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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1983

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Test was performed according to a modification of the preincubation test of Yahagi et al [1975].
Yahagi T. Degawa M. Seino Y. Matsushima T. Nagao M. Sugimura T. Hashimoto Y (1975): Mutagenicity of carcinogenic azo dyes and their derivatives. Cancer Lett 1 :91-96.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
-Analytical purity: As a rule, if a chemical was mutagenic or gave a questionable response, it was analyzed by Radian for identity and purity.
- Lot/batch No.: CI7430
- Chemical source: Pfaltz & Bauer. They were supplied to the testing laboratories by a chemical repository (Radian Corporation, Austin, TX), which was responsible for purchase and inventory of the chemicals, collection of physical, toxicological and safety data for each chemical, coding each test sample, shipment to the testing laboratories, and chemical analyses (when requested). Each sample sent out by the repository carried a unique, six-digit code number (Aliquot number) so that it could be tested under code as an unknown. The laboratories were also supplied with available information on the volatility, density, solubility, flammability. And stability of each chemical; Radian only performed solubility tests. Also sent, but in a sealed envelope coded with the Aliquot number, was the chemical name(s) along with the available information on its toxicological effects and decontamination procedures.
The laboratories were instructed to open this envelope only in the event of a spill or exposure to the chemical and to treat all coded chemicals as potential mutagens and carcinogens. After completion of the testing, the unopened envelopes were returned to the Radian Corporation.
-All chemicals were stored at the testing laboratories as recommended by the chemical repository.

Method

Target gene:
histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from male Sprague-Dawley rats and male Syrian hamsters induced with Aroclor 1254
Test concentrations with justification for top dose:
-To select the dose range for the mutagenesis assay, the test chemicals were checked for toxicity to TA100 up to a concentration of 10 mg/plate or the limit of solubility, both in the presence and absence of S-9 mix.
-Mutagenesis assay (preincubation): At least five doses of test chemical, in addition to the concurrent solvent and positive controls, were tested on each strain in the presence of S-9 mix or buffer. If toxicity was not apparent in the preliminary toxicity determination, the highest dose tested was 10 mg/plate; otherwise the upper limit of solubility was used. If toxicity was observed, the doses of test chemical were chosen so that the high dose exhibited some degree of toxicity. Occasionally, in the earlier tests, the high dose was greater than 10 mg/plate.
-Three plates were used, and the experiment was repeated no less than 1 week after completion of the initial test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Each chemical was dissolved and diluted immediately prior to testing.
- Justification for choice of solvent/vehicle: The solvent of choice was distilled water; dimethyl sulfoxide (DMSO) was used if the chemical was insoluble or not sufficiently soluble in water. Ethanol (95 % ) or acetone was used if the chemical was not soluble or stable in DMSO.
Controlsopen allclose all
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2-AA) on all strains with or without rat and hamster S9
Positive controls:
yes
Positive control substance:
sodium azide
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylenediamine (NOPD)
Positive controls:
yes
Positive control substance:
9-aminoacridine
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 hours at 37°C
- Expression time (cells in growth medium): 48 hours at 37°C
- Selection time (if incubation with a selection agent): 48 hours at 37°C

SELECTION AGENT (mutation assays): 0.5 mM L-histidine and 0.5 mM d-biotin

NUMBER OF REPLICATIONS: 3 plates and the experiment was repeated no less than 1 week after completion of the initial test.

DETERMINATION OF CYTOTOXICITY
- Method: viability on complete medium (for Monuron)
- Any supplementary information relevant to cytotoxicity: To select the dose range for the mutagenesis assay, the test chemicals were checked for toxicity to TA100 up to a concentration of 10 mg/plate or the limit of solubility, both in the presence and absence of S-9 mix. ). If toxicity was not apparent in the preliminary toxicity determination, the highest dose tested was 10 mg/plate; otherwise the upper limit of solubility was used. If toxicity was observed, the doses of test chemical were chosen so that the high dose exhibited some degree of toxicity. Occasionally, in the earlier tests, the high dose was greater than 10 mg/plate.


