Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A battery of key in vitro studies with Monuron was performed, including the Ames bacterial mutation assay, Mammalian mutagenicity assay an Chromosome Aberration test. The bacterial and mammalian gene mutation assays with Monuron were negative, whereas the Chromosome Aberration test with Monuron was positive with metabolic activation at concentrations leading to precipitation. A supporting Sister Chromatid Exchange assay with Monuron was positive with and without metabolic activation, both at concentration with and without precipitation.

Link to relevant study records

Referenceopen allclose all

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
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
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.
Target gene:
histidine
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.
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."
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

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.

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.

 

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
screening protocol for detecting chemically induced cytogenetic changes in vitro : Galloway SM, Bloom AD, Resnick M, Margolin BH, Nakamura F, Archer P, Zeiger E (1985): Development of a standard protocol for in vitro cytogenetic testing with Chinese hamster ovary cells: Comparison of results for 22 compounds in two laboratories. Environ Mutagen 7: 1-51.
GLP compliance:
not specified
Type of assay:
other: in vitro mammalian cell chromosme aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO-W-B1
Details on mammalian cell type (if applicable):
CELLS USED
- Modal number of chromosomes: We did not score aberrations in polyploid cells but used metaphases with 19-23 chromosomes (the modal number being 21).
- Normal (negative control) cell cycle time: In some tests (largely those at LBI), information on the extent of cell cycle delay seen in the SCE test was used to select a later cell harvest time for the aberration test. (Allowance was made for changes in the degree of cytotoxicity resulting from the shorter incubation time as compared with the SCE test). If little or no delay was found, the cell harvest time for the aberration test was 8-12 hr after the beginning of treatment. This yielded cells in their first mitosis. Depending on the amount of delay seen in the SCE test, later harvest times, eg, 18-26 hr, were used to allow delayed cells to reach mitosis.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
cultured in Mc-Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics
Metabolic activation:
with and without
Metabolic activation system:
The S9 mix consisted of 15 µL/mL liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/mL NADP, and 4.5 mg/mL isocitric acid in serum-free medium.
Test concentrations with justification for top dose:
-Dose selection: Initially, dose selection was based on a preliminary growth inhibition test in which cells that excluded trypan blue were counted 24 hr after treatment. The top doses selected for the cytogenetics assays were those estimated to reduce growth by 50%. This approach was subsequently modified such that toxicity estimates were made from observations of cell monolayer confluence and mitotic activity in the same cultures used for analysis of SCEs or aberrations. The aim was to obtain results at the highest dose at which sufficient metaphase cells would be available for analysis. We also used observations on cell growth and cell cycle kinetics from the SCE test to select the doses and fixation times for the chromosome aberration tests. In the first SCE test with each chemical, cells were exposed to a range of doses spanning four to five orders of magnitude, in half-log increments, up to a maximum dose of 5-10 mg/ mL or to the limits of solubility in culture medium. In some cases, test chemical precipitate was observed at the higher dose levels. Dose selection for repeat trials involved a range of doses based on observations from the first trial.
-Aberration test: Originally, doses were chosen for the aberration test based on a preliminary test of cell survival 24 hr after treatment. For most tests reported here, doses were based on observations of cell confluence and mitotic cell availability in the SCE test. The doses for the aberration tests were usually spaced more closely than the half-log series of the preliminary SCE test and extended into the toxic range. In some tests (largely those at LBI), information on the extent of cell cycle delay seen in the SCE test was used to select a later cell harvest time for the aberration test. (Allowance was made for changes in the degree of cytotoxicity resulting from the shorter incubation time as compared with the SCE test).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Test chemicals (under which Monuron) were supplied under code by the National Toxicology Program chemical repository (Radian Corp., Austin, TX) and were dissolved immediately before use in water, dimethyl sulfoxide (DMSO), ethanol, or
acetone, in that order of preference.

Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
triethylenemelamine
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
In some tests (largely those at LBI), information on the extent of cell cycle delay seen in the SCE test was used to select a later cell harvest time for the aberration test. (Allowance was made for changes in the degree of cytotoxicity resulting from the shorter incubation time as compared with the SCE test). If little or no delay was found, the cell harvest time for the aberration test was 8-12 hr after the beginning of treatment. This yielded cells in their first mitosis. Depending on the amount of delay seen in the SCE test, later harvest times, eg, 18-26 hr, were used to allow delayed cells to reach mitosis. Cells were exposed to the test chemical for 2 hr in the presence of S9 or throughout the incubation period without S9.

- Exposure duration: throughout the incubation period without S9; 2 hr in the presence of S9
- Expression time (cells in growth medium):
- Fixation time (start of exposure up to fixation or harvest of cells):for Monuron: 19.5-26 hr

SPINDLE INHIBITOR (cytogenetic assays): colcemid

STAIN (for cytogenetic assays): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were collected by mitotic shake-off. Slides were stained with Giemsa and coded.

NUMBER OF CELLS EVALUATED: 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis and from positive (triethylenemelamine, mitomycin C, or cyclophosphamide) and solvent controls.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

DETERMINATION OF CYTOTOXICITY
- Method: other: Initially, dose selection was based on a preliminary growth inhibition test in which cells that excluded trypan blue were counted 24 hr after treatment. The top doses selected for the cytogenetics assays were those estimated to reduce growth by 50%. This approach was subsequently modified such that toxicity estimates were made from observations of cell monolayer confluence and mitotic activity in the same cultures used for analysis of SCEs or aberrations. The aim was to obtain results at the highest dose at which sufficient metaphase cells would be available for analysis. We also used observations on cell growth and cell cycle kinetics from the SCE test to select the doses and fixation times for the chromosome aberration tests. In the first SCE test with each chemical, cells were exposed to a range of doses spanning four to five orders of magnitude, in half-log increments, up to a maximum dose of 5-10 mg/ mL or to the limits of solubility in culture medium. In some cases, test chemical precipitate was observed at the higher dose levels. Dose selection for repeat trials involved a range of doses based on observations from the first trial.
For most tests reported here, doses were based on observations of cell confluence and mitotic cell availability in the SCE test.

