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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan. - Jul. 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 1997
Deviations:
yes
Remarks:
For the strains TA98 (+/-S9), TA1537 (+S9) and E. coli WP2 (+S9) the required non-toxic concentrations were not sufficient for evaluation of mutagenicity and a clear result could not be derived.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-diiodopropane
EC Number:
210-993-4
EC Name:
1,3-diiodopropane
Cas Number:
627-31-6
Molecular formula:
C3H6I2
IUPAC Name:
1,3-diiodopropane
impurity 1
Chemical structure
Reference substance name:
1-chloro-3-iodopropane
EC Number:
230-088-8
EC Name:
1-chloro-3-iodopropane
Cas Number:
6940-76-7
Molecular formula:
C3H6ClI
IUPAC Name:
1-chloro-3-iodopropane
impurity 2
Reference substance name:
Unknown Impurity
IUPAC Name:
Unknown Impurity
Test material form:
liquid
Details on test material:
Batch 90423 / CME0032526
Specific details on test material used for the study:
Name: 1,3-Diiodopropane
CAS no.: 627-31-6
Purity: > 99 %
EC no.: 210-993-4
Molecular formula: C3H6I2
Molecular weight: 295.89 g/mol

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Species / strain / cell type:
E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Per bacteria strain and concentration, three plates with (+S9) and three plates without metabolic activation (-S9) were used.
S9 was obtained by Trinova Biochem GmbH, Gießen (Batch nos. 4183, 4053, 4120, 4180, 4069), produced from the livers of male Sprague-Dawley rats which were treated with Phenobarbital-5,6 Benzoflavone resp. 500 mg Aroclor 1254/kg body weight intraperitoneally.
S9-mix consisted of 22.5 mL Phosphate buffer, 1.0 mL 0.1M NADP-solution, 0.125 mL 1M G6P-solution, 0.5 mL Salt solution and 1.0 mL Rat liver S9.
Phosphate Buffer: 0.184 g Sodium di-hydrogen phosphate monohydrate NaH2PO4*H2O and 1.722 g Di-sodium hydrogen phosphate dihydrate Na2HPO4 * 2H2O; add to 100.0 mL H2O demineralized (the pH of the solution was adjusted to 7.4 with HCl 1M).
NADP-Solution for S9-mix, 0.1 M: 787.4 mg NADP disodium salt (MR = 787.4 g/mol) and add to 10.0 mL H2O demineralized.
Glucose-6-Phosphate (G6P) Solution for S9-mix, 1 M: 680.3 mg Glucose-6-phosphate disodiumsalt dihydrat (MR = 340.13 g/mol) and add to 2.0 mL H2O demineralized.
Salt Solution for S9-mix: 1.23 g Potassium chloride (KCl), 0.814 g Magnesium chloride hexahydrate MgCl2*6H2O and add to 10.0 mL H2O demineralized.
Test concentrations with justification for top dose:
The following nominal test item concentrations were prepared for experiment 1: 5; 1.5; 0.5; 0.15; 0.05 µL test item/plate.
All negative and positive controls were valid. No precipitation was seen. The test item showed cytotoxic effects towards S. tyhimurium TA98 (+/-S9), TA100 (+/-S9), TA1535 (+/-S9), TA1537 (+/-S9) and E. coli WP2 (+S9) and thus, the concentrations used for experiment 1b were adjusted as follows:
TA98 (+S9 and -S9), TA100 (-S9), TA1535 (+S9 und -S9), and E. coli WP2 (+S9): 5; 1.5; 0.5; 0.15; 0.05; 0.015; 0.005 µL/plate,
TA100 (+S9): 1.5; 0.5; 0.15; 0.05; 0.015; 0.005; 0.0015 µL/plate,
TA1537(+S9 und and -S9): 5; 1.5; 0.5; 0.15; 0.05; 0.015; 0.005; 0.0015; 0.0005; 0.00015 µL/plate.
The positive control 2-amino-anthracene of the strain E. coli WP2 was invalid and the spontaneous revertants of the solvent controls demin. water and DMSO were too high compared with the historical control data. Therefore, the experiment 1b was repeated for E. coli with metabolic activation in experiment 1c. However, experiment 1c was invalid because the spontaneous revertants of the solvent controls demin. water and DMSO were too high compared with the historical control data. Hence, the experiment 1c was repeated for E. coli with metabolic activation in experiment 1d, with a new batch of E. coli lyophilizate. The following nominal test item concentrations were prepared for experiment 1d: E. coli WP2 (+S9): 5; 1.5; 0.5; 0.15; 0.05; 0.015; 0.005 µL test item/plate.
Vehicle / solvent:
In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 mL/L in demineralized (demin.) water and dimethyl sulfoxide (DMSO). The liquid test item wass sufficiently soluble in DMSO, only. Based on the non-GLP pre-test, DMSO was chosen as solvent, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations. A stock solution containing 50 mL/L of the test item in DMSO was prepared for each experiment.
Controls
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylene diamine, benzo-a-pyrene, 2-amino-anthracene, sodium acide
Details on test system and experimental conditions:
Culture of Bacteria: On the day before the start of each experiment, a nutrient broth (Oxoid nutrient broth no. 2) was inoculated with one lyophilizate per strain at 4:00 pm. These overnight cultures were placed in the heating chamber at 37 ±1 °C for 16 hours. For the last two hours the overnight cultures were shaken on an orbital shaker (200 rpm Exp. 1 and 1b and 150 rpm Exp. 1d) at 37 ±1 °C. Afterwards, the overnight cultures were ready for use in the experiment.
During the test, the overnight cultures were stored at room temperature (20 ±5 °C) as to prevent changes in the titre.
Conduct of Experiment: Different media and solutions were prepared preliminary. On the day of the test, the bacteria cultures were checked for growth visually. The incubation chambers were heated to 37 ±1 °C. The water bath was turned to 43 ±1 °C. The table surface was disinfected. The S9-mix was freshly prepared and stored at 0 °C.
Genotype Confirmation: Confirmation of genotype is performed for each batch of lyophilized bacteria by the supplier Trinova BioChem GmbH. The batches used of lyophilized bacteria met the criteria.
