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REACH

Desmedipham

EC number: 237-198-5 | CAS number: 13684-56-5

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Studies with desmedipham are available for all endpoints (i.e. gene mutation, structural and numerical chromosomal aberration). Ames tests with desmedipham are negative; a mammalian cell mutation (HPRT) assay is also negative. A positive result is reported in a further mammalian cell mutation (mouse lymphoma) assay. A chromosomal aberration assay reports an equivocal response, whereas a micronucleus assay in human lymphocytes reports a positive response at cytotoxic concentrations.

Test method/ species	Result	Assessment	Reference
OECD 471 - Four strains of S. typhimurium + E. coli WP2 (+/- S9). Tested up to limit concentration with evidence of toxicity.	There was no evidence of mutagenicity in this study with desmedipham.	Supporting study	Poth (1990)
OECD 471 - Five strains of S. typhimurium (not TA102). Tested up to 10000 ug/plate (+/- S9) with cytotoxicity and precipitation.	There was no evidence of mutagenicity in this study with desmedipham.	Supporting study	Dillon (1990)
OECD 471 - Five strains of S. typhimurium (with TA102). Tested up to 5000 ug/plate (+/- S9) with cytotoxicity and precipitation.	There was no evidence of mutagenicity in this study with desmedipham.	Weight of Evidence	Sokolowski (2014)
OECD 473 - Investigation of chromosomal aberration in cultured human lymphocytes (+/-S9) at up to precipitating concentrations	Desmedipham (98.0% purity) did not induce structural chromosomal aberrations in cultured human lymphocytes	Supporting study	Allen et al (1985)
OECD 473 - Investigation of chromosomal aberration in CHO cells (+/-S9) at up to cytotoxic concentrations.	Equivocal result. There was some evidence of chromosomal damage and endoreduplication at cytotoxic concentrations.	Positive control responses were not satisfactory. Test material purity was not reported. - Limited reliability due to methodological limitations (positive control responses).	Leddy and Murie (1991)
OECD 487 - In vitro micronucleus assay in cultured human lymphocytes, at up to cytotoxic concentrations.	Desmedipham (98.7% purity) showed a positive response for the induction of micronuclei (+S9).	Weight of evidence	Gilby (2017)
OECD 476 - Mouse lymphoma assay performed at up to cytotoxic/precipitating concentrations (+/-S9)	Desmedipham (98.0% purity) was positive in this study (+/-S9)	Weight of evidence	Brown et al (1985)
OECD 476 - CHO HPRT assay performed at up to cytotoxic concentrations (+/-S9)	Desmedipham (98.0% purity) was negative in this study (+/-S9)	Supporting study	Cattanach and Riach (1985)
OECD 482 (deleted 2014) - Rat liver hepatocyte UDS in vitro	Desmedipham (98.0% purity) did not induce UDS under the conditions of this study	The study was performed to a deleted test method and is no longer considered valid	Cifone (1988)
OECD 482 (deleted 2014) - Rat liver hepatocyte UDS in vitro	Desmedipham (98.0% purity) did not induce UDS under the conditions of this study	The study was performed to a deleted test method and is no longer considered valid	Mohammed (1988)
NA - Rec assay	Desmedipham (98.0% purity) did not induce differential toxicity in Rec+/Rec- under the conditions of this study	The study was performed to a non-standard test method and is not considered valid	Enomoto (1988)
OECD 474 - Mouse bone marrow micronucleus assay (NMRI, m/f)	Desmedipham did not induce the formation of micronuclei in PCEs at dose level up to 5000 mg/kg bw.	Supporting study	Herbst-Guenard and Terrier (1985)
OECD 474 - Mouse bone marrow micronucleus assay (CD-1, m/f)	Desmedipham did not induce the formation of micronuclei in PCEs at dose level up to 2000 mg/kg bw.	Supporting study	Holmstrom and Innes (1991)
OECD 474 - Rat bone marrow micronucleus assay (CD, m/f)	Desmedipham did not induce the formation of micronuclei in PCEs at dose level up to 2000 mg/kg bw.	Key study	Mason (2003)
OECD 489 - Rat Comet assay (liver, stomach)	Desmedipham was negative in this Comet assay performed at dose levels up to 2000 mg/kg bw.	Key study	Bruce (2017)

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-02-16 to 2017-04-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Appearance: White solid.
Storage conditions: Ambient (15-25°C), in the dark.

Species / strain / cell type:

lymphocytes: Human lymphocytes

Cytokinesis block (if used):

Cytochalasin B, at a final concentration of 6 µg/mL, was added to all cultures

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9:
Male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver.
- method of preparation of S9 mix:
The S9 mix contained: S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors were filter sterilized before use.

Test concentrations with justification for top dose:

Preliminary toxicity test: 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL.
Main tests: -S9 mix (3 hours): 12.5, 125, 250, 275, 300, 325, 350 and 400 µg/mL.
+S9 mix (3 hours): 12.5, 125, 200, 225, 250, 275 and 300 µg/mL.
-S9 mix (20 hours): 12.5*, 65, 75*, 85, 95, 105, 115 and 125* µg/mL.
Additional Main tests: -S9 mix (3 hours): 5*, 50, 100, 125*, 150, 175*, 200, 225, 250, 275 and 300 µg/mL.
+S9 mix (3 hours): 5*, 50, 100, 125*, 150*, 175, 200*, 225, 250, 275 and 300 µg/mL.

*cultures that were analyzed for micronucleus frequency.

The maximum final concentration tested in the preliminary toxicity test was 2000 µg/mL as this is the standard limit concentration within this test system as recommended in the regulatory guidelines.

The highest concentration was intended to be that which caused a depression in the cytokinesis block proliferative index (CBPI) equivalent to 55±5% cytotoxic with the concurrent vehicle control or, where no cytotoxicity was observed, the maximum concentration as recommended in the test guidelines or the limit of solubility.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for percentage of solvent in the final culture medium: The final volume of DMSO added to the cultures was 0.5% v/v.

Untreated negative controls:

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Lymphocyte cultures were incubated for approximately 48 hours following stimulation with PHA, before addition of the test item. The test item was prepared in the vehicle and dilutions made for both sets of cultures. Single cultures were prepared for each treatment level and duplicate cultures were prepared for vehicle controls. S9 homogenate was present in appropriate cultures at a final concentration of 2% v/v.
Before treatment, all cultures were centrifuged and re-suspended in the required volume of fresh medium, taking into account the treatment volume and S9 mix volume, where required. Test item preparations were added to cultures at 0.5% v/v. Cultures were incubated at 34 to 39°C for 3 hours.
The cells were centrifuged, and the medium was replaced with fresh medium. Cytochalasin B, at a final concentration of 6 µg/mL, was then added to all cultures. The cultures were incubated for a further 17 hours until the scheduled harvest time.

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate cultures were prepared for each treatment level and positive control cultures; quadruplicate cultures were prepared for vehicle controls.
- Number of independent experiments: two slides were prepared from each culture.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 20 hours.

- The cells were harvested by centrifugation at 500 g for 5 minutes. The supernatant was removed and the cell pellet re-suspended and treated with a 4 mL hypotonic solution (0.075M KCl) at 34 to 39°C, cultures were then incubated for 3 minutes at 34 to 39°C to cause swelling. Cultures were agitated, 4 mL of ice-cold fixative (3:1 v/v methanol: acetic acid) was added slowly onto the culture surface and the cultures were slowly inverted to mix. The cultures were centrifuged at 500 g for five minutes. The supernatant was removed, and the cell pellet re suspended. A further 4 mL of fresh fixative was then added, and the cells stored at 2 to 8°C until slide preparation.

FOR MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: Cytochalasin B, at a final concentration of 6 µg/mL, was added to all cultures.

- Methods of slide preparation and staining technique used including the stain used:
The cultures were centrifuged at 500 g for 5 minutes and the supernatant removed. A homogeneous cell suspension was prepared. Pre-cleaned microscope slides were prepared for each culture by aliquoting the re-suspended cells onto the slides and allowing the slides to air-dry. Two slides were prepared from each culture. The slides were then stained using an acridine orange solution at 0.0125 mg/mL in purified water. The remaining cell cultures were stored at 2 to 8°C until slide analysis was complete.

Prior to micronucleus analysis, all slides were randomly coded. Interphase cells were examined by fluorescence microscopy and the incidence of micronucleated cells per 1000 binucleate cells per culture were scored where possible.

- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
Cells were included in the analysis provided the cytoplasm remained essentially intact and any micronuclei present were separate in the cytoplasm or only just touching the main nucleus (not connected to the nucleus by a nucleoplasmic bridge). Micronuclei should lie in the same focal plane as the cell and should possess a generally rounded shape with a clearly defined outline. The main nuclei of the binucleate cells scored for micronuclei should be of approximately equal size. The diameter of the micronucleus should be between 1/16 and 1/3 that of the main nucleus. The color of the micronuclei should be the same or lighter than the main nucleus. There should be no micronucleus like debris in the surrounding area.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Analysis of Cytotoxicity Data:
Cytotoxicity = 100-100{(CBPIT-1)/(CBPIC-1)}

Where CBPI = (No. mononucleate cells +2 x No. binucleate cells + 3 x No. multinucleate cells)/Total number of cells.
T = test item treatment culture
C = solvent control culture

Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytotoxicity.

- Criteria for Assessing Genotoxic Potential:
Providing that all of the acceptance criteria have been met, the test item was considered to be clearly positive if, in any of the experimental conditions examined:
At least one of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent vehicle control.
The increase in the frequency of micronucleated cells is dose-related when evaluated with an appropriate trend test.
Any of the results are outside the distribution of the historical vehicle control data (above the upper control limit).
If all of these criteria are met, the test item was considered able to induce chromosome breaks and/or gain or loss in the test system.
Providing that all of the acceptance criteria have been met, a negative response will be claimed if, in all of the experimental conditions examined:
None of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent vehicle control.
There is no concentration-related increase when evaluated with an appropriate trend test.
All results are inside the distribution of the historical vehicle control data (below the upper control limit).
If all of these criteria are met, the test item was considered unable to induce chromosome breaks and/or gain or loss in the test system.

Evaluation criteria:

Acceptance and assessment criteria
The following criteria were applied for assessment of assay acceptability:
- The concurrent vehicle control must be considered acceptable for addition to the laboratories historical vehicle control database (lie below or close to the upper control limit).
- Concurrent positive controls must induce responses that are compatible with the laboratories historical positive control database and produce statistically significant increases compared with the concurrent vehicle control.
- The criteria for selection of the top dose concentration are consistent with those outlined in the study plan.
Tests that did not fulfill the required criteria were rejected and therefore are not reported.

