Diquat dibromide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames, +S9 negative, -S9 negative, S. typhimurium: TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA pKM101, OECD 471, Callander 1986

- In vitro chromosome aberration study in mammalian cells, +S9 positive (at cytotoxic concentrations), -S9 equivocal, Human lymphocytes, OECD 473, Wildgoose 1986

- In vitro mammalian cell gene mutation assay (MLA), +S9 negative, -S9 negative, L5178Y mouse lymphoma cells, OECD 476, Cross 1986a

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 Dec 1985 to 20 Dec 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2000

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his- (S. typhimurium), trp- (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: induced rat liver S9 from six male rats dosed with Aroclor 1254.
- method of preparation of S9 mix: After sacrifice, each animal was dissected using sterile instruments to expose the liver. The liver was removed in lobes and placed in a beaker of ice-cold buffer (see below). After all the livers were removed, the pieces were washed in ice-cold buffer, then transferred to a pre-weighed beaker containing a measured volume of (ice-cold) buffer. The beaker was then reweighed and the liver weight calculated. The remaining volume of buffer to make a 25 % w/v homogenate was then measured out and stored on ice until required. Pieces of liver were then added, together with buffer from the beaker, to the pre-sterilised tube of a homogeniser, and homogenised with eight passes at 1070 rpm. The homogenate was poured into a sterile conical flask, and kept on ice until all the livers had been homongenised. The rest of the measured buffer was then added to the flask and the contents mixed by swirling. Equal volumes were then added to pairs of centrifuge tubes kept on ice. After being tightly capped the tubes were placed in a pre-cooled 8 x 50 mL rotor, and centrifuged at 9000 x g for 10 minutes in a refrigerated centrifuge with the temperature regulated at 4°C. The timing sequence was started once the rotor reached full speed. The resultant supernatants (S9 fraction) were combined and divided into 13 mL aliquots, rapidly frozen and stored at -70 °C. A 0.5 mL sample of the S9 fraction was added in duplicate to nutrient agar plates. The plates were incubated for 48 hours at 37 °C and assessed for contamination. When required, an aliquot of S9 fraction was thawed at room temperature until ice was no longer visible. It was then stored on ice until used.
- S9 Buffer: This was prepared as follows components with final concentrations: Sucrose 250 mM, Tris base 50 mM, EDTA tetrasodium salt (dihydrate) 1.0 mM. These were dissolved in approximately 4800 mL de-ionised H2O, the pH adjusted to 7.5 and the volume made up to 5000 mL. The solution was then stored at 4°C until required. Aliquots were sterilised by filtration through a 0.45 μm filter as needed.
- Co-factor Solution: The co-factor solution was made up from the following with their final concentration: 100 mM Na2HPO4, 33 mM KCl, 5 mM Glucose-6-Phosphate, 4 mM NADP (Na salt), 4 mM NADPH, 8 mM MgCl2. This was dissolved in 180 mL de-ionised H2O, the pH adjusted to 7.4 and the volume made up to 200 mL. The solution was then divided into 10 mL and 20 mL aliquots and stored at -20 °C as required. When required. sufficient aliquots were thawed by standing at room temperature until no ice was visible, sterilised by filtration through an 0.45 µm filter then stored on ice until used.

Test concentrations with justification for top dose:

All concentrations given as µg test substance cation
- Preliminary cytotoxicity assay: 10, 50, 100, 500, 1000, 5000 µg/plate
- Main assay (without metabolic activation): 0.1, 0.5, 1, 5, 10, 50 µg/plate, plus 0.01 and/or 0.05 µg/plate in repeat experiments
- Main assay (with metabolic activation): 0.5, 1, 5, 10, 50, 100 µg/plate, plus 0.01 and/or 0.05 µg/plate in repeat experiments

Vehicle / solvent:

- Solvent used: dionised water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

N-ethyl-N-nitro-N-nitrosoguanidine

other: 4-nitro-o-phenylenediamine (4-NOPD); 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2); N-Methyl-N´-nitro-N-nitrosoguanidine (MNNG)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

EXPERIMENTAL PERFORMANCE
The presence of the uvrB deletion (Salmonella) and the uvrA mutation (E.coli) were confirmed by testing the sensitivity of each culture to mitomycin C (10 µL of a 10 µg/mL solution) in the same manner as sensitivity to crystal violet was tested. Damage to DNA caused by mitomycin C is repaired in normal bacteria by the uvr excision repair pathway, and is thus toxic to strains deficient at either the uvrA or uvrB loci. When fresh frozen stocks were prepared (from the frozen permanent strains), the Salmonella strains were tested for histidine requirement and for reversion properties using diagnostic mutagens as described by Ames et al (1975) and Maron and Ames (1983), except that the mutagens were incorporated in the top agar layer as in a normal test (see below) rather than spot tested as stated by Ames. All experiments were conducted using frozen stock cultures prepared as above in October 1985 (S.typhimurium strains), and in November 1980 (E.Coli)

Post-mitrochondrial supernatant (S9-mix)
The variant of the routine Ames assay used for this work was conducted with and without S9-mix incorporated in the top-agar. The S9-mix was prepared as follows in expressed volumes per 30 mL S9-mix:
- 3 mL S9 fraction
- 7 mL Sucrose-Tris-EDTA Buffer
- 20 mL Co-factor solution
In tests without metabolic activation, the S9-mix was replaced by an equivalent volume of phosphate buffer. Both the S9-mix and the phosphate buffer were kept on ice until used. Unused S9-mix was discarded at the end of the day.