Spontaneous Revertants: The number of spontaneous revertants was determined for each solvent, used in the test by investigating three replicates with and without metabolic activation, incubation for 48 hours at 37 ±1 °C for each strain.
Determination of Titre: The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. It should give a density of 10⁹ cells/mL (at the least). Two replicates with and without metabolic activation, incubation for 48 hours at 37 ±1 °C.
Toxicity Control: Performed in experiment 1 only and analogously to the titre control with the maximum dose of the test item on maximal-soft agar. Two replicates with and without metabolic activation, incubation for 48 hours at 37 ±1 °C.
Sterility Control: Performed analogously to the test item but with solvent only and S9 (without adding bacteria) on top agar. Four replicates, incubation for 48 hours at 37 ±1 °C.
Solubility: Plates were checked for precipitation of test item at the end of the incubation by visual inspection.
Positive Controls: Using diagnostic mutagens. The stock solutions of the substances were diluted to achieve an application volume of 0.1 mL/plate. Three replicates with and without metabolic activation, incubation for 48 hours at 37 ±1 °C.
Per bacteria strain and concentration, three plates with (+S9) and three plates without metabolic activation (-S9) were used. The test item solutions were prepared as described above. For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ±1 °C.
Plate incorporation method: The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
500 µL S9-mix or phosphate buffer (for test without metabolic activation).
100 µL bacteria suspension
2000 µL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.
Pre-incubation method: The following materials were gently vortexed in a test tube and incubated at 37 ±1 °C for 20 minutes:
100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
500 µL S9-mix or phosphate buffer (for test without metabolic activation).
100 µL bacteria suspension
After the pre-incubation for 20 minutes, 2000 µL top agar was added and the tube was gently slewed. The mixture was poured onto the selective agar plate. The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ±1 °C.
Evaluation criteria:
The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
Five different analysable and non-toxic concentrations should be used for the evaluation of the mutagenic potential of the test item. A substance is considered to be mutagenic, if a reproducible increase with or without metabolic activation of revertant colonies per plate exceeding an increase factor of 2 for the bacteria strains TA98, TA100, TA102, TA1535 and TA1537 compared to vehicle controls in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity. A substance is not mutagenic if it does not meet these criteria. If the criteria listed above are not clearly met, the results will be assessed as equivocal and will be discussed. Tables of results and, if applicable, graphs of the values are included in this final report. It is stated, at which concentration (µL test item/plate) mutagenicity could be observed. The lowest concentration which showed mutagenicity is decisive for the assessment of the test item.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
no genotoxicity was seen but only 3 (-S9) and 4 (+S9) non-cytotoxic conc. were available for assessment
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 0.15 µL/plate (-S9) and 1.5 µL/plate (+S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5 µL/plate (+S9) and 1.5 µL/plate (-S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1.5 µL/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
not determined
Remarks:
unlikely, but due to very low cytotoxicity 5 conc. without cyctotoxicity could not be assessed
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 0.0005 µL/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1.5 µL test item/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
with
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Experiment 1
Confirmation of the Criteria and Validity
All strains met the criterion of at least 10⁹ bacteria/mL (correlating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and nearly all were within the historical control data ranges. For TA1537 without metabolic activation, the number of revertants of the positive control 4- Nitro-1,2-phenylene diamine was slightly out of the historical range, but showed a clearly positive response.
Solubility and Toxicity
In experiment 1, the test item showed no precipitates on the plates in all tested concentrations and the bacterial background lawn was visible and not affected.
However, probable toxicity could be observed, as the number of revertant colonies was reduced for S. typhimurium TA98 (+/-S9), TA100 (+S9), TA1535 (+S9), TA1537 (+/-S9) and E. coli WP2 (+S9) at the highest test item concentration (5 µL/plate). A reduction in colony number was also seen at 1.5 µL/plate for S. typhimurium TA98 (-S9), TA100 (+S9) and TA1537 (+/-S9).
On the plates with the lower test item concentrations a reduction of revertant colonies was observed for TA98 (-S9) at 0.5 and 0.15 µL/plate, for TA1537 (-S9) at 0.5; 0.15 and 0.05 µL/plate and for TA1537 (+S9) at 0.5 and 0.05 µL/plate.
Mutagenicity
A significant increase of the number of revertant colonies in the treatments with TA1535 in the presence of metabolic activation could be observed in the test item concentrations 0.5 µL/plate, 0.15 µL/plate and 0.05 µL/plate. A tendency of a concentration-related increase over the tested range was found with the highest f(I) value at 0.15 µL/plate.
To verify this result and to achieve sufficient non-toxic concentrations for the bacteria strains with toxic concentrations, a further experiment was performed.
The mean values of revertants of the three replicates of the controls and the test item are shown in Table 1 a.