Statistics:

The analysis assumed that the replicate was the experimental unit. An arcsine transformation was used to transform the data. Desmedipham technical treated groups were then compared to control using Williams’ tests (Williams 1971, 1972). Positive controls were compared to control using t tests. Trend tests have also been carried out using linear contrasts by group number. These were repeated, removing the top dose group, until there were only 3 groups. Statistical significance was declared at the 5% level for all tests. Data were analyzed using SAS (SAS Institute 2002) and Quasar (Quasar 1.5 2016).

Species / strain:

lymphocytes: Human Lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Additional 3-Hour Treatment in the Absence of S9 Mix: A reduction in CBPI compared with vehicle control values equivalent to 54.6% cytotoxicity, was obtained with Desmedipham at 175 µg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: Human Lymphocytes

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Additional 3-Hour Treatment in the Presence of S9 Mix: A reduction in CBPI compared with vehicle control values, equivalent to 60.2% cytotoxicity, was obtained with Desmedipham at 200 µg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

See the results' tables in the file attached in "Overall remarks, attachments"

1. Preliminary Toxicity Test
In all exposure conditions the highest concentration tested was 2000 µg/mL and precipitate was observed by eye at the end of treatment at 500 µg/mL and above. Therefore cultures treated at 1000 µg/mL and above were discarded.
After 3-hour treatment in the absence of S9-mix, a reduction in CBPI compared with vehicle control values, equivalent to 32.0% cytotoxicity, was obtained with Desmedipham technical at 250 µg/mL. Overt toxicity was observed at 500 µg/mL.
After 3-hour treatment in the presence of S9-mix, a reduction in CBPI compared with vehicle control values, equivalent to 98.6% cytotoxicity, was obtained with Desmedipham technical at 500 µg/mL.
After 20-hour treatment in the absence of S9-mix, a reduction in CBPI compared with vehicle control values, equivalent to 97.3% cytotoxicity, was obtained with Desmedipham technical at 250 µg/mL. Overt toxicity was observed at 500 µg/mL.
These data were used to select concentrations for the main test.

2. Main experiment:

3-Hour Treatment in the Absence of S9 Mix:
The cytotoxicity results of the main test, 3-hour treatment in the absence of S9 mix, are presented in table 1.

Concentrations of Desmedipham technical used for the main micronucleus test were 12.5, 125, 250, 275, 300, 325, 350 and 400 µg/mL. No precipitate was observed by eye at the end of treatment at any concentration tested. A reduction in CBPI compared with vehicle control values equivalent to 87% cytotoxicity, was obtained with Desmedipham technical at 275 µg/mL. As an inappropriate toxicity profile was achieved, i.e. no test item concentration affording 50-60% cytotoxicity, the test was discontinued and an additional test performed using modified concentrations.

Additional 3-Hour Treatment in the Absence of S9 Mix:

The results and statistical analysis of the additional main test, 3-hour treatment in the absence of S9 mix, are presented in tables 2 and 3.

Concentrations of Desmedipham technical used for the main micronucleus test were 5, 50, 100, 125, 150, 175, 200, 225, 250, 275 and 300 µg/mL. No precipitate was observed by eye at the end of treatment at any concentration tested. A reduction in CBPI compared with vehicle control values equivalent to 54.6% cytotoxicity, was obtained with Desmedipham technical at 175 µg/mL.
Concentrations of Desmedipham technical selected for micronucleus analysis were 5, 125 and 175 µg/mL.

Micronucleus Analysis:

Desmedipham technical caused a statistically significant increase in the number of binucleate cells containing micronuclei at 175 µg/mL (p<0.01) when compared with the vehicle controls. The increase was above the laboratory historical upper control limit and was associated with a significant linear trend (p<0.001). An increased number of mitotic figures were observed on the slides treated at 175 µg/mL.

Mean micronucleus induction in the vehicle control and Desmedipham technical treatment concentrations 5 and 125 µg/mL were below the laboratory historical upper control limit. The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei, demonstrating the sensitivity of the test system.

3-Hour Treatment in the Presence of S9 Mix
The cytotoxicity results of the main test, 3-hour treatment in the presence of S9 mix, are presented in table 4.

Concentrations of Desmedipham technical used for the main micronucleus test were 12.5, 125, 200, 225, 250, 275 and 300 µg/mL. No precipitate was observed by eye at the end of treatment at any concentration tested. A reduction in CBPI compared to vehicle control values, equivalent to 87.2% cytotoxicity, was obtained with Desmedipham technical at 225 µg/mL. As an inappropriate toxicity profile was achieved, i.e. no test item concentration affording 50-60% cytotoxicity, the test was discontinued and an additional test performed using modified concentrations.

Additional 3-Hour Treatment in the Presence of S9 Mix
The results and statistical analysis of the additional main test, 3-hour treatment in the presence of S9 mix, are presented in tables 5 and 6.

Concentrations of Desmedipham technical used for the main micronucleus test were 5, 50, 100, 125, 150, 175, 200, 225, 250, 275 and 300 µg/mL. No precipitate was observed by eye at the end of treatment at any concentration tested. A reduction in CBPI compared with vehicle control values, equivalent to 60.2% cytotoxicity, was obtained with Desmedipham technical at 200 µg/mL.
Concentrations of Desmedipham technical selected for micronucleus analysis were 5, 125, 150 and 200 µg/mL.

Micronucleus Analysis
Desmedipham technical caused statistically significant increases in the number of binucleate cells containing micronuclei at 150 and 200 µg/mL (p<0.001) when compared with the vehicle controls. The increases were above the laboratory historical upper control limit and were associated with a significant linear trend (p<0.001).

Mean micronucleus induction in the vehicle control and Desmedipham technical treatment concentrations 5 and 125 µg/mL were below the laboratory historical upper control limit. The positive control compound (cyclophosphamide) caused a statistically significant increase in the number of binucleate cells containing micronuclei, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

20-Hour Treatment in the Absence of S9 Mix
The results and statistical analysis of the main test, 20-hour treatment in the absence of S9mix, are
presented in tables 7 and 8.

Concentrations of Desmedipham technical used for the main micronucleus test were 12.5, 65, 75, 85, 95, 105, 115 and 125 µg/mL. No precipitate was observed by eye at the end of treatment at any concentration tested. A reduction in CBPI compared to vehicle control values, equivalent to 50.6% cytotoxicity, was obtained with Desmedipham technical at 125 µg/mL. Concentrations of Desmedipham technical selected for micronucleus analysis were 12.5, 75 and 125 µg/mL.

Micronucleus Analysis
Desmedipham technical did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls. Mean micronucleus induction in the vehicle control and all Desmedipham technical treatment concentrations were below the laboratory historical upper control limit.
The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei, demonstrating the sensitivity of the test system.

Conclusions:

It was concluded that Desmedipham technical showed evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described.

Executive summary:

This study was designed to assess the potential of Desmedipham technical to cause an increase in the induction of micronuclei in cultured human peripheral blood lymphocytes in vitro.
The study consisted of a preliminary toxicity test, a main micronucleus test and an additional test for both 3-hour treatments.

Human lymphocytes in whole blood culture, stimulated to divide by addition of phytohaemagglutinin (PHA) 48 hours prior to treatment, were exposed to Desmedipham technical for 3 hours in both the absence and presence of exogenous metabolic activation (S9 mix) and for 20 hours in the absence of S9 mix. The maximum final concentration to which the cells were exposed was 2000 μg/mL, dosed at 0.5% v/v, in order to test up to the maximum concentration as recommended in the guidelines that this assay follows. Vehicle (DMSO) and positive control cultures were included in all appropriate test conditions.

Three Desmedipham concentrations were assessed for determination of induction of micronuclei for the 3-hour and 20-hour treatments in the absence of S9-mix. Four concentrations were assessed for the 3-hour treatment in the presence of S9-mix. The highest concentration selected for all exposures, was that which caused a reduction in Cytokinesis-block proliferative index (CBPI) equivalent to approximately 55±5% cytotoxicity.

Following 3-hour treatment in the absence of S9 mix, a reduction in CBPI equivalent to 54.6% cytotoxicity was obtained with Desmedipham at 175 μg/mL. Concentrations of Desmedipham technical selected for micronucleus analysis were 5, 125 and 175 μg/mL. Following 3-hour treatment in the presence of S9 mix, a reduction in CBPI equivalent to 60.2% cytotoxicity was obtained with Desmedipham technical at 200 μg/mL.

Concentrations of Desmedipham technical selected for micronucleus analysis were 5, 125, 150 and 200 μg/mL.
In the absence of S9 mix following 20-hour treatment, a reduction in CBPI equivalent to 50.6% cytotoxicity was obtained with Desmedipham technical at 125 μg/mL. Concentrations of Desmedipham selected for micronucleus analysis were 12.5, 75 and 125 μg/mL.

In the absence of S9 mix, following 3-hour treatment, Desmedipham technical caused a statistically significant increase in the number of binucleate cells containing micronuclei at 175 μg/mL (p<0.01) when compared with the vehicle controls. The increase was above the laboratory historical upper control limit and was associated with a significant linear trend (p<0.001).

In the presence of S9 mix, following 3-hour treatment, Desmedipham technical caused statistically significant increases in the number of binucleate cells containing micronuclei at 150 and 200 μg/mL (p<0.001) when compared with the vehicle controls. The increases were above the laboratory historical upper control limit and were associated with a significant linear trend (p<0.001).

In the absence of S9 mix, following 20 hour treatment, Desmedipham technical did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls.

The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1990-11-05 to 1991-03-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The test was conducted according to OECD TG 471. This guideline was revised in 1997 with new recommendations. Two were not meet in this test: Only strains with GC base pair at the primary reversion site were included in the assay which may not detect certain oxidising mutagens, cross-linking agents and hydrazines. 2- Aminoanthracene was used as the only indicator of the efficacy of the S9-mix. Each batch of the S9 should also be characterised with a mutagen that requires metabolic activation by microsomal enzymes, e.g., benzo(a)pyrene, dimethylbenzanthracene.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

See "Rationale for reliability incl. deficiencies" for more information

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

White powder, stored in the dark at ambient temperature.