Agar Plates
9 cm diameter vented Petri-dishes pre-poured with Vogel Bonner minimal medium and containing 1.5 % w/v agar and 2 % w/v glucose. The plates were stored at ambient temperature until needed to dry them and ensure sterility.

TYPE OF BACTERIAL ASSAY
Standard plate test (with and without S9). Toxicity was seen in the first main experiment and so lower dose levels were assessed in Experiment 2. Due to contamination seen in this experiment, repeat data were generated for strain TA98 in a 3rd experiment.

PROTOCOL
Bacterial cultures were prepared from frozen stocks by incubating overnight at 37°C in a shaking incubator. The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 μL Test solution at each dose level, solvent and positive controls;
- 500 μL S9 mix or phosphate buffer;
- 2 mL Overlay agar containing 0.5 μM histidine / 0.5 μM biotin or 1.03 μM tryptophan, as appropriate.
The mixture was poured onto the surface of a prepared plate and allowed to gel. After the agar was set the plates were incubated upside down for 64 - 68 hours at 37 °C in the dark. For each strain and dose level, including the positive controls three plates were used. For negative controls 2 or 5 plates were used. Following incubation all plates were counted using an automated colony counter with the discriminators adjusted to a standard setting which only permits the counting of mutant colonies.

Evaluation criteria:

A positive response in a (valid) individual experiment is achieved when one or both of the following criteria are met:
- a significant, dose-related increase in the mean number of revertants is observed;
- a two-fold or greater increase in the mean number of revertant colonies (over that observed for the concurrent solvent control plates) is observed at one or more concentrations

A negative result in a (valid) individual experiment is achieved when:
- there is no significant dose-related increase in the mean number of revertant colonies per plate observed for the test substance; and
- in the absence of any such dose response, no increase in colony numbers is observed (at any test concentration) which exceeds 2x the concurrent solvent control.

For a positive response in an individual experiment to be considered indicative of an unequivocal positive, i.e. mutagenic, result for that strain/S9 combination, then the observed effect(s) must be consistently reproducible.

Statistics:

One-tailed Student’s t-test. The corresponding probability for each dose level was determined from a t-table using the appropriate degrees of freedom.

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

PRELIMINARY CYTOTOXICITY ASSAY
The test substance induced significant toxicity in strain TA100 (both with and without S9) at doses of 50 μg test substance cation/plate or greater. At 100 μg/plate, the observed toxicity was more extreme in the absence of S9 than in the presence of S9. The doses ranges selected for the first full experiment were therefore 100 – 0.5 μg test substance cation/plate (+S9) and 50 – 0.1 μg test substance cation/plate (-S9).

MUTAGENICITY ASSAY
In the first full experiment, the test substance did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the tester strains used, either in the presence or absence of S9-mix, at any sub-toxic dose. In view of the range of toxic effects seen in this experiment, the repeat experiment was conducted using a range of lower dose levels in the presence or absence of S9-mix (5.0 – 0.01 μg/plate (-S9) and 10 – 0.05 μg/plate(+S9). Contamination was seen with strain TA98 in the second experiment and so repeat data were generated for this strain in a third experiment. No significant increases in revertant colony numbers were observed in any strain, with significant toxicity being observed in (at least) the top dose tested in each case.

The positive controls for each experiment induced the expected responses, indicating the strains were responding satisfactorily in each case. Revertant colony numbers for the solvent control plates were within acceptable ranges.

Conclusions:

Under the conditions of this assay, the test substance gave an unequivocal negative, i.e. non-mutagenic response, in both the presence and absence of an auxiliary metabolising system (S9) in all five Salmonella typhimurium tester strains used (TA1535, TA1537, TA1538, TA98 and TA100), and also in one strain of Escherichia coli (WP2 uvrA pKM101).

Executive summary:

In a reverse gene mutation assay in bacteria, according to OECD TG 471 and in compliance with GLP, strains TA1535, TA1537, TA1538, TA98 and TA100 of S. typhimurium and WP2uvrA pkM101 of E. coli were exposed to the test substance at concentrations of 0.05, 0.1, 0.5, 1.0, 5.0, 10, 50, or 100 µg test substance cation/plate in the presence of mammalian metabolic activation (plate co-incubation) and at concentrations of 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10, or 50 µg test substance cation/plate in the absence of mammalian metabolic activation, S9 (plate co-incubation). The test substance was tested up to cytotoxic concentrations (up to 5000 µg/plate) in an initial dose-ranging study.

In two separate experiments, the compound did not induce any significant increase in the observed numbers of revertant colonies in any of the tester strains used, neither in the presence or absence of an auxiliary metabolising system (S9). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

In conclusion, the test substance gave an unequivocal negative, i.e. non-mutagenic, response in this assay.  