Experiment 1b
Confirmation of the Criteria and Validity
All strains met the criterion of at least 10⁹ bacteria/mL (correlating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. Nearly all determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and all were within the historical control data ranges. The mean revertant value for the solvent control demin. water for TA100 (+S9) was slightly out of the range.

Solubility and Toxicity
In experiment 1b, the test item showed no precipitates on the plates in all tested concentrations. At a test item concentration of 5 µL/plate the number of revertants was decreased and the bacterial background lawn was absent or only slightly visible for TA98 (+/-S9), TA1535 (+S9) and TA1537 (+/-S9). For TA100 (-S9) the number of revertants is slightly reduced at 5 µL test item/plate but the background lawn is visible.
At a concentration of 1.5 µL test item/plate the number of revertant colonies was clearly reduced for TA98 (+S9), TA100 (+S9) and TA1537 (+/-S9). For TA98 (+S9), TA100 (+S9) and TA1537 (-S9) the background lawn was not affected, for TA1537 (+S9) it was not visible. On the plates with the lower test item concentrations a clear reduction of revertant colonies was observed for TA98 (-S9) at 0.5 and 0.15 µL/plate, for TA98 (+S9) at 0.5 and 0.015 µL/plate. For the strain TA1537 (+S9) a clear reduction was observed at a concentration of 0.5; 0.05 and 0.0005 µL/plate.
For TA100 (+/-S9) and TA1535 (+/-S9) the number of revertant colonies was not reduced in a test item concentration lower than 1.5 µL/plate.
Mutagenicity
A significant increase of the number of revertant colonies in the treatments with TA1535 in the presence of metabolic activation could be observed in the test item concentrations 0.5 µL/plate, 0.15 µL/plate, 0.05 µL/plate, 0.015 µL/plate and 0.005 µL/plate. A concentration-related increase over the tested range was found with the highest f(I) value at 0.15 µL/plate. In the treatment with TA1537, the number of revertants was clearly increased at a concentration of 0.005 µL/plate, but in absence of significant increases in the next higher and next lower concentration, this finding is considered incidental. Therefore, due to findings with TA1535 the test item is stated as mutagenic under the conditions of this experiment.
To achieve a valid experiment for E. coli with sufficient non-toxic concentrations a further experiment was performed (experiment 1c).
The mean values of revertans of the controls and the test item are shown in Table 2 a.