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Livers of rats induced by the polychlorinated biphenyl mixture.
- method of preparation of S9 mix: Ice-cold 0.05 M phosphate buffer, pH 7.4, was added to the following pre-weighed reagents to give final concentrations in the S9 mix of:

NADP di-Na salt 4 mM
Glucose-6-phosphate di-Na salt 25 mM
MgCl2.6H2O 8 mM
KCl 33 mM

This solution was immediately filter-sterilised by passage through a 0.45 urn Millipore filter and mixed with the liver 9,000 g supernatant fluid in the following proportion:

co-factor solution 9 parts liver preparation 1 part
This combination of co-factors and liver preparation was called the S9 mix.

- quality controls of S9 (enzymatic activity, metabolic capability, total protein concentration)

Test concentrations with justification for top dose:

33, 100, 333, 1000, 3333 and 10000 mg/plate. The dose levels used selected on the basis of the results of the toxicity test.

Vehicle / solvent:

DMSO:
Desmedipham technical and the positive control substances, except sodium azide, were dissolved in dimethylsulphoxide. Sodium azide was dissolved in sterile, ultra-pure water.

Untreated negative controls:

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate.
- Number of independent experiments: 2.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2 days.

Evaluation criteria:

A test article was considered positive if there was either a dose-related response or a reproducible effect in independent tests. A significant mutagenic response was recorded, if at least a doubling of the mean concurrent vehicle control values was observed in strains TA 1535, TA 1537, TA 1538 and TA 98 at some concentrations of the test substances, and a 1.5 fold increase over the control value in strain TA 100.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight toxicity to bacteria was observed at 10000 µg/plate with and without S9. Precipitation of the test substance was noted at 10000 mg/plate in all strains with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True 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:

cytotoxicity

Remarks:

Very slight toxicity was also noted at 10000 mg/plate. Precipitation of the test substance was noted at 10000 mg/plate in all strains with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Very slight toxicity was also noted with and without S9 at 10000 mg/plate. Precipitation of the test substance was noted at 10000 mg/plate in all strains with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Very slight toxicity was also noted with and without S9 at 10000 mg/plate. Precipitation of the test substance was noted at 10000 mg/plate in all strains with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True 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:

cytotoxicity

Remarks:

Slight toxicity to bacteria was observed at 10000 µg/plate with and without S9. Precipitation of the test substance was noted at 10000 mg/plate in all strains with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No mutagenic activity was observed in any of the 5 bacterial strains used, with or without S9.

See the Results' tables in the attched file in "Overall remarks, attachments"

Conclusions:

Desmedipham was not mutagenic in the Salmonella typhimurium strains used in the study at doses up to 10000 µg/plate. The study was acceptable.

Executive summary:

Desmedipham technical was tested for mutagenic activity in Salmonella tvphimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 at concentrations ranging from 33 µg to 10000 µg per plate.

The tests were conducted on agar plates in the presence and absence of induced rat liver preparation and co-factors (S9 mix) required for mixed-function oxidase activity.

Concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the S9 mix.

Desmedipham technical did not induce mutagenic activity in any of the 5 bacterial strains used, either in the presence or absence of S9 mix.

A slightly thin background lawn of microcolonies was noted in Salmonella typhimurium TA 1535 and TA 100 at 10000 µg per plate in the presence and absence of S9 mix. A thin lawn was noted in TA 1537, TA 1538 and TA 98 at 10000 µg per plate in the presence and absence of S9 mix.

Precipitation of the test substance was observed at 10000 µg per plate in all 5 bacterial strains, in both activation conditions.

It was concluded that Desmedipham technical was not mutagenic to Salmonella typhimurium when tested in dimethylsulphoxide at concentrations extending into the toxic range.

Reference 3

Endpoint:: in vitro gene mutation study in mammalian cells
Type of information:: experimental study
Adequacy of study:: supporting study
Study period:: 1990-07-16 to 1991-04-22
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: guideline study with acceptable restrictions
Qualifier:: according to guideline
Guideline:: OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Version / remarks:: 1984
Deviations:: yes
Remarks:: The study was conducted according to OECD Guideline 476 (1984). This TG was revised in 1997 and latest in July 2015. The study is deviating from current TG in the lack of historical control data.
GLP compliance:: yes (incl. QA statement)
Type of assay:: in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:: hypoxanthine-guanine-phosphoribosyl transferase (HPRT)
Species / strain / cell type:: Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):: Cell line K1BH4.
Metabolic activation:: with and without
Metabolic activation system:: S9 from the livers of Aroclor 1254 induced rats.
Test concentrations with justification for top dose:: 10, 20, 40, 80, 160, 320 µg/ml
Vehicle / solvent:: DMSO
Negative solvent / vehicle controls:: yes
Positive controls:: yes
Positive control substance:: 3-methylcholanthrene; ethylmethanesulphonate
Details on test system and experimental conditions:: Desmedipham technical (purity not reported) was tested for mutagenic activity using the Chinese hamster ovary (CHO) hypoxanthine-guanine-phosphoribosyl transferase (HPRT) locus assay in the presence and absence of S9 from the livers of Aroclor 1254 induced rats. Preliminary toxicity tests demonstrated that desmedipham induced complete killing of the cell population and precipitation at 500 and 5000 µg/ml both with and without S9. The positive control substances used were ethyl methanesulphonate (EMS 200 µg/ml, -S9) and 3-methylcholanthrene (3-MC 3 µg/ml, +S9). Desmedipham, EMS and 3-MC were dissolved and diluted in DMSO, which was used as solvent control.
Four independent mutation assays were performed (2 with S9, 2 without S9) and the final concentrations of desmedipham ranged from 10 to 320 µg/ml. Each experiment consisted of a vehicle control (4 cultures), positive control (3 cultures) and desmedipham (5 or 6 concentrations, 2 cultures per concentration). The cells were exposed to the test substance for 5 hours, after which the cells were washed, cultured, and incubated for a total period of 7 days to allow for expression of the mutant phenotype. The results were expressed as the number of mutants per 10^6 surviving cells.
The purity of the test substance was not specified. 2.5 x 10^7 cells were treated and allowed expression, and 1 x 10^6 cells plated per dish per culture in the mutation assay.
Evaluation criteria:: The test result at a single dose was considered positive if the mutant fraction was at least doubled compared to the vehicle control value and the cloning efficiency was at least 10% of the vehicle control. An experiment was positive if the response in at least one of the 3 highest acceptable doses was significant and was associated with an increase in mutant numbers and/or there was a dose related trend. A compound was mutagenic or non-mutagenic if either a significant or insignificant response, respectively, was reproducible under the same activation conditions.
: Key result
Species / strain:: Chinese hamster Ovary (CHO)
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Positive controls validity:: valid
Additional information on results:: No significant evidence of mutagenic activity was obtained at any tested concentration (10, 20, 25, 40, 55, 70 µg/ml). The higher concentrations (80, 85, 100, 160, 320 µg/ml) were toxic in all 4 assays. There was an increase in mutant frequency at 55 µg/ml (with S9, assay 3) and at 70 µg/ml (without S9, assay 2), but in these cases the relative total growth (RTG) was very low (4% and 0.1%) compared to the control value (100-116%). The solvent and positive control values were within the normal ranges expected for the test laboratory and reported in the literature. Data is summarised in the tables attached below.
Remarks on result:: other: negative
Conclusions:: Study was deemed unacceptable but valid.
Test result is negative. Under the test conditions the desmedipham was not mutagenic in Chinese hamster ovary cell HPRT locus mutation test.
Executive summary:: Desmedipham was tested for mutagenic activity at the hypoxanthinein guanine-phosphoribosyl transferase (hqprt) locus Chinese hamster ovary (CHO) cells in both the presence and absence of a rat liver preparation and co-factors required for mixedactivity function oxidase activity (S9 mix).

Preliminary toxicity tests demonstrated that Desmedipham induced complete killing of the cell population in both the absence and presence of S9 mix at 500 and 5000 μg.ml-1.

In 4 independent mutation assays (2 in the presence and 2 in the absence of S9 mix), the final concentration of Desmedipham in the medium ranged from 10 to 320 μg.ml-1. The higher concentrations of Desmedipham were toxic in all 4 assays.

No significant evidence of mutagenic activity of Desmedipham was obtained in either the presence or absence of S9 mix in any experiment. Concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the S9 mix.

It is concluded that Desmedipham is not mutagenic to Chinese hamster ovary cells at the hqprt locus, in either the presence or absence of S9 mix when tested in dimethylsulphoxide at concentrations extending into the toxic range.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989-12-05 to 1990-07-14

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

OECD TG 471 and 472. TG 471 was revised in 1997 with new recommendations. One was not meet in this assay: 2-Aminoanthracene was used as the only indicator of the efficacy of the S9-mix. Each batch of the S9 should also be characterised with a mutagen that requires metabolic activation by microsomal enzymes, e.g., benzo(a)pyrene, dimethylbenzanthracene.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

May 26, 1983

Deviations:

yes

Remarks:

See "Rationale for reliability incl. deficiencies" for more information

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

1983-05-26

Deviations:

yes

Remarks:

See "Rationale for reliability incl. deficiencies" for more information

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 84/449, L 251, B 14, p. 143-145

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Environmental Protection Agency, Code of Federal Regulations, "The salmonella typhimurium reverse mutation assay"

Version / remarks:

Title 40, Subpart F-Genetic Toxicity, Revision July 1, 1986

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Agricultural chemicals laws and regulations, Japan (II), 1985, "Bacterial Reverse Mutation Assay"

Version / remarks:

1985

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: "Standards for Mutagenicity Tests Using Microorganism", Japan Industrial Safety and Health Association

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

solid, colourless

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: from the livers of rats.

- method of preparation of S9 mix:
S9 mix preparation was performed according to Ames et al.
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution in a ratio 3:7.

- concentration or volume of S9 mix and S9 in the final culture medium:
The composition of the cofactor solution was concentrated to yield the following concentrations in the S9 mix:

8 mM MgCl2
33 mM KC1
5 mM glucose-6-phosphate
5 mM NADP

in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

During the experiment the S9 mix was stored in an ice bath.

Test concentrations with justification for top dose:

Experiment I /TA 1535 + TA 1537:
10.0, 33.3, 100.0, 333.3, 666.6, 1000.0 and 5000.0 µg/plate

Experiment I/TA 98, TA 100 + WP2:
10.0, 33.3, 100.0, 333.3, 1000.0 and 5000.0 µg/plate

Experiment II/ all strains:
10.0, 33.3, 100.0, 333.3, 666.6, 1000.0 and 5000.0 µg/plate

According to the results of this pre-experiment the concentrations applied in the main experiments are chosen. The concentration range include at least two decadic logarithms.

The maximum concentration is 5000.0 (µg/plate, unless limited by toxicity or solubility of the test article. The concentration range includes at least two decadic logarithms. In this study at least five adequate spaced concentrations are tested. Two independent experiments are performed.