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Sep 1985 to 6 Dec 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1983

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Remarks:

Human

Details on mammalian cell type (if applicable):

- The lymphocytes were obtained on the days of culture initiation from healthy donors. Equal volumes of blood from 2 donors (male for Experiment 1 and female for Experiment 2). All donors had a previously established low incidence of chromosomal aberrations in their peripheral blood lymphocytes.
- At least 25 mL of blood was collected into lithium/heparin anticoagulant tubes and mixed well.
- At 0 hours, 0.5 mL whole blood was added to a sterile plastic bottle containing 9.5 mL of RPMI 1640 tissue culture medium supplemented with 10 % FCS, 0.1 mg/mL PHA and 100 units/mL penicillin and streptomycin. 100 cultures, (50 from each donor) were initiated in this way, and maintained at 37 °C.

Metabolic activation:

with and without

Metabolic activation system:

- S9 derived from induced, Aroclor 1254 rat liver.
- The metabolic activation system (S9-mix) was prepared as required (on each day of culture treatment) as a 1:1 mixture of S9 fraction and cofactor solution. The cofactor solution was prepared as a single stock solution of Na2HPO4 (150 mM), KCl (49.5 mM), glucose-6-phosphate (7.5 mM), NADP (Na salt) (6 mM) and MgCl2 (12 mM) in 1 litre of deionised water and adjusted to a final pH of 7.4.

Test concentrations with justification for top dose:

- Without S9 mix: Experiment 1: 26.7, 107.0, 267.4 and 534.8 µg/mL. And Experiment 2: 13.4, 53.5, 107.0, 267.4 and 534.8 µg/mL.
- With S9 mix: Experiment 1 and 2: 26.7, 107.0, 267.4 and 534.8 µg/mL.

Vehicle / solvent:

- Solvent used: physiological saline (0.85 %)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Test performance
-  Without activation: At 44 hours after culture initiation, the test sample of test substance was administered to duplicate cultures for Donors 1 and 2, at ten concentrations ranging from 5.3 - 5347.6 µg/mL test substance ion. The test sample was dissolved in physiological saline, and dosed in a volume of 100 µL. The highest dose level of this dose range was determined by the limit of solubility of the test sample in the dosing volume at room temperature. Solvent controls of 10 µL saline were also included. The final concentration of mitomycin C used in the culture medium was 0.5 µg/mL, dosed in a volume of 50 µL saline. In Experiment 3 the dose range was between 5.3 and 534.7 µg/mL the highest dose selected from the dose-ranging study, from which three appropriate dose levels were chosen for chromosomal analysis.
-      With activation: At 43.5 hours (prior to dosing at 44 hours), the test sample at dose levels ranging from 5.3 - 5347.6 µg/mL growth medium in 100 µL saline, and positive and negative controls were pre-incubated at 37 °C for 30 minutes with the auxiliary metabolic enzymes; 200 µL 1:1 S-9 and co-factor mix. The solvent control was 10 µL saline, the positive control 50 µg/mL cyclophosphamide, dosed in 100 µL saline. At the end of the 30 minute pre- incubation period (44 hours), the blood cultures were added to compound +S-9 mixtures and maintained at 37 °C for a further 3 hours.
In experiment 4 the dose range was between 5.3 and 534.7 µg/mL the highest dose selected from the dose-ranging study, from which three appropriate dose levels were chosen for cytogenetic analysis. In the case of Donor 2 an additional higher dose level was also included.
 
Exposure duration:
Exposure for the blood cultures, to the test sample and controls was 3 hours (44 - 47 hours) at 37 °C. At 47 hours the growth medium was removed by centrifugation at approximately 300 x g for 5 mins and replaced with fresh medium.
In experiment 4, exposure for all the blood cultures, to the test sample and controls was 44 hours 20 minutes – 47 hours 20 minutes. All cultures were maintained at 37 °C for the remainder of the 72-hour growth period.
Two hours prior to harvesting, the cultures were treated with colchicine at a final concentration of 10 µg/mL in each sample.
In experiment 3 colchicine treatment was at two and half hours prior to harvesting at 72 hours.
At 71 hours the cultures were transferred to 15 mL conical centrifuge tubes and spun at approximately 300 x g for 5 minutes. The blood cultures were then washed three times in HBSS to prevent any staining artefacts that may have been caused by the presence of residual compound. Colchicine was present at a concentration of 10 µg/mL throughout this procedure.
At 72 hours, the cultures were suspended in 5 mL of 0.075 M potassium chloride and mixed, the timing sequence was started at this point, a further 5 mL of KCl 0.075 M was then added. The cultures were allowed to stand at room temperature for 12 minutes.
After centrifugation at approximately 300 x g for 5 minutes the cultures were fixed in glacial acetic acid/methanol fixative (1 part:3 parts), added dropwise, this was then made up to a volume of 10 mL. After three subsequent changes of fixative, slides were prepared by dropping cell suspension on to clean, dry microscope slides. The slides were air dried. Four slides were prepared for each culture selected for analysis.
 
