Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non mutagenic in bacterial reverse mutation assay (Ames test).

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from July 7 to August 26, 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(2000)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Standard plate test
1) TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA: 0, 60, 300, 1500, 7500, 15000 µg/plate.
2) TA 100: 0, 200, 400, 600, 800, 1000 µg/plate
3) TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA: 0, 1000, 2000, 3000, 4000 and 5000 µg/plate.

Preincubation test
4) TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA: 0, 30, 100, 300, 1000, 3000 µg/plate.
5) TA 100: 0, 500, 1000, 1500, 2000, 2500 µg/plate.
Vehicle / solvent:
- Vehicle: water.
- Justification for choice of solvent/vehicle: good solubility of test substance in water.
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-aminoanthracene, 4-nitro-o-phenylendiamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation method

Test tubes containing 2-mI portions of soft agar (overlay agar, i.e. 100 ml agar (0.8 % agar + 0.6 % NaCI) and 10 ml amino acid solution (minimal aminoacid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45 °C, and the remaining components are added in order:
0.1 ml test solution or vehicle
0.1 ml fresh bacterial culture
0.5 ml S9 mix (in tests with metabolic activation)
or
0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within ca. 30 seconds.

Composition of the minimal glucose agar for Salmonella Typhimurium:
980 ml aqua dest.
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37 °C for 48 - 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Composition of the minimal glucose agar for Escherichia coli:
300 ml solution B (agar)
100 ml solution A (saline solution)
8 ml solution C (glucose solution)
10 ml solution D (casein solution)
After incubation at 37 °C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.

METHOD OF APPLICATION: preincubation test
0.1 ml test solution or vehicle, 0.1 ml bacterial suspension and 0.5 ml S9 mix or phosphate buffer are incubated at 37 °C for the duration of about 20 minutes using a shaker. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the agar plates within ca. 30 seconds.
After incubation at 37 °C for 48 - 72 hours in the dark, the bacterial colonies are counted.
Evaluation criteria:
The test substance is considered positive in this assay if a dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either with or without S9 mix, is noted.

A test substance is generally considered nonmutagenic in this test if the number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in standard plate test (from 3000 µg/plate), in preincubation test (from 1000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- precipitation: no test substance precipitation was found. As long as precipitation does not interfere with the colony scoring, 5 mg/plate is selected and analysed (if not toxic) as the maximum dose.

COMPARISON WITH HISTORICAL CONTROL DATA: at non cytotoxic levels, test results are in the range of historical control data, except for TA 100 strain with metabolic activation. Further investigation on ths specific strain did not confirm this results, which was then considered as accidental.

ADDITIONAL INFORMATION ON CYTOTOXICITY
- bacteriotoxic effect (reduced his- and trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in titer) was observed in the standard plate test depending on the strain and test conditions from about 3000 to 5000 µg/plate onward.
- in preincubation assay, batteriotoxicity (reduced his- and trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in titer) was obserevd depending on the strain and test conditions at doses ≥ 1000 µg/plate.

Standard plate test

historical data TA 1535 TA 100 TA 1537 TA 98  WP2 uvrA
without S9-mix 11-25 82-155 6-16 17-44 25-51
with S9-mix 12-25 88-156 6-19 20-49 25-56

µg/plate TA 1535 TA 100 TA 1537 TA 98 WP2 uvrA
without S9-mix with S9-mix without S9-mix with S9-mix without S9-mix with S9-mix without S9-mix with S9-mix without S9-mix with S9-mix
0 18 17 111 112 12 13 28 43 34 40
60 18 16 106 168 13 8 28 45 36 46
300 18 16 105 212 11 15 26 44 40 43
1500 15 18 103 170 9 15 30 35 40 44
7500 2 2 13 11 0 2 4 13 14 18
15000 0 0 0 0 0 0 0 1 3 5

µg/plate TA 100
with S9-mix
0 112
200 152
400 138
600 150
800 149
1000 119

µg/plate TA 1535 TA 100 TA 1537 TA 98 WP2 uvrA
without S9-mix with S9-mix without S9-mix with S9-mix without S9-mix with S9-mix without S9-mix with S9-mix without S9-mix with S9-mix
0 18 19 103 124 10 12 31 38 36 38
1000 17 20 111 130 11 9 28 38 32 41
2000 17 21 105 147 11 8 26 36 34 39
3000 13 22 62 78 4 6 23 39 35 33
4000 7 19 14 30 1 2 17 22 29 26
5000 1 8 9 10 0 1 8 12 21 18