- OTHER: All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaks and terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total. ” Gaps and endoreduplications were recorded but were not included in the totals. We did not score aberrations in polyploid cells but used metaphases with 19-23 chromosomes (the modal number being 21).
Evaluation criteria:
We used the scheme presented in Table 1 to combine the results of the trend test with the evaluation at individual doses for both SCE and aberration analyses. This scheme differs somewhat from that used in our previous report [Galloway et al, 1985]: The P values have been modified, and a test with one dose that is positive but without a positive trend test is now considered “?” instead of ‘‘ - ,” as was previously used. The designation “weak-positive’’ (“ + w”) was used in cases for which there was weak evidence for a positive response and is not an indication of potency. However, if there was a very strong trend as a result of a large increase in aberrations at a single dose, we called the result ‘‘w + *” to denote the fact that the level of aberrations was high.
In combining the conclusions from two or more trials for a compound, the emphasis was on determining whether any positive indications were repeatable. We gave more weight to the trial that had a more appropriate experimental design because of higher dose levels (if soluble), more appropriate dose levels, and/or a harvest time selected to be optimal based on information on cell cycle delay from the SCE test. Other factors, such as a reduced number of cells scored or an unusually high solvent control level of aberrations, usually reduced the weight given to a result.
Examples of combining results are discussed more fully in Results for individual compounds. Also presented in Table 1C is the scheme for combining results obtained with and without S9, in which the more positive result dominates.
The decision to repeat tests was not based on formal criteria throughout the study. Usually a test was repeated if there was a single elevated point, if the controls were unusually high, or if the range of toxicity was inadequate. In some cases, results were positive. When the statistical tests were subsequently applied, the increases were not always significant.
Statistics:
For chromosome aberrations, linear regression analysis of the percentage of cells with aberrations vs the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binomial sampling assumption (as opposed to Poisson) was used, and the test was that described by Margolin et al [1983, Statistical analysis and sample-size determinations for mutagenicity experiments with binomial responses. Environ Mutagen 5:pp 714-715]. The P values were adjusted by Dunnett’s method to take into account the multiple dose comparisons. For data analysis, we used the “total” aberration category, and the criterion for a positive response was that the adjusted P value be < 0.05.

Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
CHO-W-B1
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
at 1300-1600 µg/mL (precipitation!)
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
CHO-W-B1
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
at 800-1000 µg/mL
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The aberration test was positive with activation. The negative slope in the dose-response relation is likely to be the result of insolubility, since precipitate of test compound Monuron was visible at all the dose levels scored

RANGE-FINDING/SCREENING STUDIES:
Initially, dose selection was based on a preliminary growth inhibition test in which cells that excluded trypan blue were counted 24 hr after treatment. The top doses selected for the cytogenetics assays were those estimated to reduce growth by 50%. This approach was subsequently modified such that toxicity estimates were made from observations of cell monolayer confluence and mitotic activity in the same cultures used for analysis of SCEs or aberrations. The aim was to obtain results at the highest dose at which sufficient metaphase cells would be available for analysis. We also used observations on cell growth and cell cycle kinetics from the SCE test to select the doses and fixation times for the chromosome aberration tests. In the first SCE test with each chemical, cells were exposed to a range of doses spanning four to five orders of magnitude, in half-log increments, up to a maximum dose of 5-10 mg/ mL or to the limits of solubility in culture medium. In some cases, test chemical precipitate was observed at the higher dose levels. Dose selection for repeat trials involved a range of doses based on observations from the first trial.
For most tests reported here, doses were based on observations of cell confluence and mitotic cell availability in the SCE test. The doses for the aberration tests were usually spaced more closely than the half-log series of the preliminary SCE test and extended into the toxic range. In some tests (largely those at LBI), information on the extent of cell cycle delay seen in the SCE test was used to select a later cell harvest time for the aberration test. (Allowance was made for changes in the degree of cytotoxicity resulting from the shorter incubation time as compared with the SCE test). If little or no delay was found, the cell harvest time for the aberration test was 8-12 hr after the beginning of treatment. This yielded cells in their first mitosis. Depending on the amount of delay seen in the SCE test, later harvest times, eg, 18-26 hr, were used to allow delayed cells to reach mitosis. Cells were exposed to the test chemical for 2 hr in the presence of S9 or throughout the incubation period without S9.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
We have not attempted to include the historical controls in the formal statistical analysis because of the known variability in aberration frequencies from day to day and from reader to reader. The control data were, however, valuable in forming our decision scheme for data evaluation and are useful in deciding whether to repeat an assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: For most tests reported here, doses were based on observations of cell confluence and mitotic cell availability in the SCE test.
Remarks on result:
other: LEC < 1300 µg/mL (Least effective concentration tested (LEC) is the lowest dose to give a statistically significant increase (P ≤ 0.05) in aberrations )

Table 2. Chemicals Tested and Summary of Sister Chromatid Exchange (SCE) and Chromosome Aberration (ABS) Results With Ranges, Within Which Results Were Obtained and Least Effect Concentrations Tested (LEC µg/mL)*

 

 

SCE

ABS

 

 

-S9

+S9

Summary

-S9

+S9

Summary

LBI

Result

+d

-/+d

+

-d

+d

+

Range

50-200

100-1500a

800a-1000a

1300a-1600a

 

LEC

100

-/1200

-

<1300

Least effective concentration tested (LEC) is the lowest dose to give a statistically significant increase (P≤0.05) in aberrations or a 20% increase in SCEs. For chemicals with which the lowest dose tested gave a positive response, the LEC is preceeded by ‘‘ < ”.

Laboratories: COL, Columbia University; LBI, Litton Bionetics, Inc.

Individual trial results are separated by “/”. For definitions of +, +W, +W*, ?, -, and summary, see Table 1A-C under Material and methods; “-”, no LEC observed. a, Precipitate or immiscibility (noted for LBI results only); b, endoreduplication; c, tetraploidy ; d, delayed harvest (For SCE tests, standard harvest time was about 26 hr in BrdUrd; for LBI data marked “d” the harvest was after 28.5 to 37.3 hr in BrdUrd. For aberrations, standard harvest time was 14 hr at COL; harvest was 10.5 from beginning of treatment at LBI unless marked “d,” in which case it was 19.5-26 hr); e, one trial, two harvest times.

 

Conclusions:
The chromosome aberration test for Monuron was positive with activation. The negative slope in the dose-response relation is likely to be the result of insolubility, since precipitate of test compound was visible at all the dose levels scored. Seiler [Seiler JP (1978): Herbicidal phenylalkylureas as possible mutagens. I. Mutagenicity tests with some urea herbicides. Mutat Res 58:353-359.] reported that monuron was negative in the mouse bone marrow micronucleus test. The summary conclusion of this chromosome aberration test with Monuron is positive.
Executive summary:

Among 108 coded chemicals, Monuron was tested in Chinese hamster ovary (CHO) cells for the induction of chromosome aberrations with and without exogenous metabolic activation, using protocols designed to allow testing up to toxic doses. Monuron was tested at Litton Bionetics, Inc.

Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc-Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics. Tests were carried out with and without an in vitro metabolic activation system (S9 mix). In tests without metabolic activation, the test chemical was left in culture until colcemid addition, whereas with activation the test chemical was added along with S9 mix for only 2 hr at the beginning of the test period. The S9 mix consisted of 15 µg/mL liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/mL NADP, and 4.5 mg/mL isocitric acid in serum-free medium.

Test chemicals were supplied under code by the National Toxicology Program chemical repository (Radian Corp., Austin, TX) and were dissolved immediately before use in water, dimethyl sulfoxide (DMSO), ethanol, or acetone, in that order of preference.

Initially, dose selection was based on a preliminary growth inhibition test in which cells that excluded trypan blue were counted 24 hr after treatment. The top doses selected for the cytogenetics assays were those estimated to reduce growth by 50%. This approach was subsequently modified such that toxicity estimates were made from observations of cell monolayer confluence and mitotic activity in the same cultures used for analysis of SCEs or aberrations. The aim was to obtain results at the highest dose at which sufficient metaphase cells would be available for analysis. We also used observations on cell growth and cell cycle kinetics from the SCE test to select the doses and fixation times for the chromosome aberration tests. In the first SCE test with each chemical, cells were exposed to a range of doses spanning four to five orders of magnitude, in half-log increments, up to a maximum dose of 5-10 mg/ mL or to the limits of solubility in culture medium. In some cases, test chemical precipitate was observed at the higher dose levels. Dose selection for repeat trials involved a range of doses based on observations from the first trial.

For most tests reported here, doses were based on observations of cell confluence and mitotic cell availability in the SCE test. The doses for the aberration tests were usually spaced more closely than the half-log series of the preliminary SCE test and extended into the toxic range. In some tests (largely those at LBI), information on the extent of cell cycle delay seen in the SCE test was used to select a later cell harvest time for the aberration test. (Allowance was made for changes in the degree of cytotoxicity resulting from the shorter incubation time as compared with the SCE test). If little or no delay was found, the cell harvest time for the aberration test was 8-12 hr after the beginning of treatment. This yielded cells in their first mitosis. Depending on the amount of delay seen in the SCE test, later harvest times, eg, 18-26 hr, were used to allow delayed cells to reach mitosis. Cells were exposed to the test chemical for 2 hr in the presence of S9 or throughout the incubation period without S9.

Cells were collected by mitotic shake-off. Slides were stained with Giemsa and coded, and 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis and from positive (triethylenemelamine, mitomycin C, or cyclophosphamide) and solvent controls. All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaks and terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total. ” Gaps and endoreduplications were recorded but were not included in the totals. We did not score aberrations in polyploid cells but used metaphases with 19-23 chromosomes (the modal number being 21).

For chromosome aberrations, linear regression analysis of the percentage of cells with aberrations vs the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binomial sampling assumption (as opposed to Poisson) was used, and the test was that described by Margolin et al [1983, pp 714-715]. The P values were adjusted by Dunnett’s method to take into account the multiple dose comparisons. For data analysis, we used the “total” aberration category, and the criterion for a positive response was that the adjusted P value be < 0.05.

For Monuron the aberration test was positive with activation. The negative slope in the dose-response relation is likely to be the result of insolubility, since precipitate of test compound was visible at all the dose levels scored. Seiler [1978] reported that monuron was negative in the mouse bone marrow micronucleus test.

The protocol we have developed allows us to test chemicals at doses up to toxic levels and to adjust harvest times based on cell cycle kinetics assessed in the initial SCE test. The protocol is still evolving; in particular, a reassessment of the analysis of the aberration data strongly suggests that the resolution of the assay would be improved by scoring 200 instead of 100 cells per dose level [Margolin et al, 1986].

The highest dose(s) tested frequently extended into a cytotoxic range, and the maximum dose was determined by the availability of sufficient analyzable cells. Since some agents yield positive responses only at doses that are cytotoxic, inducing cell death and/or delay in cell cycle progression, it is important to consider the possible relationships between the induction of SCEs or chromosome aberrations and cytotoxicity .

In tests for aberration induction, nontoxic doses were not always examined, since the doses for aberration testing were often selected to be in the toxic range based on a prior SCE test. Also, because we sampled only 100 cells per dose, the sensitivity with which we could detect an increase in aberrations was rather low [Margolin et al, 1986]so only the highly cytotoxic doses might yield statistically significant effects. In eight of 24 positive chromosome aberration tests that could be evaluated (with or without S9), increases in aberrations were detected at doses that did not induce obvious toxicity (reduction in confluence); monuron was one of these chemicals.

 

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
L5178Y tk+/-mouse lymphoma cell forward mutation assay based upon procedures described by Clive and Spector (Mutat Res 44:269-278, 1975) and Clive et al. (Mutat Res 59:61-108, 1979).
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
Monuron was supplied from the National Toxicology Program Chemical Repository, Radian Corporation, Austin, TX 78766. Monuron was identified only by a code number until the completion of all testing and evaluation, at which time the code was broken.
Target gene:
thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
L5178Y clone 3.7.2C mouse lymphoma cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: obtained from Dr. D. Clive, Burroughs Wellcome Co., Research Triangle Park, NC 27709
- Methods for maintenance in cell culture if applicable:
L5178Ytk+/tk mouse lymphoma cells were stored in liquid nitrogen. Thawed samples were cultured and used for up to 3 months, then discarded. Laboratory cultures were confirmed as free from mycoplasma by cultivating or Hoechst staining techniques and maintained in Fischer's medium at 37 °C on gyratory tables. Fischer's medium (designated F0) was supplemented with 2 mM L-glutamine, sodium pyruvate, 110 µg/mL, 0.05% pluronic F68, antibiotics, and 10% heat-inactivated donor horse serum (v/v) (designated F10P). On a single occasion, within 1 week of the start of an experiment, cultures were purged of tk-/tk- mutants by exposure for 1 day to F10P containing THMG (thymidine, 6 µg/mL hypoxanthine, 5 µg/mL, glycine, 7.5 µg/mL and methotrexate, 0.1 µg/mL), then for 3 days to F10P containing THG only, (i.e., THMG without methotrexate).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Periodically checked for Mycoplasma contamination: yes. Laboratory cultures were confirmed as free from mycoplasma by cultivating or Hoechst staining techniques and maintained in Fischer's medium at 37 °C on gyratory tables.
- Periodically 'cleansed' against high spontaneous background: yes. On a single occasion, within 1 week of the start of an experiment, cultures were purged of tk-/tk- mutants by exposure for 1 day to F10P containing THMG (thymidine, 6 µg/mL hypoxanthine, 5 µg/mL, glycine, 7.5 µg/mL and methotrexate, 0.1 µg/mL), then for 3 days to F10P containing THG only, (i.e., THMG without methotrexate).
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 (induced (RLI) or uninduced (RLN) rat livers)
Test concentrations with justification for top dose:
-Preliminary toxicity test: Ten-fold differences in test compound concentrations were used in the toxicity test, the highest being 5 mg/mL unless a much lower concentration was indicated by the poor solubility of a compound.
-Mutagenicity test: Test compound concentrations were primarily two-fold dilutions from the highest testable concentration, as estimated from the toxicity test.
For Monuron following concentrations were used:
First trial :without S9: 0, 50, 100, 200, 400, 800 and 1600 µg/mL; with S9: 0, 150, 350, 550, 750 and 950 µg/mL (precipitation at 950 µg/mL).
Second trial: without S9: 0,350, 550, 750 and 950 µg/mL; with S9: 0, 150, 350, 750 and 950 µg/mL.
Third trial: with S9: 0, 300, 500, 700, 900 and 1100 µg/mL