Rationale for test conditions:
Salmonella strains TA98, TA100, TA1535, and TA1537 were used in a modification of the preincubation test of Yahagi et al [1975]. The preincubation procedure was selected because of reports that it was no less sensitive than the plate test, and was more effective than the plate test for various chemicals such as aliphatic nitrosamines [Prival et al, 1979; Yahagi et al, 1975], pyrrolizidine alkaloids [Yamanaka, et al, 1979], and volatile chemicals [Rosenkranz et al, 1980]. Liver S-9 was prepared from male Sprague-Dawley rats (RU) and Syrian hamsters (HU) that were induced with Aroclor 1254 [Ames et al, 1975].
Hamster liver was used because of indications from a prior study (unpublished) and reports that the use of hamster S-9 would detect a number of chemicals undetected with rat S-9 [Prival and Mitchell, 1981; Bartsch et al, 1975; Sugimura, personal communication]. The protocol was standardized among the three laboratories, as discussed below. Each chemical was coded and tested as an unknown; the primary purpose of each test was to determine whether or not the chemical was mutagenic.
This is why, in the case of a positive result, only the strains and activation systems that gave the positive results were repeated, not the entire series. The protocol was designed to allow the individual investigators the flexibility to change doses based on their interpretations of the results of the initial experiment. To monitor the performance of each laboratory, a set of positive and negative control chemicals was chosen.
These chemicals were included, on a random basis, in batches of coded test chemicals sent to the testing laboratories. In addition to these controls, a small number of test chemicals selected, at random, were resubmitted to the same laboratory or sent to a second laboratory to determine interlaboratory reproducibility.
This publication is a presentation of Salmonella testing results on 250 coded chemicals, encompassing 370 tests.
Evaluation criteria:
Prior to statistical analysis no formal rules were used; however, a positive response was indicated by a reproducible, dose-related increase, whether it be twofold over background or not. The matrix of test strains and activation systems used allowed the investigators to detect trends or patterns that might not be as evident if only one strain and activation system were examined. In addition to the standard "positive" and "negative" categories, there is also "questionable" (or "inconclusive"). This applied to low-level responses that were not reproducible within the laboratory or to results that showed a definite trend but with which the investigator did not feel comfortable in making a "+" or "-" decision.
It also included tests in which an elevated revertant colony yield occurred at only a single dose level. After a decision on the mutagenicity of a sample was made, a request to decode the sample was sent to the repository, and the code was broken.
Statistics:
The data were subsequently evaluated using an analysis based on the models presented by Margolin et al [1981]; this analysis will be described elsewhere [Risko et al, manuscript in preparation]. As a result of these statistical analyses, a number of calls were changed from the original "negative" to "equivocal. " The statistical analysis did not result in any "positive" or "equivocal" calls being called "negative."

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Table1. Sources, Purities and Mutagenicities of 250 Chemicals in Salmonella

Chemical name

CAS number

Chemical Source

Lot number

Label purity

Analyzed purity

Testing Lab

Test result

Monuron

150-68-5

Pfaltz & Bauer

CI7430

-

 

EGG

negative

EGG: EG&G Mason Research Institute

Table2. Concentrations of Positive Control Chemicals (µg/plate) for Monuron

TA98

TA100

TA1535

TA1537

-S9

(NOPD)a

+S9

(2-AA)

-S9

(SA)

+S9

(2-AA)

-S9

(SA)

+S9

(2-AA)

-S9

(9AAD)

+S9

(2-AA)

12.0

RLI 1.5b

2.5

RLI 1.5

2.5

RLI 1.5

80.0

RLI 1.5

 

HLI 0.75

 

HLI 0.75

 

HLI 0.75

 

HLI 0.75

aNOPD. 4-nitro-o-phenylenediamine; 2-AA. 2-aminoanthracene; SA. sodium azide; 9AAD, 9-amino-acridine.

bDifferent concentrations for each S-9 source.

Applicant's summary and conclusion

Conclusions:
Monuron tested negative in a Salmonella Mutagenicity test in Salmonella typhimurium strains TA1535, TA1537, TA98 , and TA100 with and without S9-mix obtained from male rats or male hamsters induced with Aroclor 1254.
Executive summary:

All chemicals selected for genetic toxicology screening are initially tested in Salmonella using a preincubation procedure which is a modification of the Salmonella/mammalian microsome test of Ames et al.. The Salmonella tests were performed by three laboratories. Monuron was tested at Microbiological Associates (formerly EG&G Mason Research Institute (EGG)) by Dr. Steve Haworth.

Salmonella strains TA98, TA100, TA1535, and TA1537 were used in a modification of the preincubation test of Yahagi et al.. Liver S-9 was prepared from male Sprague-Dawley rats (RLI) and Syrian hamsters (HLI) that were induced with Aroclor 1254. In the case of a positive result, only the strains and activation systems that gave the positive results were repeated, not the entire series.

Each chemical was dissolved and diluted immediately prior to testing. The solvent of choice was distilled water; dimethyl sulfoxide (DMSO) was used if the chemical was insoluble or not sufficiently soluble in water. Ethanol (95 % ) or acetone was used if the chemical was not soluble or stable in DMSO.

Salmonella typhimurium strains TA1535, TA1537, TA98 , and TA100 were obtained by the individual laboratories from Dr. Bruce Ames, University of California, Berkeley. Frozen cultures were stored in liquid nitrogen (EGG) in 0.2-mL aliquots (EGG), in steriIe, screw-cap vials. EGG transferred a loopful of the thawed cultures into Oxoid Nutrient Broth #2 (CM 67) and discarded the unused portion of the thawed culture. All overnight cultures (late log phase) were obtained by incubation at 37°C on a shaker for 12-15 hr and were routinely checked for genetic integrity as recommended by Ames et al..