Analysis of metaphase cells:
The slides were stained in a 10 % solution of buffered Giemsa stain for 7 minutes, rinsed in distilled water, blotted dry and mounted in DPX. The mitotic index (MI), (the percentage of cells in c-metaphase) was determined for each culture selected for analysis. The mitotic index (MI), (the percentage of cells in c-metaphase) was determined for each culture selected for analysis. The slides were coded to avoid observer bias. Where possible, one hundred cells in metaphase were analysed from each blood culture for the incidence of chromosomal damage. With positive control cultures, only those numbers of cells sufficient to confirm a positive response were analysed.
Parameters include:
- No. of cells examined per dose: 100 (for positive controls only those numbers of cells sufficient to confirm a positive response were analysed)
- Scored for structural: Yes
- Scored for numerical: Yes (polyploidy noted if observed)
- Coded prior to analysis: Yes

Statistics:

The values obtained in all of the test groups were compared with the values from negative controls using the Fisher's Exact test one-sided.

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

Significant chromosome damaging effects were only observed in cultures which showed marked cytotoxicity, i.e. at dose levels which inhibited mitosis by approximately 40% or more.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Significant chromosome damaging effects were only observed in cultures which showed marked cytotoxicity, i.e. at dose levels which inhibited mitosis by approximately 40% or more.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

CYTOGENETIC ASSAY
Results for cultures with auxiliary metabolic activation: The highest dose level of test substance selected for cytogenetic analysis was the maximum tolerated dose (MTD) determined by its effect on mitosis. A concentration of test compound which inhibited cell division by approximately 50 - 80 % was considered appropriate, and in the case of Donor 1 this was 267.4 µg/mL test substance with 107.0 µg/mL being the MTD for Donor 2.
The dose levels of test substance chosen for cytogenetic analysis for Donor 1 were 267.4, 107.0 and 26.7 µg/mL, and for Donor 2 107.0, 53.5 and 13.4 µg/mL.
In the case of Donor 1, mitotis was inhibited by approximately 65 % at the MTD of test substance, and with Donor 2 mitosis was inhibited by approximately 53 % at the MTD.
Chromosome aberration levels were elevated to statistical significance of p< 0.01 in cultures from Donor 1 treated with 267.4 and 107.0 µg/mL test substance with no increase above solvent control values seen in cultures treated with 26.7 µg/mL. A slight increase above solvent control values was seen at the 107.0 µg/mL MTD in cultures from Donor 2, however, this increase was not statistically significant nor was it considered to be of biological importance. No increases in chromosomal aberration were observed in cultures from Donor 2 treated with 53.5 and 13.4 µg/mL.

Results for cultures with auxiliary metabolic activation:
- The highest dose level of test substance selected for this phase of the study was the MTD, which was 267.4 µg/mL in the case of Donor 1 and 534.8 µg/mL for Donor 2.
- The lower dose levels of test substance which were considered appropriate for cytogenetic analysis were 107.0 and 26.7 µg/mL for Donor 1 and 267.4, 107.0 and 26.7 µg/mL for Donor 2.
- In the case of Donor 1 mitosis was inhibited by approximately 55 % at the MTD of test substance, and with Donor 2 mitosis was inhibited by approximately 76 % at the MTD.

Chromosome aberration levels were elevated to statistical significance of p < 0.01 in cultures from both donors treated with 267.4 µg/mL test substance. A statistically significant difference of the same order was also observed at the 534.8 µg/mL dose level of test substance which was included for Donor 2. In cultures from Donor 1 treated with 107.0 µg/mL of the test sample chromosomal damage was statistically significantly increased above the solvent control values at the p < 0.05 level, at the 107.0 µg/mL dose in Donor 2 chromosome aberration levels were not increased. No other increases in chromosomal damage of either statistical significance or biological importance were seen in cultures treated with 26.7 µg/mL. The test system was shown to be sensitive as demonstrated by the response obtained with mitomycin C and cyclophosphamide respectively.

Table 1: Chromosomal Abnormalities and Mitotic Index

Treatment	With S9		Without S9
	Mean % of Abnormal Cells Excluding Gaps	Mean Mitotic Index (%)	Mean % of Abnormal Cells Excluding Gaps	Mean Mitotic Index (%)
Donor 1 Saline control  10 µL/ mL Cyclophosphamide 50 µg/mL Mitomycin C      0.5 µg/mL Test substance  as cation speices 534.8 µg/mL 267.4 µg/mL 107.0 µg/mL 26.7 µg/mL	2.5 68.75 -   Not analysed 14.79** 6.5* 3.5	11.0 3.0 -   <0.5 5.0 6.5 8.0	1.0 - -48.0   Not analysed 16.00** 6.0** 0.0	11.5 - 6.0   <0.5 4.0 7.0 9.5
Donor 2  Saline control  10µL/ mL Cyclophosphamide 50 µg/mL Mitomycin C      0.5 µg/mL Test substance as cation species 538.8 µg/mL 267.4 µg/mL 107.0 µg/mL 26.7 µg/mL 13.4 µg/mL	1.5 67.86 -      24.29** 7.38** 3.50 3.50 -	10.5 3.0 -   2.5 5.5 6.5 10.5 -	1.0 -   30.19   Not analysed Not analysed 3.94 0.45 0.50	9.5 - 6.0 0.0 1.5 4.5 7.5 10.0

**Statistically significantly different from solvent control values at the p < 0.01 level using Fisher’s Exact Test

*Statistically significantly different from solvent control values at the p < 0.05 level using Fisher’s Exact Test

Conclusions:

The test substance was found to induce chromosomal damage in the in vitro human lymphocyte cytogenetic assay, but only at dose levels where there were obvious cytotoxic effects.