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: medium without serum, distilled water, or dimethyl sulphoxide (DMSO; analytical grade). For Monuron DMSO was used as solvent.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO analytical grade for Monuron
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension (exposure); plate incorporation (cloning efficiency, mutant selection)
- Cell density at seeding (if applicable): 3 aliquots of 10E6 cells were used for mutant selection

DURATION
- Preincubation period:
- Exposure duration: 4 hr incubation
- Expression time (cells in growth medium): 2-day expression period (cell population density being adjusted after 24 hr)
- Selection time (if incubation with a selection agent): incubation with 3µg/TFT/mL for 11-14 days in 5% CO2:95% air at 37°C
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: 3 aliquots of 10E6 cells were used for mutant selection. The chemicals were tested at least twice (duplicate)


METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED:

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Toxicity was expressed as either a reduction of cell population growth in suspension during the expression period or a reduction in cloning efficiency. A measure of the overall toxicity was the relative total growth (RTG), which is defined as

RTG = (total suspension growth X cloning efficiency) in dosed culture
(total suspension growth x cloning efficiency) in control culture


OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:
Evaluation criteria:
Compliance with predetermined quality control criteria (Table 1) was required before the response of a cellular population to the test chemical was evaluated. The basis for this choice of criteria is described elsewhere [Caspary et al., 1988] and is the outcome of evaluation of historical data in two laboratories (Litton Bionetics, Inc., and SRI International). Four response categories were defined (Table 2). Primary judgments were made at the level of individual experiments, but judgment on the mutagenic potential of a chemical was made on a basis of consensus of all valid experimental results (Table III). The statistical analysis was based upon the mathematical model proposed for this system [Lee and Caspary, 1983] and consisted of a dose-trend test [Barlow et al., 1972] and a variance analysis of pair-wise comparisons of each dose against the vehicle control. In the result tables, significant differences from concurrent vehicle control values at the 5% level are indicated by underlines of the average mutant fractions at the appropriate concentrations. Where a statistically significant response occurred, the lowest observed effective dose (LOED) was noted. There is a report that non-physiological pH levels can produce increases in mutant fraction [Cifone et al., 1987]. Consequently, pH shifts observed from phenol red color changes to yellow or purple were noted and are recorded in the tables.
According to Cifone et al. [1987], such changes impacted upon data evaluation only in the presence of S9 mix. However, studies at this laboratory [McGregor, unpublished] have failed to confirm this interaction of pH and S9 mix. It is possible that the lack of such an effect is due to the lower S9 concentration used in this laboratory. Osmotic pressure was not measured, but, again, osmotic effects were unlikely because the highest concentrations tested were never greater than 5 mg/mL.
Statistics:
The statistical analysis was based upon the mathematical model proposed for this system [Lee and Caspary, 1983] and consisted of a dose-trend test [Barlow et al., 1972] and a variance analysis of pair-wise comparisons of each dose against the vehicle control. In the result tables, significant differences from concurrent vehicle control values at the 5% level are indicated by underlines of the average mutant fractions at the appropriate concentrations. Where a statistically significant response occurred, the lowest observed effective dose (LOED) was noted.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
up to 950 µg/mL
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
first trial: result discounted because precipitation; second trial: no concentration dependent response over the range; third trial: negative from 300-1100 µg/mL
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Table 1. Test results Monuron

Without S9 TRIAL 1: negative

Concentration

µg/mL

CE

cloning efficiency %

RTG

relative total growth %;

MC

mutant colony

count

MF

mutant fraction (mutant colonies per 10E6 clonable cells)

AVG

MF

group average mutant fraction

DMSO

dimethylsulphoxide

95

99

81

29

 

0.

93

96

97

35

 

107

96

81

25

 

94

109

109

39

32

50.

86

88

73

28

 

92

87

95

35

31

100.

92

82

110

40

 

95

92

69

24

32

200.

99

79

102

34

 

90

83

130

48

41

400.

101

59

114

38

 

84

75

106

42

40

800.

120

29

126

35

 

100

15

152

50

43

1600.

LETHAL

 

 

 

 

LETHAL

 

 

 

 

EMS (ethyl methanesulphonate)

250. µg/mL

70

74

351

168

 

85

72

369

146

157

MMS (methyl methanesulphonate)

15. µg/mL

51

22

241

159

 

66

33

288

145

152

underline, P< 5%

 

Without S9 TRIAL 2: negative

Concentration

µg/mL

CE

cloning efficiency %

RTG

relative total growth %;

MC

mutant colony

count

MF

mutant fraction (mutant colonies per 10E6 clonable cells)

AVG

MF

group average mutant fraction

DMSO

57

99

112

65

 

0.

64

107

94

49

 

48 R

74

111

78

 

54

94

107

66

60

350.

70

82

125

60

 

87

107

197

75

68

550.

78

80

149

64

 

76

92

139

61

62

750.

61

52

111

61

 

71

67

101

47

54

950.

80

22

134

56

 

83

37

79

32

44

EMS

250. µg/mL

71

96

448

211

 

71

108

576

272

742

MMS

15. µg/mL

37

27

421

378

 

38

34

391

343

360

R = Rejected when 50% > CE > 120%

underline, P< 5%

 

Induced S9 TRIAL 1: nontoxic, negative

Concentration

µg/mL

CE

cloning efficiency %

RTG

relative total growth %;

MC

mutant colony

count

MF

mutant fraction (mutant colonies per 10E6 clonable cells)

AVG

MF

group average mutant fraction

DMSO

104

96

110

35

 

0.

84

107

103

41

 

79

100

112

47

 

81

96

119

49

43

150.

84

74

114

45

 

62

69

123

66

56

350.

65

55

99

51

 

65

51

93

48

49

550.

76

76

126

55

 

79

78

130

55

55

750.

