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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:
June-October 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: in accordance with guidelines

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
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): Algea Fert Solid K+
- Substance type: fertilizer ( algal extract)
- Physical state: solid, black micro-flakes
- Lot/batch No.: 00680
- Expiration date of the lot/batch: March 2012
- Stability under test conditions: Stable under suitable storage conditions.
- Storage condition of test material: original tightly-closed containers in well- ventilated place.

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
- Type and identity of media: Oxide nutrient broth No. 2. Minimal glucose agar contained agar, Vogel-Bonner minimal medium E and 40% glucose. The overlay agar contained 0.6% agar, 0.5% NaCl and 0.05 mM histidine - biotin (Maron and Ames, 1983).
- Properly maintained: yes
Species / strain / cell type:
S. typhimurium TA 97
Details on mammalian cell type (if applicable):
- Type and identity of media: Oxide nutrient broth No. 2. Minimal glucose agar contained agar, Vogel-Bonner minimal medium E and 40% glucose. The overlay agar contained 0.6% agar, 0.5% NaCl and 0.05 mM histidine - biotin (Maron and Ames, 1983).
- Properly maintained: yes
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
- Type and identity of media: Oxide nutrient broth No. 2. Minimal glucose agar contained agar, Vogel-Bonner minimal medium E and 40% glucose. The overlay agar contained 0.6% agar, 0.5% NaCl and 0.05 mM histidine - biotin (Maron and Ames, 1983).
- Properly maintained: yes
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: Oxide nutrient broth No. 2. Minimal glucose agar contained agar, Vogel-Bonner minimal medium E and 40% glucose. The overlay agar contained 0.6% agar, 0.5% NaCl and 0.05 mM histidine - biotin (Maron and Ames, 1983).
- Properly maintained: yes
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
- Type and identity of media: Oxide nutrient broth No. 2. Minimal glucose agar contained agar, Vogel-Bonner minimal medium E and 40% glucose. The overlay agar contained 0.6% agar, 0.5% NaCl and 0.05 mM histidine - biotin (Maron and Ames, 1983).
- Properly maintained: yes

Metabolic activation:
with and without
Metabolic activation system:
S9 mammalian liver fraction
Test concentrations with justification for top dose:
5000 µg, 1500 µg, 500 µg, 150 µg and 50 µg/plate, both with and without metabolic activation system (S9)

Test concentrations for this study were selected from the results obtained with solubility/ precipitation test and preliminary cytotoxicity test of the test article employing only TA100, with and without the metabolic activation system.

ALGEA FERT SOLID K+ was found completely soluble in Analytical grade water at a concentration of 50 mg/ml. No precipitation was observed in the final reaction mixture up to the concentrations of 1.85 mg/ml, corresponding to final test concentrations of 5000 µg/plate. 5000 µg/plate was the highest concentration selected for the preliminary cytotoxicity study.

Before commencing the study, the test article was assessed for cytotoxicity to the tester bacteria using tester strain TA100. Five concentrations viz. 5000 µg, 1500 µg, 500 µg, 150 µg, and 50µg/plate of the test article, suspended in analytical grade water, were tested for toxicity to bacterial cells.
ALGEA FERT SOLID K+ was not found to be cytotoxic to the bacterial cells at the maximum concentration of 5000 µg/plate. Hence 5000 µg/plate was selected as the maximum test concentration for main study.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water, analytical grade
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
overlay agar supplemented with histidine, served as a control to check spontaneous revertants.
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
2 µg/plate, without S9 mix

Migrated to IUCLID6: for TA1535
Untreated negative controls:
yes
Remarks:
overlay agar supplemented with histidine, served as a control to check spontaneous revertants.
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR 191, for TA97a
Remarks:
1 µg/plate, without S9 mix
Untreated negative controls:
yes
Remarks:
overlay agar supplemented with histidine, served as a control to check spontaneous revertants.
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.5 µg/plate, without S9 mix