Experiment 1c and 1d
Confirmation of the Criteria and Validity
The E. coli WP2 strain met the criterion of at least 10⁹ bacteria/mL (correlating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. The determined values for the spontaneous revertants of the negative control in Experiment 1c were outside of the normal range of the test laboratory. Thus, the experiment was performed with a new batch of bacteria (Experiment 1d) that shows a low number of colonies in principal, what could be reproduced in numerous non-GLP pre-tests.
The positive control (diagnostic mutagen) showed a clearly mutagenic effect with metabolic activation but was not within the historical control data ranges, resulting from the low spontaneous revertants in DMSO.
Solubility and Toxicity
In experiment 1d, the test item showed no precipitates on the plates in all tested concentrations and the bacterial background lawn was visible and not affected. No reduction in the number of revertant colonies compared with the negative control DMSO could be observed.
Mutagenicity
A significant increase in the number of revertant colonies in the treatments with E. coli WP2 in the presence of metabolic activation could be observed in the test item concentrations 1.5 µL/plate, 0.5 µL/plate and 0.15 µL/plate. A tendency of a concentration-related increase over the tested range was found with the highest f(I) value at 0.5 µL/plate. To what extent this effect is only due to the low number of spontaneous revertants or whether mutagenicity is actually present cannot be determined with certainty, since a comparison of the new batch with the historical data may not be meaningful. Therefore, the result of experiment 1d is not assessed as clearly positive but considered as inconclusive.
The mean values of revertants of the controls and the test item are shown in Table 3 a.

CONCLUSION
Mutagenicity of Test item
The study was performed with the plate incorporation (experiments 1, 1b and 1d) in the absence and presence of a metabolic activation system (S9). Under these conditions the influence of the test item on bacterial test strains was evaluated. The test item 1,3-diiodopropane showed a relevant increase in the number of revertants in the Salmonella typhimurium test strain TA1535 with metabolic activation in the experiments 1 and 1b.
Based on the results of this study it is concluded that the test item 1,3-diiodopropane is mutagenic in at least the Salmonella typhimurium strain TA1535 in the presence of metabolic activation under the experimental conditions of the present study.

VALIDITY
Nearly all negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
All positive controls showed f(I) values > 2 which demonstrated the mutagenic potential of the diagnostic mutagens.
In experiment 1 the mean revertant value of the positive control 4-nitro-1,2-phenylene diamine (for TA1537, -S9) was slightly out of the historical range, but shows a clearly positive response. The mean revertant value for the solvent control demin. water for TA100 (+S9) was also slightly out of the range.
As the results for E. coli WP2 in experiment 1b and 1c were not considered valid an experiment 1d was performed with a new batch of E. coli WP2 which showed a lower number of revertants. As a result, the values were not inside of the historical control data range but were confirmed in several non-GLP pre-tests and by the supplier of the lyophilizates.
The confirmation tests of the genotype performed by Trinova BioChem GmbH did not show any irregularities. The control of the titre was above the demanded value of 10⁹ bacteria/mL. In the sterility control no growth of bacteria could be detected. Since all criteria for acceptability have been met, the study is considered valid.