71

56

134

63

 

79

58

160

68

65

950.

87

61

173

66

 

68

50

146

72

69

MCA (3-methylcholanthrene)

2.5 µg/mL

55

47

566

341

 

50

34

615

409

375

Precipitation at 950. µg/mL

underline, P< 5%

 

Induced S9 TRIAL 2: questionable

Concentration

µg/mL

CE

cloning efficiency %

RTG

relative total growth %;

MC

mutant colony

count

MF

mutant fraction (mutant colonies per 10E6 clonable cells)

AVG

MF

group average mutant fraction

DMSO

79

107

59

25

 

0.

81

99

64

26

 

83

102

91

37

 

94

92

85

30

30

150.

81

103

96

40

 

69

72

117

56

48

350.

59

75

77

44

 

62

83

88

47

46

550.

58

77

112

65

 

72

79

114

53

59

750.

71

42

114

54

 

63

33

106

57

55

950.

91

34

115

42

 

79

20

115

48

45

MCA

2.5 µg/mL

62

51

520

280

 

65

52

456

234

257

underline, P< 5%

 

Induced S9 TRIAL 3: negative

Concentration

µg/mL

CE

cloning efficiency %

RTG

relative total growth %;

MC

mutant colony

count

MF

mutant fraction (mutant colonies per 10E6 clonable cells)

AVG

MF

group average mutant fraction

DMSO

74

96

130

58

 

0.

75

107

166

74

 

79

92

160

68

 

73

105

139

64

66

300.

73

88

123

56

 

95

136

173

61

58

500.

70

84

158

75

 

72

74

148

69

72

700.

74

42

159

72

 

78

40

178

76

74

900.

58

16

117

68

 

72

12

176

82

75

1100.

58

10

131

75

 

60

9

128

71

73

MCA

2.5 µg/mL

41

27

454

374

 

33

22

427

438

406

underline, P< 5%

Conclusions:
Monuron was clearly not mutagenic at concentrations up to 950 µg/mL in the absence of S9 mix. The RTG was reduced to 30% at this dose level, where precipitation also occurred. In the presence of S9 mix, however, the results were ambiguous. A statistically significant response occurred in the first experiment at 950 µg/mL, but this should be discounted because of compound precipitation. In the second experiment, statistically significant increases in mutant fractions occurred at concentrations of 150, 550, and 750 µg/mL but not at 350 or 950 µg/mL. There was no concentration dependent response over the range, i.e., the lack of significance at two concentrations was not due to large variations in response between cultures in the same group. This questionable response was investigated in a third experiment where no statistically significant responses were observed at any concentration.
Overall conclusion: Monuron was not identified as mutagen.
Executive summary:

Seventy-two chemicals were tested for their mutagenic potential in the L5178Y tk+/-mouse lymphoma cell forward mutation assay, using procedures based upon those described by Clive and Spector (Mutat Res 44:269-278, 1975) and Clive et al. (Mutat Res 59:61-108, 1979). Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3µg/mL. The chemicals were tested at least twice.

Monuron was not identified as mutagens.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Monuron was tested in a key Micronucleus study and a key Chromosome Aberration Test in mice after single intraperitoneal dosing up to 250 mg/kg bw. The Micronucleus study was positive by trend analysis at 125 and 250 mg/kg bw, whereas the Chromosome Aberration at same dose levels was negative.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
three-treatment, single-sample micronucleus test protocol.
- Principle of test: The mouse bone marrow micronucleus assay is a commonly used test for detecting the genetic toxicity of chemicals in laboratory rodents. Considerable effort has gone into developing protocols which maximize the likelihood of detecting genetic toxicity while minimizing the human and animal resources required to conduct a test. In studies designed to assess the relative effectiveness of protocols in which the numbers of daily treatments and sample times varied from one to three , three test chemicals were used: (1) 7, 12-dimethylbenzanthracene, which is slowly absorbed and metabolized and was the primary basis for testing guidelines requiring multiple sample times out to 72 hours posttreatment; (2) benzidine, which is an in vivo micronucleus inducer but has proven difficult to detect; and (3) mitomycin C, which is water soluble, direct acting, and has an in vivo half life of less than an hour. A protocol involving three daily treatments and a single sample time 24 hr following the final treatment was found to be the most effective for detecting all three chemicaIs.
- Short description of test conditions: Preliminary dose determination experiments were performed, after which groups of 5 male B6C3F1 mice were injected intraperitoneally three times at 24 hr intervals with Monuron dissolved in com oil. The total dosing volume per mouse was 0.4 mL. Solvent control animals were injected with 0.4 mL of solvent only. A concurrent positive control group of mice was included in each of the micronucleus tests Twenty-four hours after the final, smears of the bone marrow cells from femurs were prepared. Air-dried smears were fixed and stained with acridine orange; 2000 polychromatic erythrocytes (PCE) were scored per animal for frequency of micronucIeated cells. In addition, the percentage of PCEs among the total erythrocyte population in the bone marrow was scored for each dose group as a measure of toxicity. The results were tabulated as the mean of the pooled results from all animals within a treatment group, plus or minus the standard error of the mean. Repeat tests were performed for Monuron, based on results of the initial micronucleus test.
Type of assay:
other: in vivo micronucleus test in mouse bone marrow cells
Specific details on test material used for the study:
Monuron was received from the NTP chemical repository (Radian Corporation, Austin, TX).
Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: obtained from the National Toxicology Program production facility at Taconic Farms
- Age at study initiation: common age between 9 and 14 weeks
- Weight at study initiation: weighing within a 2 g range of a mean weight between 25 and 33 g
Additional information on animal husbandry can be found in Tice et al. [Tice RR, Erexson G. Hilliard CJ. Huston JL, Boehm RM. Gulati D, Shelby MD (1990a): Effect of treatment protocol and sample time on frequencies of micronucleated cells in mouse bone marrow and peripheral blood. Mutagen 5:313-321.].
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil (for Monuron)
- Justification for choice of solvent/vehicle: corn oil was used for for water-insoluble chemicals
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Each chemical was prepared in the appropriate solvent (corn oil for water-insoluble chemicals (Monuron)) and suspended using a Tek-Mar Tissumizer® for chemicals in com oil All test chemicals were administered within 30 min of preparation. Suspended DMBA (positive control in case of Monuron) was stored at room temperature and dissolved MMC was stored at 0-5°C between treatments within an experiment. All treatments were by intraperitoneal (IP) injection at a volume of 0.4 mL per mouse. Identification numbers were randomly assigned to mice prior to euthanasia.
Duration of treatment / exposure:
3 consecutive days
Frequency of treatment:
3 intraperitoneal injections at 24 h intervals (3 consecutive days)
Post exposure period:
24 hr after the third treatment
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
MN Induction Experiment
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Remarks:
MN Induction Experiment
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
MN Induction Experiment
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
MN Induction Experiment
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Repeat study
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
Repeat study
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Repeat study
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
7,12-dimethylbenzanthracene (DMBA) in corn oil (in case of Monuron); DMBA was purchased from Eastman Kodak (Rochester, NY)
- Route of administration: intraperitoneal
- Doses / concentrations: a weakly active dose of DMBA in corn oil (12.5 mg/kg DMBA)
Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose Determination Studies were performed. The test doses for Monuron were based on toxicity/mortality observed in the dose determination studies. Groups of 5 mice were administered the test chemical by IP injection on three consecutive days. Animals were monitored twice daily, and 48 hr after the third treatment, the surviving mice were euthanized by CO2 asphyxiation. Bone marrow and peripheral blood smears (two slides/tissue/mouse) were prepared by a direct technique [Tice et al., 1990a]. Air-dried smears were fixed using absolute methanol and stained with acridine orange [Tice et al., 1990a). Bone marrow smears from each animal were evaluated at 1000 x magnification using epi-illuminated fluorescence microscopy (450-490 nm excitation, 520 nm emission) for determination of the percentage of PCE among 200 erythrocytes. Based on the results obtained, the maximum administered dose was estimated or additional dose determination experiments were conducted to more accurately estimate the maximum dose to be tested in the primary MN test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): For the initial MN test, groups of 5-7 mice were injected IP on three consecutive days with either the test chemical (at 1 x, 1/2 x , and 1/4 x , where x is the maximum dose determined in the dose determination experiments), a weakly active dose of the positive control chemical (DMBA (7, 12-dimethylbenzanthracene) in corn oil or MMC (Mitomicyn C) in PBS), or the appropriate solvent. Mice were euthanized with CO2 24 hr after the third treatment. For Monuron also a repeat (second) bone marrow test was conducted.