In case the results of the pre-experiment are in accordance with the criteria described above, these data are reported as a part of the main experiments.

Vehicle / solvent:

- Solvent used: ethanol

- Justification for choice of solvent: The solvent was chosen because of its solubility properties and its relative non-toxicity for the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-aminoanthracen, 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 100 µl Bacteria suspension (cf. test system, pre-culture of the strains).
- Test substance added in agar.
- Exposure duration: 72 hours at 37°C in the dark.

Evaluation criteria:

A test article was considered positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration was induced. A test article was considered mutagenic if the number of reversions in strain TA 100 was at least twice as high, and in strains TA 1535, TA 1537 and TA 98 at least three times higher than the spontaneous revertant count.

Statistics:

No appropriate statistical method is available

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The plates incubated with the test article showed normal background growth up to 5000 mg/plate with and without S9 in both independent experiments. When compared to the solvent control, no significant reproducible and dose-dependent increases in the numbers of revertants were observed in any strains tested up to and including 5000 mg/plate. The positive controls showed a distinct increase of induced revertant colonies.

Conclusions:

Desmedipham was not mutagenic in the Salmonella typhimurium and Escherichia coli strains used in the experiment at doses up to 5000 µg/plate. The study was acceptable.

Executive summary:

This study was performed to investigate the potential of Desmedipham to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100 and in addition to the Escherichia coli strain WP2. The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations:

Experiment I /TA 1535 + TA 1537:
10.0, 33.3, 100.0, 333.3, 666.6, 1000.0 and 5000.0 µg/plate

Experiment I/TA 98, TA 100 + WP2:
10.0, 33.3, 100.0, 333.3, 1000.0 and 5000.0 µg/plate

Experiment II/ all strains:
10.0, 33.3, 100.0, 333.3, 666.6, 1000.0 and 5000.0 µg/plate

The plates incubated with the test article showed normal background growth up to 5000.0 (µg/plate with and without metabolic activation in both independent experiments. When compared to the solvent control, no significant, reproducible and dose-dependent increase in the number of revertants was observed in any of the strains tested up to and including 5000 p,g/ plate, either with or without metabolic activation. The presence of liver microsomal activation did not influence these findings. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, Desmedipham is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 5

Endpoint:: in vitro gene mutation study in mammalian cells
Type of information:: experimental study
Adequacy of study:: weight of evidence
Study period:: 1984-07-30 to 1985-02-18
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: guideline study with acceptable restrictions
Qualifier:: according to guideline
Guideline:: OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:: 1984
Deviations:: yes
Remarks:: This study is deviating from current TG in the lack of historical control data.
GLP compliance:: yes (incl. QA statement)
Type of assay:: in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:: thymidine kinase locus
Species / strain / cell type:: mouse lymphoma L5178Y cells
Metabolic activation:: with and without
Metabolic activation system:: S9 from livers of Aroclor 1254 induced Fisher male rats was used for metabolic activation.
Test concentrations with justification for top dose:: two independent sets of tests conducted at 6.3 - 200 µg/ml (first set) and 20 - 200 µg/ml (second set)
Vehicle / solvent:: DMSO
Untreated negative controls:: no
Negative solvent / vehicle controls:: yes
True negative controls:: no
Positive controls:: yes
Positive control substance:: 3-methylcholanthrene; methylmethanesulfonate
Details on test system and experimental conditions:: Before exposure spontaneous homozygous tk-tk- mutants were removed by a cleansing procedure. Cultures containing 6 x 10^6 exponentially growing tk+tk- cells in Fisher’s medium were exposed to desmedipham for 4 hours. Exposed cultures were washed and incubated for 2 days after exposure. On day 2 after exposure the cells were adjusted to 2 x 10^5/ml for mutant selection using TFT (trifluorothymidine). Following incubation for 11 days, the numbers of colonies were counted. The results were expressed as the number of mutants per 10^5 surviving cells.
6 x 10^6 cells were treated and allowed expression, and 1 x 10^6 cells plated per dish per culture in the mutation assay.
Evaluation criteria:: Test compound dilutions were rejected if the relative cell growth was less than 1%, or the cloning efficiency after the expression period was less than 10%. The response at a single dose was considered significant if the cloning efficiency was at least 10% of the concurrent vehicle and the mutation frequency was at least 10 x 10-^5. An experiment was positive if the response in at least one of the 3 highest acceptable doses was positive by the criteria described above and was associated with an increase in mutant numbers. A compound was positive if 2 positive experiments were recorded under the same activation conditions.
: Key result
Species / strain:: mouse lymphoma L5178Y cells
Metabolic activation:: with and without
Genotoxicity:: positive
Vehicle controls validity:: valid
Positive controls validity:: valid
Additional information on results:: Preliminary toxicity tests (0.48, 4.8, 48, 480, 4800 µg/ml) demonstrated that desmedipham killed all cells at 480 µg/ml and above with and without S9. Precipitation also occurred at these concentrations.
In the first assay a dose-related increase in mutation frequencies at dose levels of 6.3, 12.5, 25, 50, 100 and 200 µg/ml with S9 was observed; the mutation frequencies were 4, 6, 5, 8, 11 x 10^-5 , respectively, compared to the negative control value (3 x 10^-5). A similar effect was also observed without S9. Desmedipham induced mutagenic responses at the highest clonable level, 100 µg/ml, with and without S9. The relative total growth values at this dose were 31% and 39% of the vehicle control values, with and without S9. The mutation frequencies and absolute mutant numbers for the positive control, MMS was lower than the acceptable value (16 x 10^-5 survivors) at 100 µg/ml without S9. However, since a positive response was obtained with desmedipham at 100 µg/ml without S9, the assay was not rejected. At 50 µg/ml with S9 the mutation frequency (8 x 10^-5) was 2.7 fold higher than the vehicle control value (3 x 10^-5) but below the used critical value (10 x 10^-5).
In the second assay, a clear dose related increase in mutation frequencies was obtained at dose levels of 20, 40, 60, 80 and 100 µg/ml with S9; the mutation frequencies were 7, 8, 9, 19 and 44 x 10^-5 , respectively, compared to the negative control value (5 x 10^-5). Significant increases in both mutation frequencies and absolute mutant numbers over vehicle control values were obtained at 80 and 100 µ g/ml with S9. Increases in the mutation frequencies were 3.8 and 8.8 fold higher than the control value, and the relative total growths compared to the vehicle control value were 37%, 2%, at 80 and 100 µg/ml with S9, respectively.
An increase in mutation frequencies at dose levels of 20, 40, 60, 80 and 100 µg/ml without S9 was recorded; the mutation frequencies were 4, 7, 6, 10 and 37 x 10^-5 , respectively, compared to the negative control value (7 x 10^-5). At 80 and 100 µg/ml without S9 the increases in mutation frequencies were 1.5- and 5.4-fold higher than the control value, and the relative total growths were 50% and 6% of the vehicle control value, respectively. Significant increases in both mutation frequencies and absolute mutant numbers over vehicle control values were obtained at 100 µg/ml without S9.
The solvent control values were within the normal ranges expected for the test laboratory and reported in the literature. The mutation frequencies obtained with the positive controls, MMS in the second assay (but not in the first) and 3-MC in both assays, were within the normal acceptable ranges for this laboratory.
Remarks on result:: other: positive
Conclusions:: Desmedipham was mutagenic in the mouse lymphoma L5178Y thymidine kinase locus (TK+/-) assay, with and without S9, at concentrations between 50 and 100 µg/ml. Dose-related mutagenic responses were observed also at lower dose levels between 20 to 60 µg/ml, with or without S9. The study was acceptable.
Executive summary:: Technical desmedipham was tested for mutagenic activity in the mouse lymphoma L5178Y assay in both the presence and absence of a rat liver preparation and co-factors required for mixed-function oxidase activity (S9 mix}.
Preliminary toxicity tests showed that technical desmedipham killed all the exposed cells at concentrations of 480 μg.mlm1-1 and above in both the presence and absence of S9 mix. Precipitation also occurred at these concentrations.
Technical desmedipham was tested for mutagenic activity in 2 independent assays in the presence and absence of S9 mix at concentrations ranging from 6.25 to 200 ug.m1-1.
In both the presence and absence of S9 mix, technical desmedipham induced significant mutagenic responses at concentrations between 50 and 100 μg.m1-1, where relative total growth ranged between 39 and 2% of the vehicle control values.
It was concluded that technical desmedipham was mutagenic in mouse lymphoma L5178Y cells in both the presence and absence of S9 mix when tested in dimethylsulphoxide at concentrations extending into the toxic range.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1984-10-18 to 1985-01-14

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study was conducted according to OECD Guideline 473 (1983). This TG was revised in 1997 and latest in September 2014. The study is deviating from 1997 TG in the lack of historical control data.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1983

Deviations:

yes

Remarks:

The study was conducted according to OECD Guideline 473 (1983). This TG was revised in 1997 and latest in September 2014. The study is deviating from 1997 TG in the lack of historical control data.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

white powder, was dissolved in ethanol.

Species / strain / cell type:

lymphocytes: human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

Preparation of liver homoqenate S-9:
All steps were at 0-4°C using sterile solutions and glassware. The livers were placed in beakers containing 0.15 M KC1. After weighing, livers were transferred to a beaker containing 3 volumes of 0.15 M KC1 and homogenised in an MSE homogeniser. This homogenate was centrifuged for 10 minutes at 9000 x 'g' and the supernatant divided into 5 ml aliquots. These were stored at -80°C and tested before use, with the carcinogen, 7,12-dimethylbenz(a)anthracene.

Type and composition of metabolic activation system:
Each ml S-9 mix contained:

S-9 fraction 0.1 ml
0.4 M MgCl2 0.02 ml
0.2 M Na2HP04 (pH 7.4) 0.5 ml
1.0 M glucose-6-phosphate 0.005 ml
0.1 M NADP 0.04 ml
Distilled water 0.335 ml

All the above solutions were mixed and then filter-sterilised (apart from the S-9 fraction which was added after filter-sterilisation of the other components).

Test concentrations with justification for top dose:

three different concentrations; 10, 50 and 100 µg/ml, with and without exogenous metabolic activation. The concentrations were selected on the basis of the results from a preliminary toxicity test (0.24 - 125 µg/ml), where desmedipham caused slight precipitation at the highest dose level.

Vehicle / solvent:

- Solvent used for Desmedipham: Ethanol
- Other solvents: Ethanol (+S9, -S9), DMSO (-S9) and sterile water (+S9) were used as solvent controls.

