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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:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline sudy

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
other: Commission Directive 2000/32/EC, Ll 362000, Annexe 4D, dated May 19, 2000
Qualifier:
according to guideline
Guideline:
other: Japanese Guidelines: Kanpoan No. 287 - Environment Protection Agency; Eisei No. 127 - Ministry of Health & Welfare; Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
2,4-di-tert-butylphenyl diphenyl phosphite; 2-tert-butylphenyl diphenyl phosphite; 4-tert-butylphenyl diphenyl phosphite; triphenyl phosphite
EC Number:
941-802-9
Molecular formula:
Unspecified
IUPAC Name:
2,4-di-tert-butylphenyl diphenyl phosphite; 2-tert-butylphenyl diphenyl phosphite; 4-tert-butylphenyl diphenyl phosphite; triphenyl phosphite
Details on test material:
- Physical state: liquid
- Stability under test conditions: stable for hours in water, saline, polyethylene glycol and CMC
- Storage condition of test material: room temperature

Method

Species / strain
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:
rat liver S9 fraction
Test concentrations with justification for top dose:
33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Remarks:
with and without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Each concentration and the controls were tested in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: backgrond growth, reduction in revertants

POSITIVE CONTROLS
- Without metabolic activation
TA 1535, TA100: sodium azide dissolved in water, 10 µg/plate
TA98: 4-nitro-o-phenyiene-diamine dissolved in DMSO, 10 µg/plate
TA1537: 4-nitro-o-phenyiene-diamine dissolved in DMSO, 50 µg/plate
WP2 uvrA: methyl methane sulfonate dissolved in water, 5 µl/plate
- with metabolic activation
TA 1535, TA 1537, TA 98, TA 100: 2-aminoanthracene dissolved in DMSO, 2.5 µg/plate
WP2 uvrA: 2-aminoanthracene dissolved in DMSO, 10 µg/plate
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 WP 2uvrA) 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.

Results and discussion

Test results
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:
starting from 2500 µg/plate in the preincubation assay
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES:
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. In the pre-experiment the concentration range of the test item was 3 - 5000 pg/plate. The pre-experiment is reported as experiment I since no relevant toxic effects were observed and 5000 pg/plate were chosen as maximal concentration.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant toxic effects, evident as a reduction in the number of revertants below 50 % of the corresponding solvent control, were observed during the preincubation assay (Experiment II) at the following concentrations:
TA 1535: 5000 µg/plate (with and without S9)
TA 1537: 2500-5000 µg/plate (with and without S9 mix)
TA 98: 2500 µg/plate (without S9) and 5000 µg/plate (with S9)
TA 100: 2500-5000 µg/plate (without S9)
WP2 uvrA: 5000 µg/plate (with S9)
The toxicity observed in the second experiment followed a rather shallow gradient leading to revertant colony counts around 50 % of the corresponding solvent control over broad dose ranges (e.g. in strain TA 1535 without metabolic activation). However, toxicity was not severe as indicated by the regular bacterial background growth and the plates were analysable at all concentrations.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

SUMMARY OF RESULTS

without S9 mix
TA 1535 TA 1537 TA 98 TA 100 WP2 uvrA
Dose (µg/plate) I II I II I II I II I II
negative control 15 11 25 6 34 24 143 167 50 41
solvent control 18 9 25 6 39 23 122 127 44 42
positive control 852 665 245 47 181 200 964 908 830 173
33.3 23 8 30 7 38 25 135 146 42 41
100 21 7 41 5 32 25 136 143 41 41
333.3 20 5 27 4 27 24 142 116 47 30
1000 22 9 31 5 24 25 135 134 40 43
2500 17 6 37 2 31 10 142 54 38 37
5000 23 3 34 3 27 13 141 54 39 24

with S9 mix
TA 1535 TA 1537 TA 98 TA 100 WP2 uvrA
Dose (µg/plate) I II I II I II I II I II
negative control 23 11 28 7 46 32 152 183 47 48
solvent control 22 12 28 10 49 27 145 149 51 43
positive control 114 106 245 61 351 598 515 872 162 169
33.3 21 13 31 8 42 33 133 162 57 40
100 21 7 31 6 45 34 110 156 53 41
333.3 23 5 34 4 46 33 113 169 56 36
1000 23 6 31 9 41 30 125 167 56 45
2500 23 6 33 5 50 23 122 131 47 30
5000 23 5 34 1 50 6 113 93 40 18

I = Plate Incorporation Test

II = Preincubation Test

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used and is therefore considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of the test article to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II) using the Salmonella typhimurium strains TA1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. Distinct toxic effects, evident as a reduction in the number of revertants, occurred in the test groups of the second experiment with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any dose 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. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that 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 substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.