DETAILS OF SLIDE PREPARATION:. Bone marrow smears (two slides mouse) were prepared, fixed in absolute methanol, and stained with acridine orange.

METHOD OF ANALYSIS: For each animal, slides were evaluated at 1000 x magnification for the number of MN-PCE among 2000 PCE and for the percentage of PCE among 200 erythrocytes.

OTHER:
Evaluation criteria:
The test conclusions (see Table 1) are based on the statistical analysis of trend and of pairwise comparisons of the solvent control with individual doses, the absolute increases in MN-PCE frequency, and where appropriate, the combination of results from repeat studies.
Statistics:
The data were analyzed using the Micronucleus Assay Data Management and Statistical software package (version 1.4), which was designed specifically for in vivo micronucleus data [ILS, 1990]. The level of significance was set at an alpha level of 0.05. To determine whether a specific treatment resulted in a significant increase in MN-PCE, the number of MN-PCE were pooled within each dose group and analyzed by a one-tailed trend test. In the software package used, the trend test incorporates a variance inflation factor to account for excess animal variability. In the event that the increase in the dose response curve is nonmonotonic, the software program allows for the data to be analyzed for a significant positive trend after data at the highest dose only has been excluded. However, in this event, the alpha level is adjusted to 0.01 to protect against false positives.
The %PCE data were analyzed by an analysis of variance (ANOVA) test based on pooled data. Pairwise comparisons between each group and the concurrent solvent control group was by an unadjusted one-tailed Pearson chi-squared test which incorporated the calculated variance inflation factor for the study.
Sex:
male
Genotoxicity:
positive
Vehicle controls validity:
valid
Positive controls validity:
valid

Table 1. . MN Data Analysis: 49 NTP Chemicals: Monuron

Chemicala

Tissueb

Trendc

P value

Dosed

(mg/kg)

MN-PCE/1000e

(no. animals)

Pair-wisef

Survivalg

%PCEh

Monuron C

Positive

-/-

150-68-5

EHRT (279574)

(CO)

BM

0.310

(0.003)

0

62.5

125

250

1.10 ± 0.10 (5)

1.75 ± 0.32 (4)

2.80 ± 0.56 (5)

1.40 ± 0.29 (5)

 

0.1223

0.0032

0.2741

5/5

5/5

5/5

5/5

56.4

38.1

47.6

48.6

BM

<0.001

0

125

250

1.20 ± 0.41 (5)

3.70 ± 0.64 (5)

6.10 ± 1.02 (5)

 

0.001

<0.001

5/5

5/5

52.0

51.6

aChemical name (rodent bioassay results, C = carcinogen; NC = non-carcinogen). MOUSE-MN CALL structural alert [Tennant and Ashby, 1991] Salmonella result. CAS No. Laboratory (Radian No.) solvent: CO = com oil, PBS = phosphate buffered saline) (ILS = Integrated Laboratory Systems, EHRT = Environmental Health Research and Testing).

bTissue used (BM = bone marrow, PB = peripheral blood.)

cValue of P for trend analysisα= .05 (value of P for trend analysis omitting the high dose valueα= .01).

dChemical concentration administered i. p. , daily, to each anima!.

eMicronucleated PCEs per 1000 PCE scored (± Standard Error of the Means) (number of animals scored).

fThe value of P for pair-wise comparisons between each treatment group and the concurrent solvent control groupα= .05.

gNo. of animals surviving treatment over number of animals treated.

hPercentage of erythrocytes that were polychromatic (i.e., [No. of PCE/No. of PCE + No. of NCE] x 100).

Table 2. Summary of Solvent Control Data From Mouse Bone Marrow Micronucleus Tests

Laboratory

Solvent

No.a

MN-PCE ± SDb

Range

EHRTc

COd

14

2.12 ± 0.70

1.10-3.70

 

PBSe

9

2.10 ± 0.40f

1.60-2.70

ILSg

CO

17

2.38 ± 0.93

1.10-4.60

 

PBS

8

2.66 ± 0.36f

2.20-3.10

 

aNo. of experimental groups (4 or 5 animals per group).

bMicronucleated PCEs per 1000 PCEs ±standard deviation.

cEnvironmental Health Research and Testing.

dCorn oil (0.4 mL)

ePhosphate buffered saline (0.4 mL).

fThe ILS-PBS control value is significantly higher than the EHRT-PBS control value (P = 0.009).

glntegrated Laboratory Systems.