Untreated negative controls:

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Cultured human lymphocytes were stimulated to divide by the addition of phytohemagglutinin and incubated for 48 hours at 37°C.

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10^6 cells/ml
- Test substance added in cells cultures: 50 µl aliquots of technical desmedipham were added
to one set of duplicate lymphocytes cultures.
- The cultures were then incubated for 22 hours prior to the addition of colchicine. Two hours after dosing, the cultures with S9 were centrifuged and the cell pellets resuspended in fresh medium, after which the cells were incubated for a further 20 hours

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor (cytogenetic assays): The cells were arrested in metaphase by colchicine, incubated for a further 2 hours, treated under hypotonic conditions and fixed.

- Methods of slide preparation and staining technique used including the stain used: Five slides were prepared from each culture. A total of 100 complete metaphase figures were examined microscopically from each culture and a maximum of 25 metaphase figures were scored in each slide. The slides were stained using Giemsa solution and, when dry, mounted in DPX.

- The following types of aberrations were analysed for: chromatid breaks, interchanges, single minutes, acentric fragments, isochromatid gaps and chromatid gaps.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The stained cells were examined by light microscopy at a magnification of x 160 and the proportion of metaphase figures in each culture derived by recording the number of metaphase cells and the total number of nuclei.

Statistics:

Statistical analyses were performed using Fisher’s test.

Species / strain:

lymphocytes: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No statistically significant increases in aberrations were noted in lymphocytes treated with desmedipham with or without metabolic activation. The mean percentage of aberrant cells (excluding or including gaps) without S9 were 0.75% and 0.5% in ethanol and DMSO control groups, respectively. The mean percentages of aberrant cells (excluding or including gaps) were 1.0%, 1.5 - 2.5% and 1.5% at dose levels of 10, 50 and 100 µg desmedipham /ml, respectively. The percentages were very similar in the test groups with S9. Both positive control groups caused statistically significant increases in proportions of metaphase figures containing aberrations compared to solvent controls.

See the Results' tables in the attached file in "Overall remarks, attachments"

Conclusions:

When tested to the limit of solubility in ethanol, desmedipham did not induce chromosome aberrations in cultured human lymphocytes with or without S9 at concentrations up to 100 µg/ml. The study was acceptable.

Executive summary:

Technical desmedipham was tested for its ability to induce chromosomal aberrations in human lymphocytes cultured in vitro.

Cultured human lymphocytes, stimulated to divide by addition of phytohaemagglutinin were exposed to the test compound in both the presence and absence of a metabolic activation system (S-9 mix) derived from rat livers. Cell division was then arrested using colchicine and after hypotonic treatment, fixation and staining, metaphase spreads were examined for chromosomal damage.

A preliminary toxicity test was carried out to assess the effect of the compound on the mitotic index of cultured human lymphocytes. On the basis of this test a top dose level of 100 ug/ml was chosen for the metaphase analysis test in both the presence and absence of S-9 mix.

Final concentrations of technical desmedipham dissolved in ethanol used for the metaphase analysis were 10, 50 and 100 µg/ml. The proportion of metaphase figures showing chromosome aberrations was similar in control and technical desmedipham-treated cultures.

It was concluded that when tested to the limits of solubility in ethanol, technical desmedipham has shown no evidence of mutagenic potential in this in vitro cytogenetic assay, in either the presence or absence of metabolic activation.

Reference 7

Endpoint:: in vitro gene mutation study in bacteria
Type of information:: experimental study
Adequacy of study:: weight of evidence
Study period:: 2014-06-23 to 2014-09-24
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:: "Ninth Addendum to OECD Guidelines for Testing of Chemicals", Section 4, No. 471: "Bacterial Reverse Mutation Test", adopted July 21, 1997.
Deviations:: no
Qualifier:: according to guideline
Guideline:: EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:: May 30, 2008
Deviations:: no
Qualifier:: according to guideline
Guideline:: EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:: August, 1998
Deviations:: no
GLP compliance:: yes (incl. QA statement)
Type of assay:: bacterial reverse mutation assay
Species / strain / cell type:: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:: with and without
Metabolic activation system:: Type and composition of metabolic activation system:
- source of S9: Rat liver
- method of preparation of S9 mix:
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to
result in a final concentration of approx. 10 % v/v in the S9 mix. Cofactors were added to the S9
mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment, the S9 mix is stored in an ice bath. The S9 mix preparation is performed
according to Ames et al.
- concentration or volume of S9 mix and S9 in the final culture medium: The protein concentration of the S9 preparation was 33.2 mg/mL in Experiment I and 38.0 mg/ml in Experiment II.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2 aminoanthracene in the Ames test.
Test concentrations with justification for top dose:: In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as Experiment I, since toxic effects were observed eight concentrations were tested in Experiment II. 5000 µg/plate was chosen as maximal concentration. The concentration range included two logarithmic decades. The following concentrations were tested in both experiments: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:: - Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Untreated negative controls:: yes
Negative solvent / vehicle controls:: yes
True negative controls:: no
Positive controls:: yes
Positive control substance:: sodium azide; methylmethanesulfonate; other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)
Remarks:: The stability of the positive control substances in solution is unknown but a mutagenic response
in the expected range are sufficient evidence of biological stability.
Details on test system and experimental conditions:: NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate.
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): The bacterial pre-cultures were incubated in a shaking water bath for 4 hours at 37° C. The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (10^8 - 10^9 cells/mL).
- Test substance added in medium: Plates with selective agar (without histidine) were used. The overlay agar contains per litre:
7.0 g Agar Agar
6.0 g NaCl
10.5mg L-Histidine x HCl x H2O
12.2mg Biotin
Sterilisations were performed at 121 °C in an autoclave.

Experiment I (Plate Incorporation)
100 µL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 µL Overlay agar.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: In the pre-incubation assay 100 µL test solution (solvent or reference mutagen solution (positive control)), 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension (50 µL for strain TA 1535) were mixed in a test tube and incubated at 37 °C for 60 minutes. After preincubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates.
- Exposure duration/duration of treatment: After solidification, the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

METHODS FOR MEASUREMENTS OF GENOTOXICIY

The colonies were counted using the Petri Viewer Mk2 with the software program Ames Study Manager (v.1.21). The counter was connected to a PC with printer to print out the individual values and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). Due to precipitation of the test item, reduced background growth and widespread bacteria colony growth the colonies were partly counted manually.
Evaluation criteria:: A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment.
However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
: Key result
Species / strain:: S. typhimurium TA 1537
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5)
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
True negative controls validity:: not applicable
Positive controls validity:: valid
: Key result
Species / strain:: S. typhimurium TA 1535
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5)
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
True negative controls validity:: not applicable
Positive controls validity:: valid
: Key result
Species / strain:: S. typhimurium TA 98
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5)
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
True negative controls validity:: not applicable
Positive controls validity:: valid
: Key result
Species / strain:: S. typhimurium TA 100
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5)
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
True negative controls validity:: not applicable
Positive controls validity:: valid
: Key result
Species / strain:: S. typhimurium TA 102
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5)
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
True negative controls validity:: not applicable
Positive controls validity:: valid
Additional information on results:: HISTORICAL DATA
The data of strain TA 100 were slightly above the historical control range in Experiment I without S9 mix (untreated control). Since this deviation is rather small, this effect was considered to be based upon biologically irrelevant fluctuations in the number of colonies. In Experiment I, without metabolic activation, the number of colonies did not quite reach the lower limit of the historical control data in strain TA 98 (untreated control). Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
Conclusions:: Desmedipham was not mutagenic in this Ames test performed to OECD 471 (1997) using S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102, in the absence and presence of metabolic activation and using concentrations up to the limit concentration of 5000 µg/plate.
Executive summary:: This study was performed to investigate the potential of desmedipham to induce gene mutations according to the plate incorporation test (Experiment I) and the pre-incubation test (Experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations in both experiments: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate. The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 µg/plate in Experiment I with and without S9 mix and in Experiment II with S9 mix. In Experiment II without S9 mix, precipitation of the test item was observed at 2500 and 5000 µg/plate. The undissolved particles had no influence on the data recording. The plates incubated with the test item showed reduced background growth in all strains. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Desmedipham a.i. at any concentration, either in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, desmedipham did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, desmedipham is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:: adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A comet assay (gene mutation endpoint) with desmedipham reports a negative result. Negative results are also reported in two bone marrow micronucleus assays (structural and numerical chromosomal aberration endpoints).

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:: in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:: Mouse bone marrow micronucleus assay
Type of information:: experimental study
Adequacy of study:: supporting study
Study period:: 1985-02-11 to 1985-03-18
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: guideline study with acceptable restrictions
Qualifier:: according to guideline
Guideline:: OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:: not specified
Qualifier:: according to guideline
Guideline:: EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:: not specified
GLP compliance:: yes (incl. QA statement)
Type of assay:: mammalian erythrocyte micronucleus test
Specific details on test material used for the study:: Physical appearance light beige coloured fine powder.
Storage The test article and a reserve sample were stored at room temperature, in the dark.
Species:: mouse
Strain:: NMRI
Details on species / strain selection:: Standard laboratory species/strain used for this type of study, with background data
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Age at study initiation: males: 6 weeks, females: 7 weeks
- Weight at study initiation: males: 26-37 g, females: 26-34 g
- Assigned to test groups randomly: yes, Animals mere randomized into the different groups after delivery using a random algorithm.
- Housing: Groups of 6 in Makrolon Type 3, with wire mesh top and granulated softwood bedding.
- Diet: Pelleted standard mouse diet, ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: 7 days under test conditions, with veterinary examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±10
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12 per day
Route of administration:: oral: gavage
Vehicle:: - Vehicle(s)/solvent(s) used: 2% carboxymethylcellulose, (CMC) in distilled water.
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw
Details on exposure:: PREPARATION OF DOSING SOLUTIONS:
A suspension was prepared immediately before application by adding the test article to distilled water containing CMC (2%).
Homogeneity of the suspension was maintained during application using a magnetic stirrer.
Duration of treatment / exposure:: single dose
Frequency of treatment:: single dose
Post exposure period:: At 24-, 48- and 72-hour intervals after treatment, six mice per sex and group were sacrificed for examination. The first five animals of each sex were evaluated.
Dose / conc.:: 0 other: mg/kg bw
Remarks:: Negative Control (2% carboxymethylcellulose, sodium salt (CMC) in distilled water)
Dose / conc.:: 5 000 other: mg/kg bw
Remarks:: Desmedipham
No. of animals per sex per dose:: 18/sex/dose
Control animals:: yes, concurrent vehicle
Positive control(s):: Cyclophosphamide (reference mutagen)
- Doses / concentrations: 50 mg/kg bw
Tissues and cell types examined:: Bone marrow
Details of tissue and slide preparation:: CRITERIA FOR DOSE SELECTION:
The maximum tolerated dose was based on acute oral toxicity data. The acute oral LD50 performed with mouse is greater than 5000 mg/kg body weight. The 5000 mg/kg body weight dose was used in this study as the maximum tolerated dose.

