polymeric zinc propylenebis(dithiocarbamate);propineb (ISO)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The key studies for in vitro genotoxicity endpoints are as follows:

1- Bacterial mutagenicity:

Salmonella typhimurium reverse mutation assay with propineb technical (OECD 471, GLP, RL1), The mutagenic activity of test substance Propineb was evaluated to be negative for the reverse mutation test in bacterial system (M-437298-01-1, Sokolowski, 2012).

2- Mammalian mutagenicity:

Mutagenicity study for the detection of induced forward mutations in the CHO-HGPRT assay in vitro (Guideline not stated, GLP, RL2), A negative result is reported for propineb in an HPRT assay in CHO cells (+/-S9) (M-104115-01-1, Lehn, 1988).

3- Cytogenicity:

Chromosomal aberration test of propineb using cultured mammalian CHL cells (US EPA OPPTS 870.5375, GLP, RL2), Propineb induced structural chromosomal aberration at toxic doses, i.e. at doses inducing more that 50% inhibition of cell growth. Anyhow, the positive results of this in vitro test are discounted by the results of the in vivo chromosome aberration test (M-001021-01, Kajiwara, 1989).

 

The following supporting studies are available:

SALMONELLA/MICROSOME TEST TO EVALUATE FOR POINT MUTATION (Guidelines were not stated, no GLP, RL2), A negative result is reported for propineb in an Ames test in four strains of S. typhimurium (M-050169-01-1, Herbold, 1980)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987/06/30 to 1987/10/22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

no guideline available

Principles of method if other than guideline:

The study conforms to the OECD Principles of Good Laboratory Practice (Bundesanzeiger Nr. 42a/7of, 2nd of March 1983). The study is not fully compliant with the current guideline with respect to the number of cells exposed and background mutation rate, and is therefore of limited sensitivity.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HGPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Sprague-Dawley male rats, served as source of the S-9 fraction. A rat liver S-9 fraction buffered with 0.15M KC1. The positive control substances DMBA or 3-MCA were tested with each new batch of S-9 fraction for their ability to induce forward mutations in the CHO/HGPRT assay. Prior to use in the HGPRT test, the S-9 fraction was tested for contamination and for cytotoxicity.

Test concentrations with justification for top dose:

0.16 - 40 µg/mL (-S9)
0.11 - 60 µg/mL (+S9)
After determination of the cytotoxicity of LH 30/Z, the concentration range of LH 30/Z for the mutagenicity study was chosen ranging from approximately 0% to 90% reduction in colony forming ability.
Due to the low cytotoxicity of LH 30/Z in the mutation assays, the test article concentration was increased during the study up to 40 µg/mL without metabolic activation and up to 60.0 µg/mL with metabolic activation.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

Evaluation criteria:

An assay normally is considered acceptable for evaluation of the results only if the following
criteria are satisfied. However, the conclusion of the study will be based upon Study Director's evaluation and interpretation of the data. The activation and nonactivation assays were repeated independently in a second assay. The average cloning efficiency of the negative controls should be; at least 50%. Assays below 50% cloning efficiency will be unacceptable. The background mutant frequency (average of the negative control) should not exceed 25x10 cells. Assays with higher spontaneous mutant frequencies, however, are riot necessarily invalid if all other criteria are fulfilled.
An experimental mutant frequency is considered acceptable only if the absolute cloning efficiency is 10% or greater. The mutant frequencies for at least five treated cultures are normally determined in each assay. Mutant frequencies are normally derived from sets of 8-10 dishes for each dose level. To allow for contamination losses , an acceptable mutant frequency can be calculated from a minimum of 5 dishes. The positive control must induce a mutant frequency of at least three times that of the negative control. An assay will be considered positive if a dose-dependent and reproducible increase in mutant frequency is observed. It is desirable to obtain this dose-relation for at least 3 doses.

Statistics:

All data are presented in tabular form, descriptive statistical methods were used to calculate means and standard deviation.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Increases in mutation frequencies were observed. However, these increases were not dose-related

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Due to the low cytotoxicity of LH 30/Z in the mutation assays, the test article concentration was increased during the study up to 40 µg/mL without metabolic activation and up to 60.0 µg/mL with metabolic activation.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Remarks:

A stability test in the solvent was not done due to the non-homologous test compound.

True negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

The test material, propineb was assayed for mutagenic activity at the HGPRT locus in CHO cells from 0.16 µg/ml to 40 µg/ml without activation and from 0.11 µg/ml to 60.0 µg/ml with activation.
Under both treatment conditions, cytotoxicities were induced. The absolute cloning efficiencies for the vehicle controls varied from 80.0% to 108.8% without activation and from 73.2% to 108.8% with activation demonstrating good cloning conditions for the assays.
The vehicle control mutant frequencies were all in the normal range of background frequencies for the assay. In contrast, the positive controls EMS, DMBA and 3-MCA induced a distinct mutagenic effect in mutant frequency, which was significantly increased over the
negative controls.
From the lack of dose-related and reproducible increases in mutant frequency the test material is considered nonmutagenic in the CHO-HGPRT Forward Mutation Assay, both with and without metabolic activation, according to our evaluation criteria.

Executive summary:

Propineb was evaluated for mutagenic effects at the HGPRT locus (forward mutation assay) in CHO cell cultures after in vitro treatment at concentrations up to 40.0 µg/ml (without S-9 mix) and 60.0 µg/ml (with S-9 mix). Under both treatment conditions, LH 30/Z induced cytotoxic effects as seen by decreases in relative population growth and cloning efficiency. These results revealed a significant cytotoxicity of LH 30/Z, both with and without S-9 mix.

 

There were neither dose-related nor reproducible increases in mutant frequency which were significantly elevated over the negative controls. In contrast, the positive controls ethylmethanesulfonate (without S-9 mix), 3-inethylchola-nthrene and dimethylbenzanthracene (with S-9 mix) revealed a clear mutagenic effect in the assay. From these results, the test substance LH 30/Z can be considered, as nonmutagenic in the CHO-HGPRT Forward Mutation Assay, both with and without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989/2/6 to 1989/03/16

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1983

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Remarks:

Chinese hamster lung fibroblasts (CHL) cells

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytogenetic assay: Two hours before the cells were harvested; mitotic activity was arrested by addition of Colcemid to each culture at a final concentration of 0.1 µg/mL.

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared from the liver of 7-week old male Sprague-Dawley rats given a mixture of phenobarbital and 5,6-benzoflavone as enzyme inducers.

Test concentrations with justification for top dose:

Cytogenetic assay:
Without S9 mix, cells were exposed to propineb with concentrations of 0, 40, 50, 60, 70, 80, 90, 100 (µg/mL) for 24 hours and 0, 20, 25, 30, 35, 40, 50, 60 (µg/mL) for 48 hours.
With S9 mix, cells were exposed to propineb with concentrations of 0, 40, 50, 60, 70, 80, 90, 100 (µg/mL).

Cytogenetic assays:
Without S9 mix, cells were exposed to 17.5, 35, 70 (µg/mL) propineb for 24 hours and 10, 20, 40 (µg/mL) propineb for 48 hours.
With S9 mix, cells were exposed to 20, 40, 80 (µg/mL) propineb for 6 hours.

Vehicle / solvent:

DMSO 1%

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

In the preliminary cytotoxicity Assay (First test), without metabolic activation for 24-hour and 48-hour treatment the following concentrations were tested: 0, 40, 50, 60, 70, 80, 90 and 100 µg/mL. In the test without metabolic activation and 48-hour treatment the following concentrations were tested: 0, 20, 25, 30, 35, 40, 50 and 60 µg/mL.

In the test with metabolic activation for 6 hours with the test material followed by 18-hour incubation period the following concentrations were tested: 0, 40, 50, 60, 70, 80, 90 and 100 µg/mL. Cytotoxicity was determined by assessing the concentration that inhibited 50% of growth (IC50)

In the cytogenetic assay, cells were exposed to the test compound, solvent or positive control for 24 or 48 hours under both non-activated conditions. Under activated metabolic conditions the cells were exposed to the test compound, solvent or positive control for 6- hours and then incubated with fresh NCS/MEM for additional 18 hours. Two hours before the cells were harvested; mitotic activity was arrested by addition of Colcemid to each culture at a final concentration of 0.1 µg/mL.