 

Table 3. Summary of Positive Control Data From Mouse Bone Marrow Micronucleus Tests

Laboratory

Solvent

No.a

MN-PCE ± SDb

Range

EHRTc

DMBAd

14

6.93 ± 2.59

2.00-12.17

 

MMC

9

6.82 ± 1.24

4.55-8.50

ILSf

DMBA

17

7.93 ± 1.69

4.40-10.60

 

MMC

8

6.85 ± 2.26

3.80-11.20

 

aNo. of experimental groups (4 or 5 animals per group).

bMicronucleated PCEs per 1000 PCEs ±standard deviation.

cEnvironmental Health Research and Testing.

dDimethylbenzanthracene (12.5 mg/kg).

eMitomycin C (0.2 mg/kg).

flntegrated Laboratory Systems.

Conclusions:
For Monuron the initial test was negative to 250 mg/kg by trend analysis but the trend test was positive if the high dose group was omitted. The repeat test was positive by trend analysis with the 125 and 250 mg/kg dose groups elevated significantly above the control. Sharma et al. [1987] reported that monuron, administered intraperitoneally once, twice, or three times at a dose of 14.4 mg/kg, increased the frequencies of micronuclei and chromosome aberrations in the bone marrow cells of Lacca strain mice.
Executive summary:

Forty-nine chemicals were tested in a mouse bone marrow micronucleus test that employed three daily exposures by intraperitoneal injection.

For the initial MN test, groups of 5-7 mice were injected IP on three consecutive days with either the test chemical (at 1 x, 1/2 x , and 1/4 x , where x is the maximum dose determined in the dose determination experiments), a weakly active dose of the positive control chemical (DMBA (7, 12-dimethylbenzanthracene) in corn oil or MMC (Mitomicyn C) in PBS), or the appropriate solvent. Mice were euthanized with CO2 24 hr after the third treatment. Bone marrow smears (two slides mouse) were prepared, fixed in absolute methanol, and stained with acridine orange. For each animal, slides were evaluated at 1000 x magnification for the number of MN-PCE among 2000 PCE and for the percentage of PCE among 200 erythrocytes.

For Monuron the initial MN test was performed at 0, 62.5, 125 and 250 mg/kg bw with corn oil as solvent control and DMBA in corn oil as positive control. For Monuron a repeat study was performed as well at 0, 125 and 250 mg/kg bw.

The initial test was negative to 250 mg/kg by trend analysis but the trend test was positive if the high dose group was omitted. The repeat test was positive by trend analysis with the 125 and 250 mg/kg dose groups elevated significantly above the control. Sharma et al. (Sharma GP. Sobti RC, Chaundhry A, Gill RK, Ahluwali KK (1987): Mutagenic potential of a substituted urea herbicide: Monuron. Cytologia 52: 841 -846 ) reported that monuron, administered intraperitoneally once, twice, or three times at a dose of 14.4 mg/kg, increased the frequencies of micronuclei and chromosome aberrations in the bone marrow cells of Lacca strain mice.

Overall results for the micronucleus tests in vivo in B6C3F1 mice with Monuron was positive.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Principles of method if other than guideline:
The mouse bone marrow chromosomal aberration test protocol is found in McFee [1989], Shelby et al. [1989], and Tice et al. [1988].
-McFee A (1989): Genotoxic potency of three quinoline compounds evaluated in vivo in mouse bone marrow cells. Environ Mol Mutagen 13:325-331.
-Shelby MD, Gulati DK, Tice RR, Wojciechowski JP (1989): Results of tests for micronuclei and chromosomal aberrations in mouse bone marrow cells with the human carcinogens 4-aminobiphenyl, treosulphan, and melphalan. Environ Mol Mutagen 13:339-342.
-Tice RR, Boucher R, Luke C, Paquette DF, Melnick RL, Shelby MD (1988): Chloroprene and isoprene: Cytogenetic studies in mice. Mutagenesis 3:141-146.
Species:
mouse
Strain:
B6C3F1
Sex:
male
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil (for Monuron)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Monuron was dissolved in corn oil (injection volume = 0.4 mL).
Duration of treatment / exposure:
single intraperitoneal injection
Frequency of treatment:
once
Post exposure period:
Chemicals were tested for induction of chromosomal aberrations in mouse bone marrow cells using two different sacrifice times. The first used a harvest time of 17 hr (standard protocol) and the second, conducted when no evidence of aberration induction was seen at 17 hr, used a harvest time of 36 hr (extended protocol).
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
8 per dose per group
Control animals:
yes, concurrent vehicle
Positive control(s):
not specified
Tissues and cell types examined:
bone marrow cells of femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: As with the micronucleus test, preliminary dose determination experiments were conducted.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):Chemicals were tested for induction of chromosomal aberrations in mouse bone marrow cells using two different sacrifice times. The first used a harvest time of 17 hr (standard protocol) and the second, conducted when no evidence of aberration induction was seen at 17 hr, used a harvest time of 36 hr (extended protocol). Male B6C3F1 mice (8 animals/dose group) received a single intraperitoneal injection with the chemical dissolved in corn oil or PBS (injection volume = 0.4 mL). Isoprene exposure was by inhalation for 14 days. Solvent control mice received equivalent injections of the solvent alone. Concurrent positive control groups were run for each test (data not presented). Mice were subcutaneously implanted with a BrdUrd tablet [McFee et al., 1983] 18 hr before the scheduled harvest (for the 17 hr harvest, this required BrdUrd implantation to precede injection with the test chemical by 1 hr). The use of BrdUrd allowed selection of first metaphase cells for scoring. Two hours prior to sacrifice, the mice received an intraperitoneal injection (IP) of coIchicine in saline.

DETAILS OF SLIDE PREPARATION:The animals were euthanized 17 or 36 hr after injection . One or both femurs were removed and the marrow was flushed out with phosphate-buffered saline (pH 7.0). Cells were treated with a hypotonic salt solution, fixed, and dropped onto chilled slides. After a 24 hr drying period, differential chromatid staining was accomplished by the technique of Goto et al. [1982].

METHOD OF ANALYSIS: Fifty well-spread first-division metaphase cells from each animal per treatment group were scored for presence of chromosomal aberrations. Responses were evaluated as the percent aberrant cells, excluding gaps, and the results were tabulated as the mean from all animals within a treatment group, plus or minus the standard error of the mean.