Migrated to IUCLID6: for TA98
Untreated negative controls:
yes
Remarks:
overlay agar supplemented with histidine, served as a control to check spontaneous revertants.
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 3-Methylmethane sulphonate, for TA100 and TA102
Remarks:
1 µg/plate, without S9 mix
Untreated negative controls:
yes
Remarks:
overlay agar supplemented with histidine, served as a control to check spontaneous revertants.
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene, for TA1535
Remarks:
10 µg/plate, with S9 mix
Untreated negative controls:
yes
Remarks:
overlay agar supplemented with histidine, served as a control to check spontaneous revertants.
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
20 µg/plate, with S9 mix

Migrated to IUCLID6: for TA97a, TA98 and TA100
Untreated negative controls:
yes
Remarks:
overlay agar supplemented with histidine, served as a control to check spontaneous revertants.
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Danthron, for TA102
Remarks:
30 µg/plate, with S9 mix
Details on test system and experimental conditions:
All the tester strains are defective in DNA repair capacity (uvrB), except TA102 and all tester strains have a defective lipopolysaccharide barrier on the cell wall (rfa). These two properties confer extra sensitivity to DNA damage and also increase permeability to large molecules that do not penetrate the normal cell wall.
Strains TA97a, TA98, TA100 and TA102 also contain resistance transfer factor (plasmid pKM 101). This factor, which confers resistance to ampicillin, enhances the operation of an error prone repair system.
In case of TA102, it contains plasmid pAQ1, which confers resistance to tetracycline.
Strains TA1535 and TA100 are highly sensitive to base - pair substitution mutation and TA97a, TA98 are highly sensitive to frame - shift mutation.
The strain TA102 contains ochre mutation.

The strains are tested routinely for other genotypic characters such as:
- Histidine requirement – The His - character of all tester strains was confirmed by demonstrating the histidine requirement for growth on selective agar plates.
- rfa mutation, i.e. cell membrane permeability was checked through sensitivity to crystal violet;
- uvrB mutation was checked through sensitivity to ultra-violet light (TA1535, TA98, TA100 and TA97a);
- R- factor plasmid (pKM101) - Presence or absence of R- factor plasmid was checked through ampicillin resistance (TA98, TA100 and TA97a), and
- pAQ1 plasmid - The pAQ1 strain (TA102) was tested for both ampicillin and tetracycline resistance (TA102).

Frozen permanent copies of the tester strains were stored in liquid nitrogen cylinders (cryocan) at about -196°C. They were prepared from fresh overnight culture to which DMSO was added as a cryoprotective agent.
In addition to frozen permanents, the tester strains were routinely maintained in the laboratory on master plates, which were stored at 2-8°C.
Master plates were used as the source of bacteria for inoculating the overnight culture for frequently used strains.

Maximum dose of 5000 µg/plate, which was not found to be cytotoxic to TA100 in the preliminary cytotoxicity, was selected as the highest dose level, with four lower dose levels viz. 1500 µg, 500 µg, 150 µg and 50 µg/plate being selected for the main assay. Triplicate plating was performed at each dose level for an adequate evaluation of the variation.

Pre-Incubation Assay without Metabolic Activation:
Aliquots of 0.1 ml cell suspension of the tester strains, containing about 108 viable cells, were added to sets of sterile tubes. Sterile phosphate buffer (0.5 ml) and freshly prepared test product / control sample / positive control (0.1 ml) were added to the tube. All the constituents of reaction mixture were introduced into sterile test tubes on an ice bath. The tubes with reaction mixture were mixed by shaking gently and were incubated at 37 ± 1 °C for 20-30 minutes in laboratory incubator.
Finally 2 ml of overlay agar with histidine-biotin, previously melted and cooled to about 45 °C, was added. The contents will be mixed and poured over the surface of the petri plates containing minimal glucose VB agar. The overlay agar will be allowed to solidify before incubation.