DISCUSSION
In all experiments, no precipitation of the test item 1,3-diiodopropane was observed at any of the tested concentrations up to 5 µL/plate. The tested strains showed different behavior after incubation with the test item
S. typhimurium TA98: In experiment 1 the test item showed toxicity towards the strain TA98 as the number of revertant colonies was reduced at the highest test item concentration (5 µL/plate) with and without metabolic activation. Without metabolic activation, a reduction could also be observed at 1.5; 0.5 and 0.15 µL/plate. Therefore, the required five non-toxic concentrations for evaluation could not be achieved. In the repetition (experiment 1b), probable toxicity with reduced revertant numbers was observed also in lower test item concentrations, at 5; 0.5, 0.15 µL/plate without metabolic activation and at 5; 1.5; 0.5, 0.015 µL /plate with metabolic activation, respectively. As this is no clear toxicity and without a dose dependency, this effect might be caused by a variation in bacterial growth, not allowing to determine a lowest toxic concentration. Thus, under the conditions of the study, not enough concentrations (according to guideline 5 non-toxic concentrations below the lowest toxic concentration) could be evaluated for mutagenicity.
S. typhimurium TA100: In experiment 1, toxicity towards TA100 in the approach with metabolic activation could be observed at 5 and 1.5 µL test item/plate. Without metabolic activation no toxicity could be observed in the main experiment, but as the cytotoxicity control shows a probable effect, both approaches were repeated in experiment 1b. In experiment 1b, toxic effects could be observed at a test item concentration of 5 µL without metabolic activation and at 1.5 µL with metabolic activation. Sufficient non-toxic concentrations were evaluated and it could be stated, that the test item has no mutagenic effect towards the S. typhimurium strain TA100 with and without metabolic activation.
S. typhimurium TA102: The test item showed no cytotoxicity towards the strain TA102 with and without metabolic activation in experiment 1. Therefore, five test item concentrations were evaluated in experiment 1 and no mutagenic effects could be observed.
S. typhimurium TA1535: In experiment 1, no cytotoxic effects towards TA1535 without metabolic activations could be observed in the main test, although the toxicity control showed signs for probable cytotoxicity. At a test item concentration of 1.5 µL/plate, the number of revertants was increased, but not exceeding a f(I) value of 2.
With metabolic activation no colonies were observed at a concentration of 5 µL test item/plate, providing a sign for toxicity. A significant increase of the number of revertant colonies in the treatments with TA1535 in the presence of metabolic activation could be observed in the test item concentrations 0.5 µL/plate, 0.15 µL/plate and 0.05 µL/plate. A tendency of a concentration-related increase over the tested range was found with the highest f(I) value at 0.15 µL/plate. To verify this result and to achieve sufficient non-toxic concentrations for evaluation of mutagenicity, a further experiment was performed with and without metabolic activation.
In experiment 1b, the toxicity without metabolic activation was not verified, sufficient test item concentrations were evaluated and showed no mutagenic effects.
With metabolic activation, the results of experiment 1 could be verified. At 5 µL test item/plate, observation of no colonies were evidence for toxicity. At a test item concentration of 1.5 µL/plate, the f(I) value was nearly one, because toxic and mutagenic effects interfere with each other. From 0.5 µL to 0.005 µL test item/plate the number of revertants was clearly increased. A concentration-related increase over the tested range was found with the highest f(I) value at 0.15 µL/plate. At a test concentration of 0.5 µL/plate, the number of revertants was lower, because toxic effects still reduced the mutagenic effect.
The experiment clearly showed a positive result and thus, the mutagenic effects from experiment 1 were reproducible; the number of revertants showed a concentration related increase and were clearly higher than the historical control data of the solvent control DMSO.
S. typhimurium TA1537: In experiment 1, the test item showed toxic effects at 5; 1.5; 0.5; 0.15 and 0.05 µL/plate without metabolic activation and at 5; 1.5; 0.5; 0.5 and 0.05 µL/plate with metabolic activation.
Therefore, the required five non-toxic concentrations for evaluation of mutagenicity could not be achieved. As there was no definite lowest toxicity concentration and no dose dependency, the repetition of the experiment (experiment 1b) was performed with the same concentrations and additionally five lower concentrations.
In experiment 1b, toxic effects could be observed in the two highest concentrations (5 and 1.5 µL/plate) without metabolic activation. At 0.5 and 0.15 µL/plate the f(I) value was exactly 0.5; therefore, there was a growth reduction but no definite toxicity and, sufficient concentrations could be evaluated. At a test item concentration of 0.005 µL/plate, the number of revertants was increased with an f(I) value exceeding 2, but the number of revertants was not higher than the historical control data of the solvent DMSO and may to be due to normal variation in bacterial growth. With metabolic activation, in experiment 1b the number of revertants was clearly reduced at 5; 1.5; 0.5, 0.05 and 0.0005 µL/plate. As the number of revertants of TA1537 is really low in general and there was no dose dependency of toxic effects, this might also be due to normal variation in bacterial growth. As there were not enough clearly non-toxic concentrations no evaluation of the mutagenic potential could be made according to OECD 471 with TA1537 (+S9).
E. coli WP2: Experiment 1 was valid for the approach without metabolic activation. In the absence of cytotoxicity, five concentrations were available for assessment of mutagenicity, all showing f(I) values lower than 2. Thus, the test substance is considered non mutagenic to E. coli without metabolic activation.
With metabolic activation, the number of revertants was reduced in the highest test item concentration (5 µL/plate) in experiment 1. Therefore, repetitions were performed in experiments 1b and 1c but both repetitions were invalid. Thus, in experiment 1d, a new batch of E. coli Lyophilizate was used.
The new batch showed a low number of colonies, compared to the batch used for experiment 1, but this could be reproduced and verified in numerous non-GLP pre-tests. As a consequence, the number of spontanous revertants of the solvent controls demin. water and DMSO and also the revertants of the positive control 2-Amino-anthracene were much lower than the historical control data. Therefore, the f(I) value seemed to be increased for the test item at a concentration of 1.5; 0.5 and 0.05 µL/plate. As the significant increase probably is a consequence of the low spontaneous revertant rates and the f(I) values were rather low (2.36, 2.39 and 2.19, respectively), this cannot be stated as a clear mutagenic effect.

In summary, the approaches with TA100 (+/-S9), TA102 (+/-S9), TA1535 (+/-S9), TA1537(-S9) and E. coli WP2 (-S9) could be evaluated with sufficient non-toxic test item concentrations. Only the S. typhimurium strain TA1535 (+S9) showed a reproducible significant dose-related increase in revertants. According to the guideline OECD 471, the criteria for a positive result needs to be fulfilled for at least one strain.
Therefore, the result of the study is clearly positive by definition, even if not enough test item concentrations could be evaluated for TA98 (+/-S9), TA1537 (+S9) and E. coli WP2 (+S9).