Statistics:
The data for both endpoints were analyzed using the Micronucleus Assay Data Management and Statistical software package (version 1.4) that employed a one-tailed Cochran-Armitage trend test across exposure groups and pairwise comparisons between each exposure group and the concurrent control [ILS, 1990]. The level of significance was set at an alpha level of 0.05. If significant interanimal variability (overdispersion) was determined to exist by a binomial dispersion test [Cochran, 1954], the Cochran-Armitage test statistic was rescaled downward by a coefficient proportional to the binomial dispersion statistic . Because both a trend test and multiple pair-wise comparisons are conducted, the P values required for a significant effect are adjusted as follows. With the alpha level set at 0.05, a P value ≤ 0.025 is considered a significant effect in the trend test (half of the alpha level). For a pairwise comparison, a P ≤ 0.008 is required for significance in a test that includes 3 exposure levels (approximately one-third of 0.025).
A trial may be considered positive based either on a significant trend or a significant pair-wise comparison. The final call on a chemical's activity is based on consideration of results of all the trials conducted and is not limited to outcomes of individual statistical analyses.
In the event that the increase in the dose response curve was not monotonic, the software program permitted trend reanalysis after data at the highest dose only was excluded; in these cases, the alpha was adjusted to 0.01 to protect against false positives.
Sex:
male
Genotoxicity:
negative

Table 1. Monuron (CAS No. 150-68-5) (MN+/ABS-)

Test (Solvent)a

Tissue

Trend

P value

Dose

(mg/kg)

MN-PCE/1000

Survival

(No. of scored)

Micronucleusa(CO)

BM

0.310

(0.003)c*

0

62.5

125

250

1.10 ± 0.10

1.75 ± 0.32

2.80 ± 0.56 *

1.40 ± 0.29

5/5

4/5

5/5

5/5

BM

<0.001*

0

125

250

1.20 ± 0.41

3.70 ± 0.64 *

6.10 ± 1.02 *

5/5

5/5

 

Harvest

time (hr)

Trend

P value

Dose

(mg/kg)

% Cells with ABS

Survival

Chromosome

aberrationsb

(CO)

17

0.726

0

125

250

500

1.25 ± 0.75

2.75 ± 0.84

1.25 ± 0.37

1.25 ± 0.53

8/8

8/8

8/8

8/8

36

0.926

0

125

250

500

1.60 ± 0.75

2.00 ± 0.93

0.75 ± 0,53

0.75 ± 0.53

5/5

8/8

8/8

8/8

aData published in Shelby et al. [1993]. Test performed at Environmental Health Research and Testing (EHRT).

bTest performed at Brookhaven National Laboratory (BNL).

cTrend test P value after dropping high dose group.

*Significant positive effect.

CO corn oil.

Table 2. Summary Results of In Vivo Mouse Bone Marrow Micronucleus and Chromosomal Aberration Tests on 65 Chemicals* (results for Monuron)

Micronucleus test results

Positive

Negative

Monuron (C, 266)

 

Chromosome Aberration test results

Positive

Negative

 

Monuron

* C = carcinogen and NTP technical report number [see Selkirk and Soward, 1993].

Conclusions:
For Monuron no significant elevations in chromosomal aberrations occurred at 17 or 36 hr sampling times, using same and higher doses as in the micronucleus tests.
Executive summary:

Tests for the induction of chromosomal aberrations (ABS) and micronuclei (MN) in bone marrow cells of mice have been conducted on 65 chemicals. Although these tests were not conducted with the purpose of comparing the outcomes of these two in vivo genetic toxicity endpoints, the availability of these test results permits such a comparison.

With Monuron a positive result in the first MN test was reproduced with higher effects observed at the same dose levels. Using the same and higher doses, no significant elevations in ABS occurred at 17 or 36 hr sampling times.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Mode of Action Analysis / Human Relevance Framework

No mutagenicity was observed in the bacterial and mammalian gene mutation study. The positive findings with Monuron in the in vitro Chromosome Aberration Test and Sister Chromatid Exchange assay and in the in vivo Micronucleus assay in mice indicate this compound to be a clastogen. However, as the effect was not dose proportional, questions were raised about the mode of action of this chemical and the nature of its clastogenicity (Sharma et al., 1987: Mutagenic potential of a substituted urea herbicide, Monuron, Cytologica 52: 841-846, 1987) . 

Additional information

Genotoxicity in vitro

Monuron was tested in a key Ames mutation tests in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 in a modified preincubation test in the absence and presence of metablic activation.up to 10000 µg/plate (Haworth et al., 1983). The positive control chemicals were tested concurrently with each test chemical. Monuron tested negative with and without metabolic activation under the conditions of this experiment.

Monuron was tested in a key Chromosome Aberration Test in Chinese hamster ovary (CHO) cells for the study of induction of chromosome aberrations with and without exogenous metabolic activation up to a maximum dose of 5-10 mg/ mL or to the limits of solubility in culture (Galloway et al., 1987). For Monuron the aberration test was positive with metabolic activation. The negative slope in the dose-response relation is likely to be the result of insolubility, since precipitate of test compound was visible at all the dose levels scored.

Monuron was tested in a key Sister Chromatid Exchange (SCE) assay in Chinese hamster ovary (CHO) cells up to toxic doses with and without metabolic activation (Galloway et al., 1987). The outcome with and without S9 was positive, showing a weak effect in the presence of marked cell cycle delay. Of the 70 positive SCE tests for which information on cell kinetics was recorded, there were 28 (41%) cases in which increases in SCEs were observed at doses lower than those causing cell cycle delay. In the remaining 42 tests, increases in SCEs were detected only at doses that resulted in cell cycle delay. The importance of increases in SCEs at toxic levels may be questioned. 

Monuron was tested in a key Mammalian mutagenicity assay for its mutagenic potential in the L5178Y tk+/-mouse lymphoma cell forward mutation assay, in the absence (up to 1600 µg/mL) and presence of metabolic activation (up to 1100 µg/mL). Monuron was not identified as a mutagen (McGregor et al., 1988).

 

Genotoxicity in vivo

Monuron tested in a key mouse bone marrow micronucleus test that employed three daily exposures by intraperitoneal injection (Shelby et al., 1993). The initial test was performed at 0, 62.5, 125 and 250 mg/kg bw with corn oil as solvent control and DMBA in corn oil as positive control. For Monuron a repeat study was performed at 0, 125 and 250 mg/kg bw. The initial test was negative to 250 mg/kg by trend analysis but the trend test was positive if the high dose group was omitted. The repeat test was positive by trend analysis with the 125 and 250 mg/kg dose groups elevated significantly above the control. Overall results for the micronucleus test in vivo in B6C3F1 mice with Monuron was positive.

Finally a, key in vitro chromosomal aberration TEST (CAT) in bone marrow cells of mice was also conducted with Monuron injected intraperitoneally in mice at 0, 1250, 250 and 500 mg/kg bw. For Monuron no significant elevations in chromosomal aberrations occurred at 17 or 36 hr sampling times, using same and higher doses as in the micronucleus tests (Shelby and Witt, 1995).

Justification for classification or non-classification

Despite the positive results for Monuron in eukaryotic systems, there is no evidence that Monuron causes germ cell damage. A classification is therefore not warranted according to CLP (No. 1272/2008 of 16 December 2008).