Cultured cells were detached from the incubation dish by adding 025% trypsin and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded. Then , 0.075 M KCl was added and the cells kept at 37°C for 15 minutes, followed by fixation with methanol:acetic acid solution. Cell were suspended in methanol:acetic acid solution to obtained a suspension.

A few drops of the harvested cell suspensions were dropped onto pre cleaned microscope slides which were then allowed to air dry. The slides were then stained in 2% Giemsa.

The selected slides were coded. One hundred metaphase figures were examined, from each culture and were scored for structural aberrations and for numerical aberrations (polyploidy).

Evaluation criteria:

The test substance was considered to cause a positive response if the following conditions were met: Statistically significant increases (P<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) were observed at one or more test concentration. The increases were reproducible between replicate cultures.

Statistics:

The number of aberrant and polyploid metaphase cells in each treatment group was compared with the solvent control value using Fisher's exact test (Fisher 1973).

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

80 µg/mL.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the presence of S9 mix, Propineb caused a reduction of growth below 50% from concentrations of 70 µg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

70 µg/mL following 24-hour treatment.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the absence of S9 mix, Propineb caused a reduction of growth below 50% from 60 µg/mL and in the 48-hour treatment already at 20 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Mitotic metaphase of chromosome enough to make an assessment of chromosomal aberration were observed at doses lower than 70 µg/mL for the 24-hour treatment and lower than 40 µg/mL for the 48-hour treatment without metabolic activation and lower than 80 µg/mL with metabolic activation.

Therefore the highest concentration was decided to be 70 µg/mL for the 24-hour and 40 µg/mL for the 48-hour treatments without metabolic activation and 80 µg/mL for the metabolic activation methods. In addition to this two lower doses with a dilution ratio of 2 were added in each treatment.

In the absence of S9 mix, propineb caused a statistically significant increase in the proportion of cells with chromosomal aberrations, when compared with the solvent control (P<0.01) at the top dose tested of 70 µg/mL following 24-hour treatment. Increased incidence in the proportion of cells with chromosomal aberration was confirmed in a second test again confined at the top dose level of 70 µg/mL. There was no effect on polyploidy at any dose levels. There was no effect on structural chromosomal aberration and/or polyploidy following treatment for 48 hours.

In presence of S9 mix, propineb caused a statistically significant increase in the proportion of cells with chromosomal aberrations, when compared with the solvent control (P<0.01) at the top dose tested of 80 µg/mL.

Table two

Cytotoxicity

Without  S9 Mix	24-hour	Propineb  (µg/mL)	0	40	50	60	70	80	90	100
		Growth rate (%)	100	73.5	54.3	42.8	37.4	23.2	14.7	14.3
	48-hour	Propineb (µg/mL)	0	20	25	30	35	40	50	60
		Growth rate (%)	100	35.3	23.6	32.6	31.2	24.7	20.8	15.0
With S9 Mix		Propineb  (µg/mL)	0	40	50	60	70	80	90	100
		Growth rate (%)	100	82.6	75.8	53.0	37.0	21.0	16.0	10.7

 

Table three

Chromosme aberration test without metabolic activation (-S9)

Exposure period (hours)	Concentrations (µg/mL)		Cells with aberrations Excluding gaps Total number (%)	Cells with aberrations Including gaps Total number (%)
24	Control	Culture medium	0 (0.0)	0 (0.0)
		DMSO	0 (0.0)	2 (1.0)
	Propineb	17.5	1 (0.5)	1 (0.5)
		35	0 (0.0)	0 (0.0)
		70	23 (11.5)***	26 (13.0)***
	Control	0.05  (MMC)	81 (40.5)***	81 (40.0)***
Exposure period (hours)	Concentrations (µg/mL)		Cells with aberrations Excluding gaps Total number (%)	Cells with aberrations Including gaps Total number (%)
48	Control	Culture medium	0 (0.0)	0 (0.0)
		DMSO	0 (0.0)	0 (0.0)
	Propineb	10	1 (0.5)	1 (0.5)
		20	2 (1.0)	2 (1.0)
		40	0 (0.0)	0 (0.0)
	Control	0.05  (MMC)	65 (32.5)***	69 (43.5)***

*** = p< 0.001

 