TREATMENT AND SAMPLING TIMES: The animals were dosed once, at 24 -, 48 -, and 72 -hour intervals after treatment, six mice per sex and group were sacrificed for examination, bone marrow was collected

DETAILS OF SLIDE PREPARATION:
All mice were sacrificed by cervical dislocation. The femora were removed from each mouse and freed of adherent tissue.
The proximal end of the femur was cut with scissors. The needle of a plastic syringe containing 0.2 ml calf serum was inserted into the proximal part of the marrow canal which was closed at the distal end. The femur was submerged in 1.5 ml calf serum in a labeled centrifuge tube. The bone marrow cells mere dispersed in the calf serum as a homogeneous suspension. The tube containing the bone marrow cells of both femora was centrifuged at 1000 rpm for 5 minutes. The supernatant was removed, leaving a thin layer of serum. The cells of the sediment were suspended by aspiration in a siliconized Pasteur pipette. A small drop of the marrow serum suspension was smeared on the slide, which was identified by project code and the animal number and allowed to dry overnight. Two slides per animal mere prepared. The following day, the smears mere stained using the panoptic stain method developed by Pappenheim

METHOD OF ANALYSIS:
From each animal 1000 polychromatic erythrocytes (PCE) were scored under the microscope for the incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE), based on scoring 1000 erythrocytes per slide, measured the toxicity of desmedipham.
Evaluation criteria:: The frequencies of micronuclei of the treated male and female groups were compared with those of the negative control groups at each sampling time. A regression model assuming a Poisson distribution mas applied. Estimation and testing were performed by maximum likelihood method. If a test article produced no statistically significant and reproducible positive response at any one of the test points, it was considered non-mutagenic in this system.
Statistics:: Statistical evaluation of the data was made using a regression model assuming a Poisson distribution and the maximum likelihood method.
Sex:: male/female
Genotoxicity:: negative
Toxicity:: yes
Remarks:: Sedation was observed in all treated animals for at least 6 hours after dosing.
Vehicle controls validity:: valid
Negative controls validity:: not applicable
Positive controls validity:: valid
Conclusions:: After a single dose of 5000 mg desmedipham /kg bw by gavage, no significant increase of micronucleated polychromatic erythrocytes in the bone marrow was observed in either male or female treated groups at either 24, 48 or 72 hours post-dose.
Executive summary:: This in vivo study with mice was performed to assess the potential mutagenic activity, induced by DESMEDIPHAM, through damage to the chromosomes or to the mitotic apparatus, according to Schmid (1) with the modifications of Salomone et al.(2). The experiment was performed with three groups, each containing 18 males and 18 females, for a total of 108 mice. The negative control groups received the test article vehicle, i.e. 2% carbaoxymethylcellulose sodium salt in distilled water. The test groups received 5000 mg/kg body weight as the maximum tolerated dose of the test article. The positive control groups received 50 mg/kg body weight cyclophosphamide dissolved in 0.9% saline solution. All animals were given a single application of 20 ml/kg body weight by gavage. Twenty-four (24), 48 and 72 hours after treatment, six mice per sex and group were sacrificed and bone marraw was removed from the femora for examination. The first five animals were used for evaluation. One thousand polychromatic erythrocytes per animal were scored for micronuclei. The ratio of polychromatic to normochromatic erythrocytes was used to assess the toxicity of the test article by counting a total of 1000 erythrocytes. Data was statistically analyzed by means of a regression model assuming a Poisson distribution. Estimation and testing were performed by maximum likelihood method. Sedation was observed in all test article-treated animals for at least 6 hours after application. After single application of the test article at 5000 mg/kg body weight by gavage, no significant test article-related increase of micronucleated polychromatic erythrocytes was observed in either male and female treated groups, when compared with corresponding negative control groups. These results were found at the three examination times, 24, 48 and 72 hours post-application, respectively. The positive control groups, which received cyclophosphamide, exhibited a significant and clear increase in the number of micronucleated polychromatic erythrocytes and thus validated, the test. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced no chromosome mutations by chromosome-breaking activity or by damage to the mitotic apparatus. Therefore, DESMEDIPHAM is not considered to be mutagenic in this in vivo mouse micronucleus assay.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-01-24 to 2017-05-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29 July 2016

Deviations:

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Description: White powder
Storage Conditions: Room temperature (15 to 30°C), protected from light

Species:

rat

Strain:

Sprague-Dawley

Remarks:

(Hsd:SD)

Details on species / strain selection:

This species has been routinely used as an animal model of choice for the mammalian alkaline Comet assay. This strain was an outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to the test substance.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation: Dose range finder: 170 –187.5 (males), 141.2 –157.7 (females).
Main study: 193.4 –216.6 (males)
- Assigned to test groups randomly: yes.
- Housing: Animals of the same sex were housed up to three per Micro-Barrier cage. Cages were placed on racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system.
- Diet: A certified laboratory rodent chow (18% Protein Rodent Diet) was provided ad libitum.
- Water: free access to tap water.
- Acclimation period: 5-6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3°F = ca. 20.5 to 23.89 °C.
- Humidity: 50 ± 20 %
- Air changes: at least 10 changes of fresh HEPA-filtered air per hour.
- Photoperiod: 12-hour light/dark cycle.

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: 1% medium viscosity Carboxymethyl cellulose (CMC) in deionized water.
- Amount of vehicle: 10 mL/kg/dose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
1- Preparation of Control Articles:
The vehicle formulation was prepared by using a suitable sized disposable glass bottle calibrated
to the required batch size volume with a yellow PTFE stir bar designated for control articles only. Approximately 70% of the total volume of DI water was added to the bottle and then an appropriate amount of CMC was slowly added while stirring. The formulation was stirred magnetically until uniform and QS (quantum satis) to the final volume with DI water. The final formulation was stored at refrigerated (2 - 8°C).
The neat EMS was prepared in 0.9% saline for injection. The dosing formulation was prepared at a concentration of 20 mg/mL, just prior to use.

2- Preparation of Test Substance Dose Formulations:
The test substance dose formulations were prepared fresh on each day of use. A suitable sized amber glass vial was calibrated with PTFE stir bar to the target batch size, which holds pre-weighed amount of test substance. The appropriate test substance vial with the pre-weighed amount of test substance was selected and combined with approximately 70% of the total volume of vehicle and stirred magnetically. The suspension was QS to the final volume with the vehicle.
The suspension was stirred then sonicated (5 minutes), homogenized with a polytron (7 minutes) and mixed magnetically (16 minutes) until homogenous in appearance. The final dose formulation was stored at room temperature.
Residual dose formulations were discarded after use.

Duration of treatment / exposure:

2 days

Frequency of treatment:

All animals in the vehicle control and test groups were dosed on two consecutive days (days 1 and 2) with the vehicle and with test substance. The second dose occurred approximately 21 hours after the first dose.

Animals in the positive control group were dosed with the positive control (EMS) once approximately 3 to 4 hours prior to euthanasia on day 2.
hours prior to euthanasia on day 2.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Group 1: Vehicle control

Dose / conc.:

500 mg/kg bw/day

Remarks:

Group 2: Desmedipham

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Group 3: Desmedipham

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

Group 4: Desmedipham

No. of animals per sex per dose:

For group 1 to 4: 6 males/group.
For group 5 (positive control): 3 males

Control animals:

yes, concurrent vehicle

other: Positive control

Tissues and cell types examined:

Liver and stomach

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on a preliminary Dose Range Finder study.

TREATMENT AND SAMPLING TIMES:
The outline of the study was as follows for the Comet assay:
• All animals in Groups 1 through 4 were dosed on two consecutive days (days 1 and 2) with the vehicle and with test substance. The second dose occurred approximately 21 hours after the first dose.
• Animals in Group 5 were dosed with the positive control (EMS) once approximately 3 to 4 hours prior to euthanasia on day 2.

All animals were euthanized 3 to 4 hours after the last dose (day 2) by CO2 asphyxiation, and then, the following was performed:
• Animals were dissected and the liver and stomach were extracted (removed) and collected.
• A section of the liver and stomach were cut and placed in formalin for possible histopathology analysis.
• Another section of the liver and stomach were placed in chilled mincing solution (Hanks’ balanced salt solution with EDTA and DMSO) and was used in preparation of cell suspensions and Comet slides.

DETAILS OF SLIDE PREPARATION:
A portion of each dissected liver was placed in 3 mL of cold mincing buffer, then the liver was finely cut (minced) with a pair of fine scissors to release the cells. A portion of each dissected interior glandular stomach was placed in 1 mL of cold mincing buffer then stomach was scraped using a plastic spatula to release the cells. Each cell suspension was strained through a Cell Strainer into a pre-labeled 50 mL polypropylene conical tube and the resulting liver and stomach cell suspensions were placed on wet-ice. An aliquot of the suspensions were used to prepare the comet slides.

Preparation of Slides
From each liver and stomach suspension, an aliquot of 2.5 µL and 7.5 µL, respectively, were mixed with 75 µL (0.5%) of low melting agarose (0.5%). The cell/agrose suspension was applied to microscope slides commercially available pre-treated multi-well slides. Commercially purchased multi-well slides were used and these slides have 3 individual circular areas, referred to as wells in the text below. The slides were kept at 2 - 8°C for at least 15 minutes to allow the gel to solidify. Slides were identified with a random code that reflects the study number, group, animal number, and organ/tissue. At least two Trevigen, Inc 3-well slides were prepared per animal per tissue.
Three slides/wells were used in scoring and the other wells were designated as a backup. Following solidification of agarose, the slides were placed in jars containing lysis solution.

Lysis
Following solidification of agarose, the slides were submerged in a commercially available lysis solution supplemented with 10% DMSO on the day of use. The slides were kept in this solution at least overnight at 2-8°C.

Unwinding
After cell lysis, slides/wells were washed with neutralization buffer (0.4 M tris hydroxymethyl aminomethane in purified water, pH ~7.5) and placed in the electrophoresis chamber. The chamber reservoirs were slowly filled with alkaline buffer composed of 300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water. The pH was > 13. All slides remained in the buffer for 20 minutes at 2-10°C and protected from light, allowing DNA to unwind.