DEVIATIONS
The following deviation was observed: For the strains TA98 (+/-S9), TA1537 (+S9) and E. coli WP2 (+S9) the required non-toxic concentrations were not sufficient for evaluation of mutagenicity and a clear result could not be derived. However, TA1535 (+S9) showed a reproducible significant dose-related increase. According to the guideline OECD 471, the criteria for a positive result needs to be fulfilled for at least one strain. Therefore, the result of the study is clearly positive by definition. The deviation was assessed and signed by the study director on 05. Oct. 2020.

Any other information on results incl. tables

Table 1 a Mean Revertants Experiment 1

Strain

S. typhimurium

E. coli WP2

TA98

TA100

TA102

TA1535

TA1537

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin. water

Mean

14

19

79

76

328

328

11

8

7

7

95

109

sd

0.0

2.1

2.3

6.9

8.0

16.0

1.5

1.5

1.5

1.2

6.1

4.6

DMSO

Mean

15

13

77

79

323

323

8

8

7

7

105

109

sd

1.0

2.0

6.1

8.3

9.2

12.2

0.6

1.0

1.0

1.5

2.3

9.2

Positive Controls*

Mean

s.g.

120

s.g.

s.g.

816

s.g.

s.g.

101

61

129

s.g.

s.g.

sd

n.c.

4.0

n.c.

n.c.

16.0

n.c.

n.c.

3.1

2.1

8.3

n.c.

n.c.

f(I)

> 2

9.23

> 2

> 2

2.53

> 2

> 3

12.63

8.71

18.43

> 2

> 2

5 µL/plate

Mean

8

3

55

0

341

227

10

0

1

0

128

45

sd

1.0

1.2

5.0

0.0

28.1

20.1

5.0

0.0

0.6

0.0

12.0

9.5

f(I)

0.53

0.23

0.71

0.00

1.06

0.70

1.25

0.00

0.14

0.00

1.22

0.41

1.5 µL/plate

Mean

6

8

56

9

317

307

14

6

3

0

124

66

sd

1.0

1.2

6.0

9.5

4.6

9.2

3.6

2.0

2.6

0.0

4.0

1.5

f(I)

0.40

0.62

0.73

0.11

0.98

0.95

1.75

0.75

0.43

0.00

1.18

0.61

0.5 µL/plate

Mean

8

16

63

61

325

320

9

26

0

3

129

125

sd

0.0

2.1

1.2

4.2

9.2

8.0

1.7

3.5

0.6

0.6

2.3

6.1

f(I)

0.53

1.23

0.82

0.77

1.01

0.99

1.13

3.25

0.00

0.43

1.23

1.15

0.15 µL/plate

Mean

7

15

56

54

317

317

12

27

2

5

124

129

sd

2.1

2.0

6.0

4.0

16.7

16.7

3.8

2.6

2.0

0.6

4.0

2.3

f(I)

0.47

1.15

0.73

0.68

0.98

0.98

1.50

3.38

0.29

0.71

1.18

1.18

0.05 µL/plate

Mean

10

14

65

70

317

328

8

22

3

2

144

125

sd

1.5

1.2

6.1

3.5

12.2

13.9

2.1

0.6

1.0

1.2

8.0

8.3

f(I)

0.67

1.08

0.84

0.89

0.98

1.02

1.00

2.75

0.43

0.29

1.37

1.15

* Different positive controls were used, see chapter 6.2.4, page 15

sd = standard deviation ±

s.g. = strong growth, too strong for counting of revertants

n.c. = not calculable

f(I) = increase factor

 

 

Table 2 a Mean Revertants in Experiment 1b

Strain

S. typhimurium

E. coli WP2

TA98

TA100

TA102

TA1535

TA1537

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin. water

Mean

13

12

56

47

--

--

9

8

6

4

--

iv

sd

3.8

2.6

9.5

3.5

--

--

1.0

1.0

0.6

0.6

--

iv

DMSO

Mean

11

15

58

53

--

--

8

7

4

6

--

iv

sd

2.3

4.6

6.1

6.7

--

--

1.5

1.2

1.0

1.5

--

iv

Positive Controls*

Mean

1001

133

523

597

--

--

403

113

138

121

--

iv

sd

0.0

6.1

16.7

45.5

--

--

20.1

6.1

24.2

16.0

--

iv

f(I)

91.00

8.87

9.34

11.26

--

--

44.78

16.14

34.50

20.17

--

iv

5 µL/plate

Mean

5

0

25

--

--

--

7

0

1

0

--

iv

sd

2.0

0.0

6.1

--

--

--

0.6

0.0

1.2

0.0

--

iv

f(I)