Executive summary:

In a mammalian cell cytogenetics assay according to OECD TG 473 and in compliance with GLP, human primary lymphocyte cultures were exposed to test substance in physiological saline both in the presence and absence of auxiliary metabolic activation. The maximum dose level of test substance was different for each donor. In the instance of Donor 1, both with and without auxiliary metabolic activation the MTD of test substance was 267.4 µg test substance cation/mL. In the case of Donor 2 the MTD varied in each phase of the study; in the presence of auxiliary metabolic activation the highest dose level of test substance tested was 534.8 µg test substance cation/mL, with 107.0 µg test substance cation/mL being the highest in the absence of auxiliary metabolic activation.

Substantially elevated and statistically significant increases in chromosomal damage were observed in all cultures treated with the MTD of test substance with the exception of Donor 2 in the absence of auxiliary metabolic activation. Elevated and statistically significant increases in chromosomal damage were also observed in all cultures treated with approximate 1/2 MTD levels of test substance, with the exception of Donor 2 in the absence of S-9 mix. There were no statistically significant increases in chromosomal damage in any of the cultures treated with test substance at approximate 1/10 MTD levels or lower.

It is indicated that the clastogenic potential of the test substance is directly related to the cytotoxicity of the compound, as elevated levels of chromosomal damage were only observed at relatively high dose levels, where cytotoxicity was apparent, and by the absence of any clastogenic effect at approximate 1/10 MTD levels where cytotoxicity was minimal. This is also supported by the exceptional case of Donor 2 in the absence of S9-mix, where the cultures were more sensitive to cytotoxic effects and lower dose levels were tested, which did not lead to a clastogenic response. The test system was shown to be sensitive to chromosome damaging effects, by the response given to the positive control substances mitomycin C (a direct-acting clastogen) and cyclophosphamide (a clastogen requiring metabolic activation).

In conclusion, the test substance was found to induce chromosomal damage in the in vitro human lymphocyte cytogenetic assay at dose levels causing cytotoxic effects.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Jan 1986 to 19 May 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

August 1984

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

For cell lines:
- Absence of Mycoplasma contamination: Yes

MEDIA USED
The cells maintained in suspension culture and kept in exponential growth. For normal maintenance the culture medium used was RPMI 1640 supplemented with L-glutamine (4mM) and 200 units per cm of penicillin + streptomycin and 10% horse serum. The cultures were kept at 37* C under an atmosphere of 5% CO2 in air, either in a gassing incubator or in a hot room in roller bottles rotated on a roller apparatus.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: From the liver of Aroclor-1254-induced rat
- method of preparation of S9 mix: The co-factor solution was prepared as a stock solution of 4 mM NADP (disodium salt), 33 mM KCl, 5 mM glucose-6-phosphate (monosodium salt), 8 mM MgCl2 6H2O and 100 mM Na2HPO4 in RPMI 1640 culture medium with a final pH adjusted to 7.4. The co-factor solution was stored at -20°C until required.

Test concentrations with justification for top dose:

- Absence of S9 mix: 6.25, 12.5, 25, 50 and 100 µg test material/mL
- Presence of S9 mix: 3.125, 6.5, 12.5, 25 and 50 µg test amterial/mL

Vehicle / solvent:

- Solvent used: saline

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: N-nitrosodimethylamine

Details on test system and experimental conditions:

Cells were exposed to test compound, negative/solvent or positive controls for 2 hours in both the presence and absence of S9 mix.

After washing, cells were cultured for 72 hours (expression period) before cell selection.

Directly after treatment on day 0, samples of the post-treated samples were diluted to 8 cells/cm3 in 20% horse serum supplemented medium and then dispensed, at 200 µL/well, into 96 well plates. Cell growth in individual microwell plates was assessed using a dissecting microscope. The survival plates and viability plates were scored for the number of wells containing no cell growth (negative wells).

The remaining cells were cultured for sufficient time to allow newly induced mutations to be detected. At the end of the 72 hour expression time, samples were grown in both selective medium (TFT, 400 µg/mL) and non-selective medium and the results obtained used to determine the mutation frequency per viable cell.

Evaluation criteria:

See "Any other information on materials and methods incl. tables".

Statistics:

The data were considered by logit regression, using a complimentary log-log link function. The dependent variable was the number of empty wells. This procedure provided maximum likelihood estimates of log mutant frequencies. Variances were inflated by the between duplicate heterogeneity factor.