Electrophoresis
Using the same buffer, electrophoresis was conducted for 30 minutes at 0.7 V/cm, at 2-10°C and protected from light. The electrophoresis time was constant for all slides.

Neutralization
After completion of electrophoresis, the slides were removed from the electrophoresis chamber and washed with neutralization buffer for at least 10 minutes. The slides (gels) were then dehydrated with 200-proof ethanol for at least 5 minutes, then air dried for at least 4 hours and stored at room temperature with desiccant.

Staining
Slides were stained with a DNA stain (i.e., Sybr-gold™) prior to scoring. The stain solution was prepared by diluting 1 µL of Sybr-gold™ stain in 15 mL of 1xTBE (tris-boric acid EDTA buffer solution).

Evaluation of DNA Damage:
Three slides/wells per organ/animal were used. Fifty randomly selected, non-overlapping cells per slide/well were scored resulting in a total of 150 cells evaluated per animal for DNA damage using the fully validated automated scoring system Comet Assay IV:

The following endpoints of DNA damage were assessed and measured:
• Comet Tail Migration; defined as the distance from the perimeter of the Comet head to the last visible point in the tail.
• % Tail DNA; (also known as % tail intensity or % DNA in tail); defined as the percentage of DNA fragments present in the tail.
• Tail Moment (also known as Olive Tail moment); defined as the product of the amount of DNA in the tail and the tail length [(% Tail DNA x Tail Length)/ 100].

Each slide was also examined for indications of cytotoxicity. The rough estimate of the percentage of “clouds” was determined by scanning 150 cells per animal, when possible (percentage of “clouds” was calculated by adding the total number of clouds for all slides scored, dividing by the total number of cells scored and multiplying by 100). The “clouds”, also known as “hedgehogs”, are a morphological indication of highly damaged cells often associated with severe genotoxicity, necrosis or apoptosis. A “cloud” is produced when almost the entire cell DNA is in the tail of the comet and the head is reduced in size, almost nonexistent. “Clouds” with visible gaps between the nuclei and the comet tail were excluded from comet image analysis.

The comet slides, which are not permanent (the slides can be affected/damaged by environmental storage conditions), were discarded prior to report finalization.

Histopathology Evaluation
A portion of each dissected liver and stomach was placed in formalin (10 % neutral-buffered formalin) for possible histopathology analysis. Per the study protocol, histopathology evaluation was not performed since biologically significant increases in DNA damage were not observed.

Evaluation criteria:

A test substance was considered to have induced a positive response if
a) at least one of the group mean for the % tail DNA of the test substance doses exhibited a statistically significant increase when compared with the concurrent negative control (p = 0.05),
b) when multiple doses were examined at a particular sampling time, the increase was dose-related (p = 0.01),
c) results of the group mean or of the individual animals of at least one group were outside the distribution of the historical negative control database for that tissue.
A test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met and there was direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue.

If the response was neither clearly positive nor clearly negative, or in order to assist in establishing the biological relevance of a result, the data were evaluated by expert judgment and/or further investigations. Any additional work was only carried out following consultation with, and at the request of, the Sponsor. In some cases, even after further investigations, the data set precluded making a conclusion of positive or negative, at which time the response was concluded to be equivocal. In such cases, the Study Director used sound scientific judgment and reported and described all considerations.

Biological significance of a positive, negative and equivocal result was based on the information on cytotoxicity at the target tissue. Where positive or equivocal findings were observed solely in the presence of clear evidence of cytotoxicity (e.g. histopathology evaluation, changes in clinical chemistry measures), the study was concluded as equivocal for genotoxicity unless there was enough information that was supportive of a definitive conclusion. I n the case of a negative study outcome where there were signs of toxicity at all doses tested, further study at non- toxic doses may be advisable.

Statistics:

The median value of 150 counts of % Tail DNA, Tail moment and Tail migration were determined and presented for each animal in each treatment group for each organ. The mean and standard deviation of the median values only for % Tail DNA were presented for each treatment group. Statistical analysis was performed only for % Tail DNA.

In order to quantify the test substance effects on DNA damage, the following statistical analysis was performed:
• The use of parametric or non-parametric statistical methods in evaluation of data was based on the variation between groups. The group variances for % tail DNA generated for the vehicle and test substance groups were compared using Levene’s test (significant level of p = 0.05). If the differences and variations between groups were found not to be significant, a parametric one-way ANOVA followed by a Dunnett’s post-hoc test was performed (significant level of p < 0.05).
• A linear regression analysis was conducted to assess dose responsiveness in the test substance treated groups (p = 0.01).
• A pair-wise comparison (Student’s T-test, p = 0.05) was used to compare the positive control group to the concurrent vehicle control group.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500-2000 mg/kg/day.
- Clinical signs of toxicity in test animals: At the highest level (2000 mg/kg/day), Piloerection was observed in all test animals.
No mortality occurred at any dose level during the course of the dose range finding assay. No considerable reductions in mean group body weights were seen in the test substance treated groups during the course of the study.

As no significant difference in the mortality or observations were seen during the dose range finding assay, the maximum tolerated dose for the definitive comet assay was set at 2000 mg/kg/dose.

RESULTS OF DEFINITIVE STUDY
- No mortality occurred at any dose level during the course of the definitive assay. No appreciable reductions in mean group body weights were seen in the test substance treated groups during the course of the study. All rats appeared normal throughout the observation period.

Comet Assay
% Tail DNA, Tail moment and Tail migration (µm) for liver cells are calculated per 150 cells for each animal:
The scoring results and a statistical analysis of data indicated the following:
• The presence of ‘clouds’ in the low and mid test substance groups was = 0.7%, which was higher than the % of clouds in the vehicle control group (0.3%). The high test substance group was 0.3% which was comparable with the vehicle control group (0.3%).
• Group variances for mean of medians of the % Tail DNA in the vehicle and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.

% Tail DNA, Tail moment and Tail migration (µm) for stomach cells are calculated per 150 cells for each animal:
The scoring results and a statistical analysis of data indicated the following:
• The presence of ‘clouds’ in the low test substance groups was 30.3%, which was higher than the % of clouds in the vehicle control group (20.5%). The mid and high test substance groups were =12.8% which were lower than the % of clouds in the vehicle control group (20.5%)
• Group variances for mean of medians of the % Tail DNA in the vehicle and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.
• No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).
• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).
• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in stomach cells as compared to the vehicle control groups (Student’s t-test, p = 0.05).
• In the vehicle control group, % Tail DNA was within the historical vehicle control range for the stomach.

These results indicate that all criteria for a valid test, as specified in the protocol, were met.

Dosing Formulation Analysis
The results of the analysis indicate that the actual mean concentrations of the analyzed Definitive Day 1 formulation samples, 50, 100 a nd 200 mg/mL, were 100.8, 104.7, 121.8% of target, respectively, with = 10.0% RSD. This indicates that all formulations for Definitive Day 1, except for the 200 m g/mL formulation, were accurately prepared and homogenous. No test substance was detected in the vehicle control sample. Additionally, Desmedipham in Carboxymethylcellulose sodium salt - Medium Viscosity (1% CMC), at concentrations of 48.7 and 219 mg/mL, was stable at 2 to 8°C for at least 25.7 hours.

See the results' tables in the attached file in "Overall remarks, attachments"

Conclusions:

Under the conditions of the assay described in this report, Desmedipham was concluded to be negative for the induction of DNA damage in liver and stomach.

Executive summary:

The test substance, Desmedipham, was evaluated for its genotoxic potential in the Comet assay to induce DNA damage in liver and stomach cells of male rats. 1% medium viscosity Carboxymethyl cellulose (CMC) in deionized water was selected as the vehicle. Test and/or control article formulations were administered at a dose volume of 10 mL/kg/dose by oral gavage.

In the dose range finding assay (DRF), the maximum dose tested was 2000 mg/kg bw/day. The additional dose levels tested were 1000 and 500 mg/kg bw/day in 3 of animals/sex. Based upon the results, the high dose for the definitive assay was 2000 mg/kg/bw/day, which is the highest guideline recommended dose for this assay.

The definitive assay dose levels tested were 500, 1000, and 2000 mg/kg/dose dosed on two consecutive days (days 1 and 2).

The test substance gave a negative (non-DNA damaging) response in this assay in liver and stomach for males in % Tail DNA. None of the test substance treated animal slides had significant increases in the % Tail DNA compared to the respective vehicle controls. The vehicle control % Tail DNA was within the Testing Facility’s historical range, and the positive control had a statistically significant increase in % Tail DNA compared to the vehicle control.
Thus, all criteria for a valid assay were met for liver and stomach.

Under the conditions of this study, the administration of Desmedipham at doses up to and including a dose of 2000 mg/kg/dose did not cause a significant increase in DNA damage in liver and stomach relative to the concurrent vehicle control. Therefore, Desmedipham was concluded to be negative in the in vivo Comet Assay.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2002-08-14 to 2003-03-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

Qualifier:

according to guideline

Guideline:

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

Deviations:

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998

Deviations:

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Japanese Ministry of Agriculture, Forestry and Fisheries. Test Data for Registration of
Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24,
2000.

Deviations:

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

JMHW Genotoxicity Testing Guideline, PAB Notification No. 1604 (1 November 1999)

Deviations:

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Joint Directive of J MHLW/METI and ME (21 November 2001)
lYAKUHATSU No. 1271
SEIKYOKU No. 1 (1 November 2001)
KANPOKI No.946
Document 17 - Testing Method for Mutagenicity Studies

Deviations:

Qualifier:

equivalent or similar to guideline

Guideline:

other: ICH

Version / remarks:

1996 & 1998

Deviations:

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Appearance: White powder
Storage conditions: Room temperature

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Sprague-Dawley CD rats

Details on species / strain selection:

Standard species/strain used for this type of study, with background control data available

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: between 140 and 150 grams and females 130 to 140 grams.
- Assigned to test groups: The animals were randomly assigned to groups and tail marked.
- Housing: Each group was kept, with the sexes separated, in cages and maintained in a controlled environment.
- Diet: free access to pelleted expanded rat and mouse No.l maintenance diet.
- Water: ad libitum
- Acclimation period: a minimum period of six days

ENVIRONMENTAL CONDITIONS
- Temperature: 21±2°C
- Relativity Humidity: 55±15%
- Photoperiod: The room was illuminated by artificial light for 12 hours per day.
During the experimental and acclimatisation period, temperature and relative humidity were monitored continuously, and were found to be within the ranges of 19-21°C and 40-60% respectively.