Pre-Incubation Assay with Metabolic Activation:
Aliquots of 0.1 ml cell suspension of the tester strains, containing about 108 viable cells, were added to sets of sterile tubes. The S9 mixture (0.5 ml) and freshly prepared test product / control sample / positive control (0.1 ml) were added to the tube. All the constituents of reaction mixture were introduced into sterile test tubes on an ice bath. The tubes with reaction mixture were mixed by shaking gently and were incubated at 37 ± 1 °C for 20-30 minutes in laboratory incubator.
Finally 2 ml of overlay agar with histidine-biotin, previously melted and cooled to about 45°C, was added. The contents were mixed and poured over the surface of the petri plates containing minimal glucose VB agar. The overlay agar was allowed to solidify before incubation.

INCUBATION: Contents of all the plates were allowed to solidify after which the plates were inverted and incubated at 37±1°C for 48-72 hours.

Test for Sterility:
To ensure sterility of the vehicle, tests for evaluation of contamination were performed along with the assay. The solvent, overlay agar, S9 mix and the highest employed dose of the test article were evaluated at the same volume that was used in the assay, but in the absence of tester bacteria. Overlay agar alone was also plated on minimal glucose VB agar. Plates for laminar flow were exposed to the laminar flow during treatment. All plating was done in triplicate. Plates were incubated for 48-72 hours at 37±1°C and then assessed for presence of contamination if any, in the form of growth of colonies on the agar.


Evaluation criteria:
A test article is considered as mutagenic if it produces a concentration related increase over the range tested and /or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
The biologically significant increase would be assumed if the average colony counts are: at least twice as compared to those in vehicle control group for strain TA97a, TA98, TA100 and TA102 and at least thrice as compared to those in vehicle control group for strain TA1535.

A test article is considered as non-mutagenic, if it does not induce any increase in the number of revertants and does not show any dose response relationship, in two separate experiments, with any bacterial strain, either with or without metabolic activation.

OBSERVATION: After incubation period, the plates were checked for sterility. The plates were observed for a uniform lawn of auxotrophs (his-) and the colonies for histidine revertants as the prototrophs (his+). Histidine revertant colonies per plate were counted and the mean number of colonies at each test point was calculated.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium 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
Species / strain:
S. typhimurium TA 97
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
Species / strain:
S. typhimurium TA 98
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
Species / strain:
S. typhimurium TA 100
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
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:
valid
Positive controls validity:
valid
Additional information on results:
REVERTANT FREQUENCIES IN VEHICLE CONTROL GROUPS
Plate counts for the spontaneous histidine revertant colonies in the vehicle control groups were found to be within the frequency ranges expected from the laboratory historical control data at INTOX. They also compared well with the range reported in the published literature.

REVERTANT FREQUENCIES IN TREATMENT GROUPS
Frequencies of histidine revertant colonies observed at all concentrations of ALGEA FERT SOLID K+ in tester strains TA1535, TA97a and TA100, with and without the presence of a metabolic activation system, were comparable to those observed in the vehicle control groups, as per the criteria employed for evaluation of mutagenic potential.
In first experiment histidine revertant colonies of tester strains TA 98 at 1500 µg/plate in absence of S9 metabolic activation, were increased, while the same was not observed in second experiment. Thus, this increase was not found to be reproducible.
ALGEA FERT SOLID K+ was found to be slightly cytotoxic to strain TA102 at 5000µg/plate with metabolic activation. While frequencies of histidine revertant colonies observed for remaining concentrations of ALGEA FERT SOLID K+ in tester strains TA102 with and without the presence of a metabolic activation system, were comparable to those observed in the vehicle control groups, as per the criteria employed for evaluation of mutagenic potential.
These observations were confirmed by repetition of the experiments.

REVERTANT FREQUENCIES IN POSITIVE CONTROL GROUPS
Concurrent positive controls demonstrated sensitivity of the assay 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

    TESTS FOR Genetic Traits

Sr.

No.

