Ascophyllum nodosum, ext.

Genetic toxicity: in vitro

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

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

Test material information

Constituent 1

Reference substance name:

Ascophyllum nodosum, ext.

EC Number:

283-907-6

EC Name:

Ascophyllum nodosum, ext.

Cas Number:

84775-78-0

Molecular formula:

Not applicable; a generic molecular formula cannot be provided for this UVCB substance.

IUPAC Name:

Ascophillum nodosum, extract

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 / strain

Species / strain 1

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 2

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 3

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 4

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 5

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

Controls

Controls 1

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

Controls 2

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

Controls 3

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

Controls 4

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

Controls 5

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

Controls 6

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

Controls 7

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.