Intergroup comparisons of log mutant frequency comparing each treated group with the solvent control were performed within each experiment.

All tests were one-sided. Similar analyses were carried out separately for the positive controls.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
- Preliminary toxicity assay: In the dose range finding experiment (data not reported) zero survival was observed at the 200 µg/mL concentration in the presence of S9 mix, with little survival (4%) at this same level in the absence of S9 mix. In the two main assays reduced survival was observed at the 50 and 100 µg/mL top dose level, both with and without S9 mix respectively (1 and 2% survival (-S9), and, 5 and 5% survival (+S9 mix)). 25 and 50 µg/mL were found to be the highest dose levels examined that allowed the determination of a mutation frequency with and without S9 mix respectively.
- Survival (cytotoxicity): Dose-related cytotoxicity was achieved both in the presence and absence of auxiliary metabolic activation (S9 mix), over the 100-6.25 µg/mL range. Little difference in cytotoxicity was observed either in the presence or absence of S9 mix in two independent experiments. No metabolism of the test substance has been attributed to mammalian cells, therefore no major differences in the metabolic capabilities of these cultures either in the presence or absence of auxiliary metabolism (S9 mix) would be expected. This appears to be borne out both by the cytotoxicity results and the mutation frequency data.

STUDY RESULTS
Mutation assay: The induction of mutation frequency was assessed by cell growth in the presence of TFT in two independent experiments after a 72 hour expression time.

In the absence of S9 mix: In experiment 1, at 50 µg/mL, there was an increased mutation frequency of 3.5 times the solvent control level which was accompanied by high cytotoxicity (a mean of 23% survival, 16% survival in the main plate assay). However this isolated increase did not attain statistical significance. Such an increase (greater than 3 fold) is considered biologically significant, however this was not reproduced in the repeat assay at the same concentration of 50 µg/mL. No other statistically or biologically significant increases were observed at concentrations below this in either experiment.

In the presence of S9 mix: At the 25 µg/mL concentration of the test substance small but statistically significant increases in mutation frequencies of 2.7 and 2.3 times the control level were observed in experiments 1 and 2 respectively, although these were again associated with high levels of cytotoxicity, (20% and 16% mean survival respectively). Below this concentration no statistically or biologically significant increases in mutation frequency were observed.

The positive controls, EMS and DMN, gave the expected responses in both experiments.

The data obtained for the test substance show slightly elevated mutation frequencies only at the highest doses at which mutation could be estimated, (25 and 50 µg/mL with and without S9 respectively). Furthermore, although the increases in mutation frequency observed at the highest dose levels in experiment 1 were accompanied by a small absolute increase in the number of mutants, no such increase was observed in experiment 2, indicating that the increased mutation frequencies were caused by decreased cell viability. Overall there was no consistent evidence of biologically and statistically significant increases in mutation frequency obtained with the test substance.

Conclusions:

The test material showed no evidence of mutagenic potential both in the presence or absence of S9 mix as determined by selection in trifluorothymidine in this L5178Y mouse lymphoma cell assay.

Executive summary:

In a mammalian cell gene mutation assay, performed according to OECD 476 in compliance with GLP, L5178Y mouse lymphoma cells were treated with the test substance in form of an aqueous technical concentrate for 2 hours in the presence and absence of rat liver S9 from animals induced with Aroclor 1254. Cells were then grown for 72 hours before being divided and cultured in the presence or absence of a selective agent (trifluorothymidine) (TFT) to assess for mutation and viability respectively.

The test material showed dose-related cytotoxicity to L5178Y cells over the range 6.25 – 100 µg/mL (-S9 mix) and 3.125 – 50 µg/mL (+S9 mix), the highest dose level at which mutation frequency could be assessed. Small increases in mutant frequency were observed in both the absence and presence of S9 mix, but these were of limited magnitude, reproducibility or statistical significance and were considered not to indicate a mutagenic effect. The positive controls did induce the appropriate response.

The test material showed no evidence of mutagenic potential both in the presence or absence of S9 mix as determined by selection in trifluorothymidine in this L5178Y mouse lymphoma cell assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- in vivo mouse bone marrow micronucleus test, negative, OECD 474, Sheldon 1986

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 Dec 1985 to 10 Jan 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2000

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

C57BL

Remarks:

6J/Alp

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 - 12 weeks (phase 1 - preliminary toxicity test), 10 - 12 weeks (phase 2 - micronucleus test)
- Weight at study initiation: 17.8 - 27.6 g (micronucleus test)
- Housing: Up to 10/cage (sexes separately) in stainless steel cages 32.5 cm x 18 cm x 12.5 cm
- Diet: Porton Combined Diet (PCD) ad libitum
- Water: filtered tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 23
- Humidity (%): 42 - 69
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 02 Dec 1985 To: 10 Jan 1986

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: deionised water
- Amount of vehicle: 10 mL/kg

Frequency of treatment:

Single dose

Post exposure period:

Bone marrow cells were harvested at 24, 48 or 72 hours post-treatment. An overview of the terminations times for the different treatment and control groups can be found in Table 1 in 'Any other information on materials and methods incl. tables'.