Character

 

Results

TA97a

TA98

TA100

TA102

TA1535

1

Histidine requirement

Desired

P

P

P

P

P

Observed

P

P

P

P

P

2

rfa mutation

Desired

P

P

P

P

P

Observed

P

P

P

P

P

3

Defective DNA repair system (DuvrB)

Desired

P

P

P

A

P

Observed

P

P

P

A

P

4

R- factor

Desired

P

P

P

P

A

Observed

P

P

P

P

A

5

pAQ1 Plasmid

Desired

A

A

A

P

A

Observed

A

A

A

P

A

P - Character should be / found to be present ;        A - Character should be / found to be absent

Desired – desired status for the respective strain and the character.

Observed – observed status for the character as observed when tested.

  FREQUENCIES OF Spontaneous HISTIDINE REVERTANTS

Tester

Strain

Plate Counts of Spontaneous Histidine Revertants Colonies

Replicate 1

Replicate 2

Replicate 3

Mean

+SD

TA97a

150

188

144

160.67

23.86

TA 98

18

30

17

21.67

7.23

TA 100

142

165

143

150.00

13.00

TA 102

287

280

279

282.00

4.36

TA 1535

5

9

12

8.67

3.51

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Salmonella typhimurium, Reverse Mutation Assay of ALGEA FERT SOLID K+ was carried out in compliance with the OECD Guidelines for Testing of Chemicals (No. 471, Section 4: Health Effects) on conduct of "Bacterial Reverse Mutation Test", adopted 21 July 1997.
Under the conditions described for this study, it is concluded that, ALGEA FERT SOLID K+ is non-mutagenic in Salmonella typhimurium strains TA1535, TA97a, TA98, TA100 and TA102.
Executive summary:

Salmonella typhimurium,Reverse Mutation Assay of ALGEA FERT SOLID K+ was carried out in compliance with the OECD Guidelines for Testing of Chemicals (No. 471, Section 4: Health Effects) on conduct of "Bacterial Reverse Mutation Test", adopted 21 July 1997 and as per mutually agreed protocol.

ALGEA FERT SOLID K+ was evaluated in the/ Salmonella Pre-incubation Assay to determine its ability to induce reverse mutation at selected histidine loci in five tester strains of Salmonella typhimuriumviz. TA1535, TA97a, TA98, TA100 and TA102 in the presence and absence of metabolic activation system (S9). Based upon the preliminary tests conducted to assess the solubility / precipitation and cytotoxicity of ALGEA FERT SOLID K+, the tester strains were exposed to the test article in triplicate cultures at the doses of 5000 µg, 1500 µg, 500 µg, 150 µg and 50 µg/plate, both with and without metabolic activation system (S9). Liver S9, induced in Sprague Dawley rats by phenobarbitone withb- naphthoflavone, was used for this purpose.

Analytical grade water was used as a vehicle. The exposed bacteria were plated onto minimal glucose agar medium supplemented with L-histidine. The plates were incubated at 37+1°Cfor      48-72 hours after which the histidine revertant colonies were counted and their frequency was compared with that in the vehicle control group. Concurrent positive control groups were also included in the experiment, as specified by the test guideline. Results of this test indicated that the frequencies of histidine revertant colonies at all concentrations ofALGEA FERT SOLID K+instrains TA1535, TA97a, TA98, TA100 and TA102, with and without the presence of a metabolic activation system, were comparable to those observed in the vehicle control group, as per the criteria employed for evaluation of mutagenic potential, and this observation was confirmed by repetition of the experiments. Plate counts for the spontaneous histidine revertant colonies in the vehicle control groups were found to be within the frequency ranges expected from the laboratory historical control data at INTOX. They also compared well with the range reported in the literature. Concurrent positive controls demonstrated sensitivity of the assay with and without metabolic activation. It is concluded that, under the conditions of this study, ALGEA FERT SOLID K+ is non-mutagenic in Salmonella typhimurium strains TA1535, TA97a, TA98, TA100 and TA102.