Aluminium Bordeaux

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:

2016-11-10 till 2016-12-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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

1

Reference substance name:

disodium 2,4-dihydroxy-5-[(2-hydroxy-3-nitro-5-sulphonato-phenyl)azo]benzoate, reaction products with chromium triacetate

Molecular formula:

not applicable

IUPAC Name:

disodium 2,4-dihydroxy-5-[(2-hydroxy-3-nitro-5-sulphonato-phenyl)azo]benzoate, reaction products with chromium triacetate

Method

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-naphthoflavone induced rat liver S9 (pre-experiment/experimentI); non-induced hamster liver S9 (experiment II)

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II:
Without S9 mix: 33; 100; 333; 1000; 2500; and 5000 µg/plate
With S9 mix: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Since no relevant toxic effects were observed 5000 µg/plate were chosen as maximal concentration.

Vehicle / solvent:

Solvent used: deionized water
Justification for choice of solvent: best suitable solvent, because of its solubility properties and its relative nontoxicity to the bacteria

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

congo red

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation; pre-incubation

DURATION:
Preincubation period: 30 Minutes
exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 plates for each concentration including the controls

DETERMINATION OF CYTOTOXICITY: Evident as a reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in strains TA 1537, TA 98 and Wp2 uvrA

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
Effects of pH: none
Water solubility: soluble
Precipitation: Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 1000 µg/plate and higher concentrations in both experiments with and without metabolic activation.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations:
Experiment I: WP2 uvrA without S9 mix: 5000 µg/plate
Experiment II: TA1537 with S9 mix: 5000 µg/plate
TA 98 with S9 mix: 5000 µg/plate

Remarks on result:

other: negative with and without rat and hamster S9

Any other information on results incl. tables

Summary of Experiment I

Study Name: 1803002	Study Code: Envigo 1803002
Experiment: 1803002 VV Plate	Date Plated: 10.11.2016
Assay Conditions:	Date Counted: 16.11.2016

	Test Group	Dose Level (per plate)	TA 1535 Revertant Colony Counts (Mean ±SD)	TA 1537 Revertant Colony Counts (Mean ±SD)	TA 98 Revertant Colony Counts (Mean ±SD)	TA 100 Revertant Colony Counts (Mean ±SD)	WP2 uvrA Revertant Colony Counts (Mean ±SD)
Without	Deion. water		10 ± 3	12 ± 3	24 ± 6	159 ± 12	33 ± 6
Activation	Untreated		9 ± 2	7 ± 2	28 ± 4	158 ± 17	37 ± 3
	test item	3 µg	11 ± 4	9 ± 2	27 ± 1	167 ± 15	39 ± 5
		10 µg	11 ± 2	9 ± 3	28 ± 4	165 ± 8	32 ± 7
		33 µg	10 ± 1	9 ± 4	27 ± 6	157 ± 22	37 ± 3
		100 µg	11 ± 1	11 ± 1	28 ± 5	152 ± 19	38 ± 9
		333 µg	11 ± 5	9 ± 2	21 ± 1	159 ± 4	36 ± 10
		1000 µg	12 ± 2^P	9 ± 3^P	22 ± 6^P	156 ± 25^P	29 ± 4^P
		2500 µg	11 ± 3^P M	7 ± 1^P M	18 ± 2^P M	129 ± 7^P M	27 ± 5^P M
		5000 µg	7 ± 1^P M	6 ± 1^P M	18 ± 4^P M	129 ± 10^P M	26 ± 5^P M
	NaN3	10 µg	1414 ± 21			2698 ± 90
	4-NOPD	10 µg			521 ± 22
	4-NOPD	50 µg		89 ± 11
	MMS	2.0 µL					895 ± 60
With	Deion. water		12 ± 5	11 ± 1	34 ± 2	137 ± 14	51 ± 2
Activation	Untreated		15 ± 1	15 ± 3	30 ± 5	157 ± 5	51 ± 5
	test item	3 µg	10 ± 2	13 ± 4	38 ± 5	155 ± 5	41 ± 5
		10 µg	11 ± 4	15 ± 5	33 ± 4	164 ± 20	40 ± 11
		33 µg	9 ± 5	13 ± 4	36 ± 6	156 ± 3	51 ± 4
		100 µg	13 ± 3	12 ± 3	42 ± 4	157 ± 11	47 ± 2
		333 µg	9 ± 2	12 ± 3	31 ± 10	144 ± 6	38 ± 7
		1000 µg	12 ± 4^P	10 ± 4^P	24 ± 3^P	118 ± 11^P	36 ± 10^P
		2500 µg	10 ± 4^P M	10 ± 2^P M	20 ± 3^P M	112 ± 8^P M	28 ± 6^P M
		5000 µg	10 ± 3^P M	6 ± 2^P M	19 ± 2^P M	111 ± 9^P M	20 ± 1^P M
	2-AA	2.5 µg	387 ± 7	112 ± 6	5067 ± 298	3048 ± 92
	2-AA	10.0 µg					416 ± 13