Liver S-9 fractions were routinely prepared from male Sprague-Dawley rats and male Syrian hamsters that were injected, ip, with Aroclor 1254 (200 mg/mL in corn oil) at 500 mg/kg. Five days after injection, the animals were sacrificed by decapitation (EGG) and the livers were removed aseptically. The animals were fasted for 12-24 hr immediately preceding sacrifice.

Liver homogenates were prepared aseptically at 0-4 °C. Excised livers were rinsed with 0.15 M KCl, then minced and homogenized (3 mL of 0.15 M KCl/g wet tissue) in a Potter-Elvehjem apparatus with a teflon pestle (EEG). The homogenate was centrifuged for 10 min at 9000 g at 4 °C. The supernatant (S-9) was decanted and distributed into freezing ampules and stored at -70°C. The microsomal enzyme reaction mix (S-9 mix) was prepared immediately prior to each assay. Unused S-9 mix was discarded and not refrozen. One milliliter of S-9 mix has the following composition: S-9, 0.10 mL; 0.04 M MgCl2 , 0.02 mL; 1.65 M KCl, 0.02 mL; 0.04 M /3-nicotinamide adenine di nucleotide phosphate (NADP), 0.10 mL; 0.05 M glucose-6-phosphate, 0.10 mL; 1.0 M NaH2P04 (pH 7.4), 0.10 mL; and distilled water, 0.56 mL.

All chemicals were tested using the preincubation procedure of the Salmonella assay as described by Yahagi et al (1975). Briefly, 0.5 mL of S-9 mix or 0.1 M P04 buffer was dispensed into an appropriate number of 13 x 100 mm culture tubes maintained at 37°C in a dry-bath. Then, 0.05 mL of cells and 0.05 mL of solvent or chemical dilution were added to each tube. The mixture was vortexed and allowed to incubate with shaking in the early tests (EGG) for 20 min at 37°C. The protocol was later changed to eliminate the shaking procedure, because the commercial shakers available would not fit in the Class II Type B hoods and, for the purposes of laboratory safety, it was inadvisable to incubate the chemicals at 37°C in the open laboratory. Following the preincubation period, 2.5 mL (EGG) of molten top agar (45°C) supplemented with 0.5 mM L-histidine and 0.5 mM d-biotin was pipetted into the tubes, which were immediately vortexed, and their contents poured onto 25 mL of minimal glucose bottom agar (Vogel and Bonner, 1956) in a 15 x 100-mm plastic petri dish (Falcon Muta-Assay, 1028 (EGG). After the overlay solidified, the plates were inverted and incubated at 37°C for 48 h.

At least five doses of test chemical, in addition to the concurrent solvent and positive controls, were tested on each strain in the presence of S-9 mix or buffer.

Three plates were used, and the experiment was repeated no less than 1 week after completion of the initial test.

To select the dose range for the mutagenesis assay, the test chemicals were checked for toxicity to TA100 up to a concentration of 10 mg/plate or the limit of solubility, both in the presence and absence of S-9 mix. One or more parameters were used as an indication of toxicity: viability on complete medium was used by EGG. If toxicity was not apparent in the preliminary toxicity determination, the highest dose tested was 10 mg/plate; otherwise the upper limit of solubility was used. If toxicity was observed, the doses of test chemical were chosen so that the high dose exhibited some degree of toxicity. Occasionally, in the earlier tests, the high dose was greater than 10 mg/plate.

The positive control chemicals were tested concurrently with each test chemical.

2-Aminoanthracene (2-AA) was tested on all strains in the presence of rat and hamster S-9. 4-Nitro-o-phenylenediamine (NOPD) was tested on TA98 without S-9. Also without S-9, sodium azide (SA) was tested on TA100 and TA1535, and 9-aminoacridine (9-AAD) was tested on TA 1537. The actual concentration for each positive control chemical used for each strain and activation condition was selected by the individual laboratory based on dose-response curves generated at the beginning of the testing program.

Although procedures for the statistical analysis of Salmonella plate test data have been developed, they were not incorporated into the initial data evaluations. The data were evaluated in an ad hoc manner by each testing laboratory and by NTP personnel. Prior to statistical analysis no formal rules were used; however, a positive response was indicated by a reproducible, dose-related increase, whether it be twofold over background or not. The matrix of test strains and activation systems used allowed the investigators to detect trends or patterns that might not be as evident if only one strain and activation system were examined. In addition to the standard "positive" and "negative" categories, there is also "questionable" (or "inconclusive"). This applied to low-level responses that were not reproducible within the laboratory or to results that showed a definite trend but with which the investigator did not feel comfortable in making a "+" or "-" decision. It also included tests in which an elevated revertant colony yield occurred at only a single dose level. After a decision on the mutagenicity of a sample was made, a request to decode the sample was sent to the repository, and the code was broken. The data were subsequently evaluated using an analysis based on the models presented by Margolin et al (1981). As a result of these statistical analyses, a number of calls were changed from the original "negative" to "equivocal. " The statistical analysis did not result in any "positive" or "equivocal" calls being called "negative."

Monuron tested negative with and without metabolic activation under the conditions of this experiment.