Dose / conc.:

62.5 other: mg/kg bw/day based on test substance cation species

Remarks:

Lower dose group

Dose / conc.:

100 other: mg/kg bw/day based on test substance cation species

Remarks:

Higher dose group

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Concentration in vehicle: 65 mg/kg
- Route of administration: single oral administration

Tissues and cell types examined:

Bone marrow Polychromatic erythrocytes (PCEs)

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION
All animals designated for bone marrow smears were killed by cervical dislocation at 24, 48 or 72 hours after receiving a single dose of the test substance. Femurs were removed and stripped clean of muscle. The iliac end of the femur was removed and a fine paint brush was wetted with a solution of albumin (6% w/v in physiological saline). This was then dipped into the marrow canal and four smears were painted on an appropriately labelled clean, dry microscope slide. The slides were allowed to air dry and were stained with polychrome methylene blue and eosin using an automatic staining machine.

CRITERIA FOR DOSE SELECTION
Preliminary toxicity testing

METHOD OF ANALYSIS
Slides were coded and scored blind. One thousand polychromatic erythrocytes were examined and the number containing micronuclei scored. The slides were also examined for evidence of cytotoxicity, which may be manifest by alterations in the ratio of different cell types in the bone marrow. This was assessed by counting the ratio of polychromatic to normochromatic erythrocytes in a sample of 500 erythrocytes.

Statistics:

Analysed for significant difference from the vehicle control group using a one-sided Student’s ‘t’ test.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING STUDY
Initially diquat was dosed at 50, 150 and 250 mg/kg. Due to the mortality observed additional dose levels of 100 and 128 mg/kg were tested. The mortalities were: 0, 0, 1, 2 and 4 for males and 0, 0, 5, 3 and 5 for females at dose levels of 50, 100, 128, 150 and 250 mg/kg diquat respectively. The dose levels used in the main micronucleus test were 80 % and 50 % of the MLD (i.e. 100 and 62.5 mg/kg).

MICRONUCLEUS TEST RESULTS
No biologically or statistically significant increase in mean frequency of polychromatic erythrocytes (PCEs) containing micronuclei was seen at any dose level at any of the sampling times. A biologically and statistically significant reduction in number of PCEs to normochromatic erythrocytes (NCEs) was observed with both dose levels of diquat at all 3 sampling times with the exception of 62.5 mg/kg at 24 hours, indicating a cytotoxic effect. The test system positive control, cyclophosphamide, induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Table 2: Mean incidence of micronuclei/1000 polychromatic erythrocytes

Treatment	Dose	Time of kill
		24 hours	48 hours	72 hours
Vehicle control (deionised water)	10 mL/kg	1.7	2.0	1.6
Cyclophosphamide (positive control)	65 mg/kg	19.3**	14.7**	3.4*
Test substance	62.5 mg/kg	1.7 (9)	2.3	1.2
	100 mg/kg	2.5	1.3	2.5
all mean values based on 10 observations except where indicated in parentheses * statistically significantly different at p < 0.05 ** statistically significantly different at p < 0.01

Conclusions:

There were no significant increases in micronucleated polychromatic erythrocytes at doses of 62.5 or 100 mg test substance/kg bw. The test substance is not clastogenic in the mouse micronucleus test.

Executive summary:

In a bone marrow micronucleus assay with C57BL/6J/Alpk mice, performed according to OECD TG 474 under GLP, 5 mice/sex/group were given a single oral dose of test substance at doses of 100 and 62.5 mg test substance cation/kg, equivalent to 80% or 50% of the median lethal dose. Bone marrow cells were harvested at 24, 48 or 72 hours post-treatment, smears were prepared and stained with polychrome methylene blue and eosin. Cells were scored for the incidence of micronucleated polychromatic erythrocytes, and also for the percentage of polychromatic erythrocytes in the erythrocyte population. The vehicle was deionised water and the positive control was cyclophosphamide.There were no signs of toxicity during the study. The test substance did not induce a biologically or statistically significant increase in polychromatic erythrocytes containing micronuclei, when tested at dose levels of 00 and 62.5 mg test substance cation/kg corresponding to 186.8 and 116.75 mg/kg pure test substance and equivalent to 80% and 50% of the median lethal dose (MLD) in C57BL/6J/Alpk mice. The positive control induced the appropriate response. Test substance was not clastogenic in the mouse micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro gene mutation study in bacteria - OECD 471 (Callander 1986)

In a reverse gene mutation assay in bacteria, according to OECD TG 471 and in compliance with GLP, strains TA1535, TA1537, TA1538, TA98 and TA100 of S. typhimurium and WP2uvrA pkM101 of E. coli were exposed to the test substance at concentrations of 0.05, 0.1, 0.5, 1.0, 5.0, 10, 50, or 100 µg test substance cation/plate in the presence of mammalian metabolic activation (plate co-incubation) and at concentrations of 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10, or 50 µg test substance cation/plate in the absence of mammalian metabolic activation, S9 (plate co-incubation). The test substance was tested up to cytotoxic concentrations (up to 5000 µg/plate) in an initial dose-ranging study.

