67

10.5

209.67

8.5

715.33

7.77

1663.67

75.98

2070.67

59.07

PC-2Aa

-

-

-

-

-

-

153.0

5.29

-

-

SD: Standard Deviation, NC: Negative Control, PC: Positive Control [TA1537: 9 -aminoacridine (75 µg/plate), TA1535: Sodium azide (0.5 µg/plate), TA98: 2-Nitrofluorene (7.5 µg/plate), TA100: Sodium azide (5 µg/plate), TA102: Mitomycin-C (0.5 µg/plate)], DW: Distilled Water

Table 7.6.1/3: Trial 1: Mean count of His+ revertant colonies in negative and positive controls and treatment plates in the presence of metabolic activation

Concentration of Aluminium ammonium sulfate (mg/plate)

His+Revertant Colonies/Plate [Presence of Metabolic Activation(5% v/v S9 mix)]

TA1537

TA1535

TA98

TA100

TA102

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (DW)

6.0

1.0

14.0

0.0

27.33

1.53

149.67

2.52

276.33

4.16

0.156

5.33

0.58

14.33

1.53

25.67

0.58

149.33

2.52

275.0

1.0

0.312

5.33

0.58

14.0

1.0

27.67

1.15

149.33

2.08

281.33

3.51

0.625

6.33

0.58

14.0

1.0

26.33

1.53

151.67

5.51

276.0

4.0

1.25

5.33

0.58

13.67

0.58

26.0

1.0

150.33

2.08

276.0

2.65

2.5

5.67

0.58

14.67

0.58

25.67

0.58

148.0

4.36

279.0

7.0

5.0

3.67

0.58

14.33

0.58

25.67

0.58

148.67

2.31

278

6.56

PC

335.33

11.02

408.67

14.57

1568.67

22.72

1624.33

11.02

2134.33

6.56

SD: Standard Deviation, NC: Negative Control, PC: Positive Control (2 -Aminoanthracene 10 µg/plate for TA1537, TA1535 and TA102, and 5 µg/plate for TA98 and TA100), DW: Distilled Water

Table 7.6.1/4: Trial 2: Mean count of His+ revertant colonies in negative and positive controls and treatment plates in the absence of metabolic activation

Concentration of Aluminium ammonium sulfate (mg/plate)

His+Revertant Colonies/Plate (Absence of Metabolic Activation)

TA1537

TA1535

TA98

TA100

TA102

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (DW)

6.67

0.58

16.67

1.15

24.0

1.0

167.33

4.73

236.67

3.51

0.051

6.33

1.0

16.0

1.0

23.0

1.0

164.33

4.51

235.67

2.08

0.128

7.0

0.58

15.0

1.0

24.67

1.53

165.67

5.03

234.33

2.08

0.32

6.33

0.58

15.67

1.53

22.0

1.0

168.33

3.21

233.67

4.16

0.8

7.33

0.58

15.33

0.58

23.67

1.53

167.67

4.73

233.33

2.31

2.0

5.67

0.58

16.33

0.58

23.0

1.0

168.0

1.0

232.33

5.13

5.0

5.33

0.58

17.0

1.0

23.67

1.15

162.0

4.58

228.33

1.53

PC

446.67

14.84

205.33

12.58

761.67

43.47

1655.33

14.98

1994.67

12.66

PC-2Aa

-

-

-

-

-

-

158.67

2.52

-

-

SD: Standard Deviation, NC: Negative Control, PC: Positive Control [TA1537: 9 -aminoacridine (75 µg/plate), TA1535: Sodium azide (0.5 µg/plate), TA98: 2-Nitrofluorene (7.5 µg/plate), TA100: Sodium azide (5 µg/plate), TA102: Mitomycin-C (0.5 µg/plate)], DW: Distilled Water

Table 7.6.1/5: Trial 2: Mean count of His+ revertant colonies in negative and positive controls and treatment plates in the presence of metabolic activation

Concentration of Aluminium ammonium sulfate (mg/plate)

His+Revertant Colonies/Plate [Presence of Metabolic Activation(10% v/v S9 mix)]