0.45

0.00

0.43

--

--

--

0.88

0.00

0.25

0.00

--

iv

1.5 µL/plate

Mean

6

1

39

0

--

--

8

8

1

1

--

iv

sd

2.3

0.6

2.5

0.6

--

--

1.0

1.7

1.0

1.2

--

iv

f(I)

0.55

0.07

0.67

0.00

--

--

1.00

1.14

0.25

0.17

--

iv

0.5 µL/plate

Mean

3

7

54

39

--

--

10

31

2

1

--

iv

sd

0.6

1.5

9.0

7.6

--

--

1.0

1.5

1.0

0.6

--

iv

f(I)

0.27

0.47

0.93

0.74

--

--

1.25

4.43

0.50

0.17

--

iv

0.15 µL/plate

Mean

5

13

47

55

--

--

9

54

2

4

--

iv

sd

1.5

1.2

7.0

1.5

--

--

1.2

1.5

1.5

1.5

--

iv

f(I)

0.45

0.87

0.81

1.04

--

--

1.13

7.71

0.50

0.67

--

iv

0.05 µL/plate

Mean

6

11

62

52

--

--

10

41

3

2

--

iv

sd

1.5

3.8

10.6

11.5

--

--

1.2

1.2

3.2

1.0

--

iv

f(I)

0.55

0.73

1.07

0.98

--

--

1.25

5.86

0.75

0.33

--

iv

0.015 µL/plate

Mean

9

6

67

52

--

--

9

39

4

4

--

iv

sd

1.0

1.2

3.8

2.1

--

--

1.0

1.5

0.6

3.5

--

iv

f(I)

0.82

0.40

1.16

0.98

--

--

1.13

5.57

1.00

0.67

--

iv

0.005 µL/plate

Mean

7

10

52

52

--

--

9

14

9

3

--

iv

sd

2.5

2.6

7.6

1.5

--

--

1.5

1.7

2.6

1.5

--

iv

f(I)

0.64

0.67

0.90

0.98

--

--

1.13

2.00

2.25

0.50

--

iv

0.0015 µL/plate

Mean

--

--

--

59

--

--

--

--

6

3

--

iv

sd

--

--

--

4.6

--

--

--

--

2.6

1.2

--

iv

f(I)

--

--

--

1.11

--

--

--

--

1.50

0.50

--

iv

0.0005 µL/plate

Mean

--

--

--

--

--

--

--

--

4

2

--

iv

sd

--

--

--

--

--

--

--

--

1.2

2.0

--

iv

f(I)

--

--

--

--

--

--

--

--

1.00

0.33

--

iv

0.00015 µL/plate

Mean

--

--

--

--

--

--

--

--

2

6

--

iv

sd

--

--

--

--

--

--

--

--

1.0

2.6

--

iv

f(I)

--

--

--

--

--

--

--

--

0.50

1.00

--

iv

* Different positive controls were used, see chapter 6.2.4, page 15

sd = standard deviation ±

s.g. = strong growth, too strong for counting of revertants

n.c. = not calculable             iv = invalid              -- = not tested

f(I) = increase factor, calculation see chapter 7.4, page 23

 

 

Table 3 a Mean Revertants in Experiment 1d

Strain

E. coli WP2

Induction

-S9

+S9

Demin. water

Mean

--

39

sd

--

3.2

DMSO

Mean

--

36

sd

--

4.4

Positive Controls*

Mean

--

164

sd

--

4.0

f(I)

--

4.56

5 µL/plate

Mean

--

34

sd

--

1.7

f(I)

--

0.94

1.5 µL/plate

Mean

--

85

sd

--

2.3

f(I)

--

2.36

0.5 µL/plate

Mean

--

86

sd

--

7.2

f(I)

--

2.39

0.15 µL/plate

Mean

--

79

sd

--

7.6

f(I)

--

2.19

0.05 µL/plate

Mean

--

37

sd

--

3.6

f(I)

--

1.03

0.015 µL/plate

Mean

--

36

sd

--

1.0

f(I)

--

1.00

0.005 µL/plate

Mean

--

36

sd

--

1.5

f(I)

--

1.00

* Different positive controls were used, see chapter 6.2.4, page 15

sd = standard deviation ±

s.g. = strong growth, too strong for counting of revertants

n.c. = not calculable

-- = not tested

f(I) = increase factor

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that 1,3-diiodopropane formally according to guideline is mutagenic in the Salmonella typhimurium strain TA1535 in the presence of metabolic activation under the experimental conditions in this study. Even if not for all strains sufficient non-toxic concentrations could be evaluated for mutagenicity, the increase of revertants in TA1535 in the presence of metabolic activation was reproducible and dose-related in tendence.
Executive summary:

The study procedures described in this report were based on the most recent Guideline OECD 471 and EU Method B.13/14. The test item 1,3-diiodopropane was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537) and Escherichia coli WP2.