Summary of Experiment II

Study Name: 1803002	Study Code: Envigo 1803002
Experiment: 1803002 HV2 Pre	Date Plated: 24.11.2016
Assay Conditions:	Date Counted: 01.12.2016

	Test Group	Dose Level (per plate)	TA 1535 Revertant Colony Counts (Mean ±SD)	TA 1537 Revertant Colony Counts (Mean ±SD)	TA 98 Revertant Colony Counts (Mean ±SD)	TA 100 Revertant Colony Counts (Mean ±SD)	WP2 uvrA Revertant Colony Counts (Mean ±SD)
Without	Deion. water		8 ± 5	9 ± 5	26 ± 8	163 ± 6	44 ± 10
Activation	Untreated		11 ± 5	11 ± 3	19 ± 2	161 ± 17	43 ± 7
	test item	33 µg	10 ± 4	9 ± 3	24 ± 3	169 ± 8	44 ± 6
		100 µg	9 ± 2	12 ± 4	28 ± 7	170 ± 10	39 ± 4
		333 µg	9 ± 3	8 ± 3	19 ± 2	150 ± 5	32 ± 8
		1000 µg	7 ± 0^P	10 ± 2^P	17 ± 2^P	138 ± 23^P	38 ± 3^P
		2500 µg	7 ± 1^P M	8 ± 2^P M	15 ± 3^P M	157 ± 14^P M	24 ± 2^P M
		5000 µg	6 ± 1^P M	6 ± 1^P M	14 ± 3^P M	143 ± 18^P M	24 ± 3^P M
	NaN3	10 µg	1253 ± 59			2325 ± 107
	4-NOPD	10 µg			626 ± 42
	4-NOPD	50 µg		122 ± 9
	MMS	2 µL					1087 ± 17
With	Deion. water		12 ± 2	18 ± 2	48 ± 1	153 ± 12	34 ± 5
Activation	Untreated		11 ± 5	14 ± 4	48 ± 13	156 ± 12	36 ± 6
	test item	3 µg	13 ± 2	18 ± 4	40 ± 9	139 ± 13	34 ± 6
		10 µg	14 ± 2	15 ± 5	42 ± 5	148 ± 7	34 ± 6
		33 µg	15 ± 1	20 ± 1	40 ± 7	150 ± 6	41 ± 3
		100 µg	15 ± 1	18 ± 4	45 ± 1	161 ± 4	35 ± 6
		333 µg	16 ± 4	17 ± 3	45 ± 12	180 ± 7	27 ± 5
		1000 µg	10 ± 1^P	17 ± 4^P	36 ± 7^P	143 ± 25^P	31 ± 8^P
		2500 µg	9 ± 1^P M	9 ± 2^P M	27 ± 3^P M	153 ± 10^P M	26 ± 2^P M
		5000 µg	7 ± 2^P M	7 ± 2^P M	18 ± 3^P M	136 ± 8^P M	17 ± 2^P M
	2-AA	2.5 µg				1133 ± 94
	2-AA	2.5 µg	415 ± 39	300 ± 17
	2-AA	10 µg					663 ± 16
	Congo red	500 µg			795 ± 48

Key to Plate Postfix Codes:              

P: Precipitate

M: Manuel Count

R:  Reduced Background growth

D:  Densely Colored Plate

Key to positive controls:

NaN3: sodium azide

4 -NOPD: 4 -nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

Congo red

Applicant's summary and conclusion

Conclusions:

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item Sanodure was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

Experiment I was performed with induced rat liver S9 mix as an exogenous metabolic activation system and Experiment II was performed with non-induced hamster liver S9 mix. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:        3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                

Without S9 mix:                             33; 100; 333; 1000; 2500; and 5000 µg/plate
With S9 mix:                                   3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 1000 µg/plate and higher concentrations in both experiments with and without metabolic activation.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

 

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	/	/	/	/
TA 1537	/	/	/	5000
TA 98	/	/	/	5000
TA 100	/	/	/	/
WP2 uvrA	/	5000	/	/

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance (see Table 3 (Individual Results of Experiment I) and Table 4 (Individual Results of Experiment II)).

 

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.