Table four

Chromosme aberration test with metabolic activation (+S9)

Exposure period (hours)	Concentrations (µg/mL)		Cells with aberrations Excluding gaps Total number (%)	Cells with aberrations Including gaps Total number (%)
6	Control	Culture medium	3 (1.5)	3 (1.5)
		DMSO	1 (0.5)	2 (1.0)
	Propineb	20	1 (0.5)	1 (0.5)
		40	0 (0.0)	1 (0.5)
		80	20 (10.0)***	22 (11.0)***
	Control	10  (CPA)	177 (88.5)***	180 (90.0)***

*** = p< 0.001

Conclusions:

Propineb induced structural chromosomal aberration at toxic doses, i.e. at doses inducing more that 50% inhibition of cell growth.

Executive summary:

A chromosme aberration tests was carried out with Propineb in Chinese hamster lung fibroblasts (CHL cells). Mitomycin C (MMC)  and cyclophosphamide (CPA) were used as positive controls for the tests without and with metabolic activation, respectively. 

Tests on inhibition of cell growth and cell division were carried out to determine the dose levels of the test article. The following dose were tested for the chromosomal aberration test: 17.5, 35 and 70 µg/mL for 24-hour treatment and 10, 20 and 40 for 48-hour treatment without metabolic activation and 20, 40 and 80 µg/mL for the 24-hour treatment with metabolic activation. MMC doses were 0.05 µg/mL for both the 24-hour and 48-hour treatments. CPA concentration was 10 µg/mL.

There were no effects on chromomal aberration in the 48-hour treatment without metabolic activation. In the presence of S9 mix, propineb caused a statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at the highly toxic concentration of 10 mM in the first test, when compared with the solvent control value (P<0.001). In the second test, at the highest level scored for aberrations, 7.5 mM, the toxicity was acceptable with a reduction in mitotic index of 55% and showed a positive response.

A quantitative analysis for polyploidy was made in cultures treated with the negative control and highest dose level. An increase in the proportion of polyploid cells was seen in both tests in both the absence and presence of S9 mix and was statistically significant in the first test in the absence of S9 mix at a highly toxic concentration. All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

It was concluded that the test substance showed evidence of clastogenic activity in this in vitro cytogenetic test system, at toxic concentrations only.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012/05/21 to 2012/06/21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

2-AA was used as the sole indicator of S9 efficacy in the study; however, each batch is stated to have been checked for activity prior to the study.

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC)

Version / remarks:

No. 440/2008, B13/14 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

712-C-98-247

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Range-finding:
First assay for all strains with or without S9 mix: 3, 10, 33, 100, 333, 1000, 2500, and 5000
µg/plate


Pre-incubation assay:
For all strains with or without S9 mix: 33; 100; 333; 1000; 2500; and 5000 µg/plate.

Vehicle / solvent:

Deionised water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

methylmethanesulfonate

other:

Details on test system and experimental conditions:

To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates.
For each strain and dose level, including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
• 100 µL Test solution at each dose level (solvent 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

In the pre-incubation assay 100 µL test solution (solvent control), or reference mutagen solution (positive control), 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacteria suspension 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. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

Rationale for test conditions:

The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the laboratory historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
- a minimum of five analyzable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

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 (3).
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration (2). 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.

Statistics:

A statistical analysis of the data is not mandatory

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

only in experiment II without S9 mix at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

only in experiment II without S9 mix at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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

Remarks:

only in experiment II without S9 mix at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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

Remarks:

only in experiment II without S9 mix at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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

Remarks:

only in experiment II without S9 mix at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In experiment II without S9 mix toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5) were observed in all strains at 5000 µg/plate.