TA1537

TA1535

TA98

TA100

TA102

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (DW)

7.33

1.15

16.33

0.58

28.0

1.0

158.0

2.0

272.0

2.0

0.051

7.67

0.58

16.0

1.0

27.67

1.53

155.0

4.58

268

3.61

0.128

8.33

0.58

14.67

0.58

25.0

1.0

155.33

2.89

269.0

4.0

0.32

8.0

1.0

15.67

1.15

26.0

1.0

157.67

2.89

271.0

3.61

0.8

8.67

0.58

15

1.0

25.0

1.0

155.33

1.53

268.0

5.57

2.0

9.33

0.58

15.33

1.15

24.67

1.15

159.0

7.55

271.0

3.61

5.0

8.0

1.0

15.0

1.0

26.67

1.53

151.67

4.16

275.0

2.0

PC

375.33

12.34

341.67

11.5

1516.33

65.61

1601.67

84.33

2087.33

18.18

SD: Standard Deviation, NC: Negative Control, PC: Positive Control (2 -Aminoanthracene 10 µg/plate for TA1537, TA1535 and TA102, and 5 µg/plate for TA98 and TA100), DW: Distilled Water

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the test conditions, Aluminium ammonium sulfate was not classified as mutagenic in the Salmonella typhimurium TA1535, TA1537, TA98, TA100 and TA102.
Executive summary:

In an in vitro genetic toxicity study, Aluminium ammonium sulfate (as a powder formulation at 83% purity) was assessed for its possible mutagenic activity, by a bacterial reverse mutation test using five histidine deficient strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 and TA102). This study was performed in compliance with GPL and the OECD guideline 471 and EC guideline B.13/14.

The treatments were performed by a plate incorporation technique both in absence and presence of metabolic activation with S9 mix (Aroclor 1254 induced rat liver homogenate).

Prior to conducting a study, the test substance was diluted in water and evaluated for cytotoxicity on strain TA100 in the presence and absence of S9. Rates employed were based on solutility and precipitation tests.

The results on the cytotoxicity tests at rates of 0.009, 0.019, 0.039, 0.078, 0.156, 0.312, 0.625, 1.25, 2.5 and 5.0 mg/plate in the presence and absence of S9 showed that partial bacterial lawn inhibition was recorded at 2.5mg/plate in the presence of 5% S9, therefore a rate of 5.0 mg/plate was selected for testing.

Based on the results of cytotoxicity testing, a first trial was conducted with the five strains at dose levels of 0.009, 0.019, 0.039, 0.078, 0.156, 0.312, 0.625, 1.25, 2.5 and 5.0 mg/plate in the presence and absence of 5% S9 in triplicate with both positive and negative controls. 

A second trial was conducted in the presence of 10% S9 and test treatment rates of 0.051, 0.128, 0.32, 0.8 and 5.0 mg/plate in triplicate with positive and negative controls.

Positive and negative controls were valids in both trials.

In the trial I (5% v/v S9 mix), the results showed no positive mutagenic effect of Aluminium ammonium sulfate in strains TA1537, TA1535, TA98, TA100 and TA102 both in the presence (5% v/v S9 mix) and in the absence of metabolic activation at any of the tested dose levels when compared with the concurrent negative control. Statistical analysis also showed that there were no significant effects.

In the trial II (10% v/v S9 mix), in the absence of metabolic activation, no significant increase in the number of revertant colonies was measured in all strains. In the presence of metabolic activation no significant increase in the number of revertant colonies was recorded with strains TA 1537, TA1535, TA98 and TA 100. The statistically significant increase in revertant colonies recorded with strain TA102 in the presence of metabolic activation was not twice the concurrent negative control and therefore was not considered as biological significant.

On the basis of the results, it was concluded that at the highest rate tested (5.0 mg/plate) Aluminium ammonium sulfate is not mutagenic in Salmonella typhimurium .

Under the test conditions, Aluminium ammonium sulfate is not classified as according to the criteria of the Annex VI to the CLP Regulation and to the 67/548/EEC Directive.

This study is considered as acceptable and satisfies the requirement for the irritation study endpoint.