In two separate experiments, the compound did not induce any significant increase in the observed numbers of revertant colonies in any of the tester strains used, neither in the presence or absence of an auxiliary metabolising system (S9). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

In conclusion, the test substance gave an unequivocal negative, i.e. non-mutagenic, response in this assay. 

 

In vitro chromosome aberration study in mammalian cells - OECD 473 (Wildgoose 1986)

In a mammalian cell cytogenetics assay according to OECD TG 473 and in compliance with GLP, human primary lymphocyte cultures were exposed to test substance in physiological saline both in the presence and absence of auxiliary metabolic activation. The maximum dose level of test substance was different for each donor. In the instance of Donor 1, both with and without auxiliary metabolic activation the MTD of test substance was 267.4 µg test substance cation/mL. In the case of Donor 2 the MTD varied in each phase of the study; in the presence of auxiliary metabolic activation the highest dose level of test substance tested was 534.8 µg test substance cation/mL, with 107.0 µg test substance cation/mL being the highest in the absence of auxiliary metabolic activation.

Substantially elevated and statistically significant increases in chromosomal damage were observed in all cultures treated with the MTD of test substance with the exception of Donor 2 in the absence of auxiliary metabolic activation. Elevated and statistically significant increases in chromosomal damage were also observed in all cultures treated with approximate 1/2 MTD levels of test substance, with the exception of Donor 2 in the absence of S-9 mix. There were no statistically significant increases in chromosomal damage in any of the cultures treated with test substance at approximate 1/10 MTD levels or lower.

It is indicated that the clastogenic potential of the test substance is directly related to the cytotoxicity of the compound, as elevated levels of chromosomal damage were only observed at relatively high dose levels, where cytotoxicity was apparent, and by the absence of any clastogenic effect at approximate 1/10 MTD levels where cytotoxicity was minimal. This is also supported by the exceptional case of Donor 2 in the absence of S9-mix, where the cultures were more sensitive to cytotoxic effects and lower dose levels were tested, which did not lead to a clastogenic response. The test system was shown to be sensitive to chromosome damaging effects, by the response given to the positive control substances mitomycin C (a direct-acting clastogen) and cyclophosphamide (a clastogen requiring metabolic activation).

In conclusion, the test substance was found to induce chromosomal damage in the in vitro human lymphocyte cytogenetic assay at dose levels causing cytotoxic effects.

 

In vitro gene mutation assay in mammalian cells - OECD 476 (Cross 1986a)

In a mammalian cell gene mutation assay, performed according to OECD 476 in compliance with GLP, L5178Y mouse lymphoma cells were treated with the test material (technical) (purity 25.8% w/v ion) for 2 hours in the presence and absence of rat liver S9 from animals induced with Aroclor 1254. Cells were then grown for 72 hours before being divided and cultured in the presence or absence of a selective agent (trifluorothymidine) (TFT) to assess for mutation and viability respectively. The test material showed dose-related cytotoxicity to L5178Y cells over the range 6.25 – 100 µg/mL (-S9 mix) and 3.125 – 50 µg/mL (+S9 mix), the highest dose level at which mutation frequency could be assessed. Small increases in mutant frequency were observed in both the absence and presence of S9, but these were of limited magnitude, reproducibility or statistical significance and were considered not to indicate a mutagenic effect. The positive controls did induce the appropriate response. In conclusion, the test material showed no evidence of mutagenic potential both in the presence or absence of S9 mix as determined by selection in trifluorothymidine in this L5178Y mouse lymphoma cell assay.

These findings were supported by another OECD 476 study, performed in compliance with GLP, in L5178Y mouse lymphoma cell (Cross 1986b)

 

in vivo mammalian erythrocyte micronucleus test - OECD 474 (Sheldon 1986)

In a bone marrow micronucleus assay with C57BL/6J/Alpk mice, performed according to OECD TG 474 under GLP, 5 mice/sex/group were given a single oral dose of test substance at doses of 100 and 62.5 mg test substance cation/kg, equivalent to 80% or 50% of the median lethal dose. Bone marrow cells were harvested at 24, 48 or 72 hours post-treatment, smears were prepared and stained with polychrome methylene blue and eosin. Cells were scored for the incidence of micronucleated polychromatic erythrocytes, and also for the percentage of polychromatic erythrocytes in the erythrocyte population. The vehicle was deionised water and the positive control was cyclophosphamide.There were no signs of toxicity during the study. The test substance did not induce a biologically or statistically significant increase in polychromatic erythrocytes containing micronuclei, when tested at dose levels of 00 and 62.5 mg test substance cation/kg corresponding to 186.8 and 116.75 mg/kg pure test substance and equivalent to 80% and 50% of the median lethal dose (MLD) in C57BL/6J/Alpk mice. The positive control induced the appropriate response. Test substance was not clastogenic in the mouse micronucleus test.

Justification for classification or non-classification

Based on the available data classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.