Table 1

Mean mutants values Pre-experiment and Experiment I:

Test item	Concentration µg/plate	Revertant colony plate (Mean ± SD)
		TA1535	TA1537	TA98	TA100	TA 102
Without metabolic activation (S9)
Deionized water		15 ± 6	13 ± 3	24 ± 4	171 ± 27	425 ± 9
Untreated		12 ± 6	15 ± 3	30 ± 3	157 ± 11	409 ± 24
Propineb Technical	3	16 ± 7	11 ± 1	21 ± 5	174 ± 4	422 ± 20
	10	14 ± 2	13 ± 1	25 ± 4	185 ± 5	404 ± 26
	33	13 ± 4	18 ± 4	28 ± 11	166 ± 16	433 ± 16
	100	17 ± 2	15 ± 2	27 ± 7	175 ± 12	490 ± 11
	333	17 ± 3	15 ± 2	26 ± 2	158 ± 10	464 ± 21
	1000	13 ± 2	15 ± 1	22 ± 2	153 ± 5	404 ± 24
	2500	15 ± 5^P	15± 2^P	29 ± 4^P	144 ± 22^P	340 ± 49^P
	5000	15 ± 1^P	10 ± 3^P	27 ± 3^P	112 ± 5^P	273 ± 15^P
NaN3	10	1804 ± 46			1808 ± 108
4-NOPD	10			255 ± 14
	50		170 ± 6
MMS	2.0					3177 ± 46
With metabolic activation (S9)
Deionized water		21 ± 7	20 ± 2	42 ± 4	217 ± 23	624 ± 34
Untreated		22 ± 4	19 ± 4	51 ± 7	205 ± 2	576 ± 49
Propineb Technical	3	21 ± 6	21 ± 2	41 ± 7	173 ± 16	532 ± 16
	10	21 ± 7	24 ± 6	43 ± 11	200 ± 23	550 ± 21
	33	22 ± 9	23 ± 6	45 ± 10	206 ± 11	561 ± 17
	100	23 ± 9	21 ± 5	42 ± 2	207± 21	592 ± 23
	333	22 ± 1	29 ± 1	43 ± 2	183 ± 30	551 ± 12
	1000	20 ± 1	19 ± 6	32 ± 8	184  ± 18	538 ± 13
	2500	18 ± 4^P	22 ± 7^P	35 ± 5^P	194 ± 9^P	520 ± 16^P
	5000	16 ± 3^PM	19± 5^PM	29 ± 3^PM	189 ± 14^PM	539 ± 9^PM
2-AA	2.5	463 ± 20	291 ± 29	1983 ± 200	2740 ± 206
	10.0					2041 ± 115

P = Precipitate     M = Manual count

 

 

Table 2

Mean mutants values Experiment II:

Test item	Concentration µg/plate	Revertant colony plate (Mean ± SD)
		TA1535	TA1537	TA98	TA100	TA 102
Without metabolic activation (S9)
Deionized water		13± 3	17 ± 4	31 ± 4	155 ± 12	430 ± 24
Untreated		9 ± 1	21 ± 2	27 ± 2	143 ± 8	371 ± 8
Propineb Technical	3	13 ± 3	16 ± 4	29 ± 2	152 ± 13	419 ± 12
	100	14 ± 5	20 ± 5	27 ± 6	154 ± 9	455 ± 19
	333	16 ± 4	17 ± 4	34 ± 10	131 ± 6	456 ± 38
	1000	15 ± 4	15 ± 1	27 ± 4	167 ± 22	384 ± 21
	2500	11 ± 3^P	14± 6^P	20 ± 4^P	98± 8^P	320 ± 10^P
	5000	0 ± 0^PMR	1 ± 1^PMR	0 ± 0^PMR	0 ± 0^PMR	179 ± 16^P
NaN3	10	2016 ± 46			2237 ± 82
4-NOPD	10			381 ± 17
	50		93 ± 4
MMS	2.0					3485 ± 224
With metabolic activation (S9)
Deionized water		22 ± 6	27 ± 4	41 ± 6	186 ± 6	463 ± 10
Untreated		19 ± 2	28 ± 3	42 ± 7	170 ± 15	525 ± 34
	33	26 ± 2	29 ± 7	47 ± 14	193± 16	525 ± 43
	100	23 ± 5	29 ± 2	39 ± 7	172 ± 14	555 ± 37
	333	19 ± 4	28 ± 8	42 ± 4	151 ± 7	517 ± 35
	1000	24 ± 2	30 ± 4	41 ± 3	154  ± 13	541 ± 55
	2500	17± 4^P	28 ± 8^P	30 ± 9^P	150 ± 9^P	460 ± 8^P
	5000	10 ± 3^PM	17± 3^PM	20 ± 3^PM	109 ± 3^PM	484 ± 21^PM
2-AA	2.5	273 ± 11	184 ± 9	1338 ± 200	1513 ± 63
	10.0					1781 ± 386