Route of administration:

oral: gavage

Vehicle:

1% carboxymethylcellulose (CMC)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test substance were freshly prepared on the morning of the test (using identical methods for each phase of the test) in 1% carboxymethylcellulose (CMC).
Cyclophosphamide was used as the positive control compound. It was prepared as a solution in purified water at a concentration of 2 mg/mL just prior to administration. All animals in all groups were dosed with the standard volume of 10 mL/kg bw administered orally by gastric intubation.

Duration of treatment / exposure:

Dosed once

Frequency of treatment:

Single

Post exposure period:

Following dosing, the animals were examined regularly and any mortalities or clinical signs of reaction were recorded. Seven males from the negative control, the intermediate and high test substance groups, six animals from the low test substance treatment group and five males from the positive control group were sacrificed 24 hours after dosing. In addition seven male animals in the negative control and high level treatment groups were sacrificed 48 hours after dosing.

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

Vehicle control

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

Preliminary toxicity test: 1 group with 2 males and 2 females.
Vehicle control: 14 males.
Desmedipham (500, 1000 mg/kg): 7 males.
Desmedipham (2000 mg/kg): 14 males.
Positive control (Cyclophosphamide): 5 males.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide:
- Justification for choice of positive control(s): standard substance specified by the test guidelines
- Route of administration: Oral: gavage
- Doses / concentrations: 20 mg/kg bw

Tissues and cell types examined:

The bone marrow of both femurs and proximal epiphyses

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
From the results obtained in the preliminary toxicity study no substantial differences in toxicity were observed between the sexes and in line with current guidelines, the main test was performed using males only. Dose levels of 500, 1000 and 2000 mg/kg bodyweight were chosen for the micronucleus test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Following dosing, the animals were examined regularly and any mortalities or clinical signs of reaction were recorded. Seven males from the negative control, the intermediate and high test substance groups, six animals from the low test substance treatment group and five males from the positive control group were sacrificed 24 hours after dosing. In addition seven male animals in the negative control and high level treatment groups were sacrificed 48 hours after dosing.

The animals were killed by exposure to rising levels of carbon dioxide and both femurs dissected out from each animal. The femurs were cleared of tissue and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 10 ml Hanks' balanced salts solution by aspiration. The resulting cell suspensions were centrifuged at 1000 rpm (150 x g) for 5 minutes and the supernatant discarded. Each resulting cell pellet was resuspended in 2ml of filtered foetal calf serum before being sedimented by centrifugation. The supernatant was discarded and the final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing in the conventional manner on glass microscope slides (Schniid 1976). Three smears were prepared from each femur.

DETAILS OF SLIDE PREPARATION:
Due to the presence of mast cell granules in rat bone smears, which appear identical to micronuclei when stained using the Romanowsky methods, a modified Feulgen staining method is employed for the rat micronucleus test in this laboratory. This method specifically stains DNA-containing bodies deep purple while leaving mast cell granules unstained. The method also allows reasonable differentiation of mature and immature erythrocytes and produces permanent preparations.

The slides were fixed and stained as described in the following schedule:
1. Fixed for 10 minutes in methanol.
2. Hydrolysed in Bouin's fluid at room temperature for 28 hours.
3. Washed three times in purified water (5 minutes per wash).
4. Stained in Schiffs reagent for one hour at room temperature.
5. Washed three times in purified water (5 minutes per wash).
6. Counter-stained for ten minutes in very dilute (approximately 0.06 g/1) aqueous Eosin
yellowish.
7. Washed for five minutes in purified water.
8. Stained for 30 minutes in Mayer's Haemalum diluted 9 volumes: 1 volume with aqueous
acridine orange solution in purified water (1 mg/ml).
9. Rinsed in purified water.
10. Rinsed in running tap water.
11. Washed for 5 minutes in purified water.
12. Air-dried.
13. Slides were mounted with coverslips using DPX mountant.
14. The mountant was allowed to harden.
NB All stains and Bouin's fluid were filtered immediately prior to use to remove particulate material.

METHOD OF ANALYSIS:
The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. One smear per animal was examined and the remaining smears were held temporarily in reserve in case of technical problems with the first smear.

The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated mature erythrocytes observed during assessment of this proportion was also kept as recommended by Schmid (1976).

Evaluation criteria:

Micronuclei are identified by the following criteria:
• Large enough to discern morphological characteristics
• Should possess a generally rounded shape with a clearly defined outline
• Should be deeply stained and similar in colour to the nuclei of other cells - not black
• Should lie in the same focal plane as the cell
• Lack internal structure, ie they are pyknotic
• There should be no micronucleus-like debris in the area surrounding the cell.

Statistics:

The results for each treatment group were compared with the results for the concurrent control group using non-parametric statistics. Non-parametric statistical methods were chosen for analysis of results because:
• They are suited to analysis of data consisting of discrete/integer values with ties such as the incidence of micronucleated immature erythrocytes.
• The methods make few assumptions about the underlying distribution of data and therefore the values do not require transformation to fit a theoretical distribution (where data can be approximately fitted to a normal distribution, the results of non-parametric analysis and classical analysis of variance are very similar).
• 'Outliers' are frequently found in the proportion of immature erythrocytes for both control and treated animals; non-parametric analysis based on rank does not give these values an undue weighting.

For incidences of micronucleated immature erythrocytes, exact one-sided p-values are calculated by permutation. Comparison of several dose levels is made with the concurrent control using the Linear by Linear Association test for trend, in a step-down fashion if significance is detected (Agresti et al. 1990); for individual inter-group comparisons (ie the positive control group) this procedure simplifies to a straightforward permutation test (Gibbons 1985). For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF PRELIMINARY TOXICITY TEST
- Dose range: One group was dosed with Desmedipham at 2000 mg/kg.
- Rationale for exposure: 2000 mg/kg is the standard limit dose for the micronucleus test.
- no substantial differences in toxicity were observed between the sexes.

RESULTS OF MICRONUCLEUS TEST
- Induction of micronuclei:
Micronucleated immature erythrocyte counts (mie):
The test substance did not cause any statistically significant increases in the number of micronucleated immature erythrocytes at either sampling time [P>0.01].

Cyclophosphamide caused significant increases in the frequency of micronucleated immature
erythrocytes [P<0.01].

Micronucleated mature erythrocytes (mme)
The test substance did not cause any substantial increases in the incidence of micronucleated mature erythrocytes at either sampling time.

Proportion of immature erythrocytes (% ie/ie + me))
The test substance failed to cause any significant decreases in the proportion of immature erythrocytes [P>0.01].

Cyclophosphamide did not cause any statistically significant decreases in the proportion of immature erythrocytes [P<0.01].

- Clinical signs and mortalities:
Incidences of bodyweight loss were recorded for some animals but these were small and not considered to be specifically treatment related.

One animal dosed with Desmedipham at 500 mg/kg was found dead approximately 24 hours after treatment. At post mortem examination, some fluid and white deposits were observed in the pleural cavity indicating the possibility that during the dosing procedure a small tear had occurred in the oesophagus. Bone marrow smears were prepared from six animals only in the low dose group (500 mg/kg) as a result of this unscheduled death.

No adverse clinical signs were observed in any other animal over the duration of the test.

See "Attachments" in "Overall remarkes, attachments" for:

Table 1 gives a summary of the results of the micronucleus test and the results of statistical analysis. The results for individual animals at the 24 and 48 hour sampling times are presented in Tables 2 and 3 respectively. Appendix 3 summarises the historical control data for micronucleated polychromatic erythrocyte counts for the vehicle and positive control.

Conclusions:

No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decrease in the proportion of immature erythrocytes were observed in rats treated with Desmedipham and killed 24 or 48 hours later, compared to concurrent vehicle control values [P>0.01 in each case].

It is concluded that Desmedipham did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered orally by gastric intubation in this in vivo test procedure.

Executive summary:

This study was designed to assess the potential induction of micronuclei by Desmedipham in bone marrow cells of the rat. Animals were treated with a single oral administration of the test substance at dose levels of 500, 1000 and 2000 mg/kg bw. A preliminary toxicity test had previously shown that a dose of 2000 mg/kg bw, the limit dose for the micronucleus test, was tolerated; this level was therefore selected as an appropriate maximum for use in the micronucleus test. The test substance and negative control were administered orally by gastric intubation. The negative control group received the vehicle, 1% carboxymethylcellulose (CMC). A positive control group was dosed orally, by gastric intubation, with cyclophosphamide at 20 mg/kg bw.

Following the preliminary toxicity test, no substantial differences in toxicity were observed between the sexes, in line with current guidelines, the micronucleus test was performed using male animals only. One animal dosed with Desmedipham at 500 mg/kg bw was found dead approximately 24 hours after treatment. At post mortem examination, some fluid and white deposits were observed in the pleural cavity indicating the possibility that during the dosing procedure a small tear had occurred in the oesophagus. Bone marrow smears were obtained from seven male animals in the negative control, the intermediate and high test substance groups, six animals in the low test substance treatment group and five animals in the positive control group 24 hours after dosing. In addition bone marrow smears were obtained from seven male animals in the negative control and high level treatment groups 48 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocytes was also kept. No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decrease in the proportion of immature erythrocytes were observed in rats treated with Desmedipham and killed 24 or 48 hours later, compared to concurrent vehicle control values [P>0.01 in each case]. The positive control compound, cyclophosphamide, produced significant increases in the frequency of micronucleated immature erythrocytes. It is concluded that Desmedipham did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered orally by gastric intubation in this in vivo test procedure.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Desmedipham showed some genotoxic activity in vitro; however appropriate follow-up studies in vivo show clear negative results. Consequently, it can be concluded that desmedipham is not genotoxic. RAC (2019) did not assess the genotoxicity data in detail, but state that desmedipham is unlikely to be genotoxic. Desmedipham does not therefore require classification for germ cell mutagenicity under CLP.

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Strain	Experiment I		Experiment II
Strain	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	1000 - 5000	1000 – 5000	1000 - 5000	/
TA 1537	1000 - 5000	1000 – 5000	2500 - 5000	2500 - 5000
TA 98	1000 – 5000	1000 – 5000	2500 - 5000	2500 - 5000
TA 100	1000 – 5000	1000 – 5000	2500 - 5000	1000 - 5000
TA 102	1000 – 5000	1000 - 5000	/	/

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Desmedipham

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Link to relevant study records

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Endpoint conclusion

Genetic toxicity in vivo

Description of key information

Link to relevant study records

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Endpoint conclusion

Additional information

Justification for classification or non-classification

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