Preincubation test

historical data TA 1535 TA 100 TA 1537 TA 98  WP2 uvrA
without S9-mix 13 -25 90 -160 5 -19 18 -44 25-54
with S9-mix 11-25 92 -159 6 -20 17 -50 25-59

µg/plate TA 1535 TA 100 TA 1537 TA 98 WP2 uvrA
without S9-mix with S9-mix without S9-mix with S9-mix without S9-mix with S9-mix without S9-mix with S9-mix without S9-mix with S9-mix
0 17 17 118 111 10 13 29 36 35 43
30 18 17 110 145 12 10 24 30 47 36
100 16 17 100 144 8 9 24 41 40 35
300 15 14 118 137 9 11 31 28 42 35
1000 9 20 29 189 3 4 13 30 22 42
3000 0 10 0 56 0 3 0 20 7 23

µg/plate TA 100
with S9-mix
0 111
500 136
1000 131
1500 110
2000 99
2500 88
Conclusions:
Under these experimental conditions, the substance is not a mutagenic agent in bacterial reverse mutation assay with Salmonella typhimurium and Escherichia coli strains. The occasionally observed slight increase in the number of revertant colonies using TA 100 with S9 mix both in the standard plate test and in the preincubation assay could not be confirmed in additional studies and is therefore regarded as incidental and not as indication of a point mutagenic activity of test substance.
Executive summary:

Method

Bacterial reverse mutation assay (Ames test) with Salmonella typhimurium (TA 1535, TA 100, TA 1537 and TA 98) and Escherichia coli (WP2 uvrA) without or with metabolic activation (S9 mix). Two methods were used for the assays: standard plate and preincubation methods. Water was used as vehicle due to the high solubility of test substance in water. In standard plate test, doses ranged between 60 and 15000 µg/plate; in preincubation test, doses were between 30 and 3000 µg/plate. Solvent controls, sterility controls and positive controls were used.

Results

Based on negative responses under test conditions, the substance is considered as non mutagenic. Indeed the number of revertant colonies was found to be within historical control ranges. Depending on strain and test conditions, from ca. 3000 - 5000 µg/plate onward the substance was cytotoxic in the standard plate test, while at doses ≥ 1000 µg/plate the substance showed cytotoxicity in preincubation test. Slight increase in revertant colonies using TA 100 with S9 mix was not confirmed in further studies with this strain; therefore, it was considered as accidental.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No data on the substance in itself (phosphate salification) was available, thus a read across approach was followed, using data on a different salification (acetate). Details on the read across are available in section 13.

Bacterial reverse mutation test (AMES test) was conducted with five different strains, namely TA 1535, TA 1537, TA98 and TA100 of Salmonella typhimurium and WP2 uvrA of Escherichia coli, according to OECD guideline 471 (1997), using standard plate and preincubation methods.

No genotoxic effects, evident as substantial increase in revertant colony numbers of any of the tester strains, were seen following treatment with substance at any dose level, neither in presence nor in absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations.

Cytotoxic effects, evident as a reduction in the number of revertants, were seen at high concentrations (3000 - 5000 µg/plate onward in standard plate test and 1000 µg/plate onward in preincubation test) with and without metabolic activation, depending on the strain.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), a mutation means a permanent change in the amount or structure of the genetic material in a cell. The term ‘mutation’ applies both to heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known.

The more general terms ‘genotoxic’ and ‘genotoxicity’ apply to agents or processes which alter the structure, information content, or segregation of DNA, including those which cause DNA damage by interfering with normal replication processes, or which in a non- physiological manner (temporarily) alter its replication.

For the purpose of classification for germ cell mutagenicity, substances are allocated to one of two categories:

 

Category 1: substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans.

Category 1A: based on positive evidence from human epidemiological studies.

Category 1B: based on:

- positive result(s) from in vivo heritable germ cell mutagenicity tests in mammals; or

- positive result(s) from in vivo somatic cell mutagenicity tests in mammals, in combination with some evidence that the substance has potential to cause mutations to germ cells.

- positive results from tests showing mutagenic effects in the germ cells of humans, without demonstration of transmission to progeny; for example, an increase in the frequency of aneuploidy in sperm cells of exposed people.

 

Category 2: substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans, based on positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

- somatic cell mutagenicity tests in vivo, in mammals; or

- other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays

 

Based on negative result in the available study, relying on a read across approach, a mutagenic potential to bacteria was excluded for Basic Red 014 phosphate.

Accordingly, the substance is not classified under the CLP Regulation (EC 1272/2008).