The test was performed in four experiments in the presence and absence of metabolic activation, with +S9 standing for the presence of a metabolic activation, and -S9 standing for absence of metabolic activation.

The initial experiment (Exp.1) was repeated to achieve five analyzable non-toxic concentrations (Exp. 1b). Experiment 1b was invalid for the strain E. coli WP2 with metabolic activation and needed to be repeated in Exp. 1c. Experiment 1c was invalid too and repeated in Exp. 1d. The invalid experiments were not reported in this report, but the raw data are kept in the test facility in the GLP-archive.

 

Experiment 1: In the first experiment, the test item (dissolved in Dimethyl sulfoxide, DMSO) was tested up to concentrations of 5 µL/plate in the absence and presence of S9 mix in the strains S. typhimurium TA98, TA100, TA102, TA1535 and TA1537 and E.coli WP2 using the plate incorporation method.

All negative and positive controls were valid.

The test item showed cytotoxic effects towards S. tyhimurium TA98 (+/-S9), TA100 (+/-S9), TA1535 (+/-S9), TA1537 (+/-S9) and E. coli WP2 (+S9).

For TA1535 with metabolic activation, an increase in the number of revertants (f(I) value > 2) was observed for a test item concentration of 0.5; 0.15 and 0.05 µL/plate. A tendency of a dose-related increase of revertants was visible.

 

Experiment 1b:

Based on the toxicity results of the experiment 1, the experiment was repeated under the same conditions with additional lower concentrations.

- TA98 (+S9 and -S9), TA100 (-S9), TA1535 (+S9 und -S9), and E. coli WP2 (+S9): 5; 1.5; 0.5; 0.15; 0.05; 0.015; 0.005 µL/plate

- TA100 (+S9): 1.5; 0.5; 0.15; 0.05; 0.015; 0.005; 0.0015 µL/plate

- TA1537(+S9 und -S9): 5; 1.5; 0.5; 0.15; 0.05; 0.015; 0.005; 0.0015; 0.0005; 0.00015 µL/plate

All negative and positive controls were valid.

The test item showed cytotoxic effects towards S. tyhimurium TA98 (+/-S9), TA100 (+/-S9), TA1535 (+S9), TA1537 (+/-S9) and E.coli WP2 (+S9).

For TA1535 with metabolic activation, an increase in the number of revertants (f(I) value > 2) was observed for a test item concentration of 0.5; 0.15, 0.05, 0.015 and 0.005 µL/plate. Additionally, an increase in the number of revertants was observed for TA1537 without metabolic activation at a test item concentration of 0.005 µL/plate.

For TA98 (+/-S9) and TA1537 (+S9) not enough non-toxic concentrations could be evaluated.

The positive control 2-amino-anthracene of the strain E. coli WP2 was invalid and the spontaneous revertants of the solvent controls demin. water and DMSO were too high compared with the historical control data.

The experiment 1b was repeated for E. coli with metabolic activation in experiment 1c.

 

Experiment 1c:

Experiment 1c was performed for E. coli WP2 with metabolic activation but was invalid because the spontaneous revertants of the solvent controls demin. water and DMSO were too high compared with the historical control data.

Therefore, the experiment 1c was repeated for E. coli with metabolic activation in experiment 1d.

 

Experiment 1d:

Experiment 1d was performed for E. coli WP2 with metabolic activation. A new batch of E. coli lyophilizate was used. The new batch generally shows a low number of colonies, but this finding could be reproduced in numerous non-GLP pre-tests.

Both the results in spontaneous revertants of the solvent controls demin. water and DMSO and the revertants of the positive control 2-Amino-anthracene were much lower than the historical control data. As a result, the f(I) value seemed to be increase for the test item at a concentration of 1.5; 0.5 and 0.05 µL/plate. To what extent this effect is only due to the low number of spontaneous revertants in the solvent control or whether mutagenicity is actually present cannot be determined with certainty, since a comparison of the new batch with the historical data may not be meaningful.

General remarks: Particularly the strains TA1535, TA1537 and E.coli WPA showed cytotoxicity at low concentrations, presumably caused by iodine release in the presence of S9 metabolic activation. Thus, it was very difficult to achieve five consecutive non-toxic concentrations for evaluation of mutagenicity and it appears likely that this secondary effect lead to the deviations observed. Thus, the results from this Bacterial Reverse Mutation Test should be interpreted with caution.