P = Precipitate     M = Manual count R= Reduced background growth

Conclusions:

This study was performed to investigate the potential of Propineb Technical to induce gene
mutations in 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, the pre-Experiment/Experiment I had concentrations of 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate. In conclusion, it can be stated that during the described mutagenicity test and under the
experimental conditions reported, the test item did not induce gene mutations by base pair
changes or frameshifts in the genome of the strains used. Therefore, Propineb Technical is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

In this in vitro study, propineb technical was tested to assess its potential to induce gene mutations in 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.

 

In the pre-experiment/experiment I the test item was tested at the following concentrations: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate and in experiment II at the following concentrations: 33, 100, 333, 1000, 2500, and 5000 µg/plate. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in experiment I and with metabolic activation in experiment II. In experiment II without S9 mix reduced background growth was observed in strains TA 1535, TA 1537, TA 98, and TA 100 at 5000 µg/plate.

 

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation in experiment I and with metabolic activation in experiment II. In experiment II without S9 mixtoxic effects, evident as a reduction in the number of revertants (below the indication factorof 0.5) were observed in all strains at 5000 µg/plate.

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with propineb pechnical at any dose level, neither 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, propineb technical did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The key study for in vivo genotoxicity endpoint is:

Micronucleus test on the mouse (OECD 474, GLP, RL1), A negative result is reported for a mouse bone marrow micronucleus assay with propineb, using single ip doses of 500 and 700 mg/kg bw (sampling at 16, 24 and 48 hours) (M-105002-01-1, Herbold, 1997)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996/10/15 to 1997/05/09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

The administration was via the i.p. route, which is generally not recommended since it is not an intended route of human exposure. Based on evidence of target tissue exposure (altered NCE:PCE ratio) and clear evidence of systemic toxicity, including mortality at 700 mg/kg bw, it is concluded that dosing route was appropriate. Two thousand (2000) instead of recommended 4000 immature erythrocytes per animal were tested, however since the total number of animals tested/ concentration was 10 (instead of the recommended 5), the overall number of analysed cells was concluded to be sufficient.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

The test material was a formulation containing 70% propineb

Species:

mouse

Strain:

other: Hsd/Win

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 6 to 12 weeks.
- Weight at study initiation: males: 37-44 g, females: 28-34 g
- Housing: The animals were kept singly in type I cages.
- Water: Tap water ad libitum.
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±1.5
- Humidity (%): 40% to 70%
- Air changes (per hr): 10

Husbandry was standardized, with twelve hours of electrical lighting daily (6.00 hours to 18.00 hours, about 500 lux), 22.5-23°C room temperature, and 47-54% (1st trial) and 33-38% (2nd trial) mean relative humidity. EP IT Elb. 2 (engineering department) gives the following settings for the animal room: 22±1.5°C, 40% to 70% humidity and air change about ten times per hour.

Route of administration:

intraperitoneal

Vehicle:

Water

Details on exposure:

The treated animals received a single intraperitoneal administration of either propineb or cyclophosphamide. The femoral marrow of groups treated with propineb was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours. The doses of propineb were 500 mg/kg bw in the first trial and 700 mg/kg bw in the second experiment. For the positive control, cyclophosphamide, 20 mg/kg body weight were used for both studies.

Including both males and females, were intraperitoneally administered 250 mg/kg bw, 500 mg/kg bw and 1000 mg/kg bw of the test material.

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Once

Post exposure period:

16, 24, 48 hours

Dose / conc.:

250 mg/kg bw/day

Remarks:

First experiment: equivalent to 175 mg/kg bw/d propineb

Dose / conc.:

500 mg/kg bw/day

Remarks:

First experiment: equivalent to 350 mg/kg bw/d propineb

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

First experiment: equivalent to 700 mg/kg bw/d propineb

Dose / conc.:

500 mg/kg bw/day

Remarks:

Second experiment: equivalent to 350 mg/kg bw/d propineb

Dose / conc.:

700 mg/kg bw/day

Remarks:

Second experiment: equivalent to 490 mg/kg bw/d propineb

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes

Positive control(s):

20 mg/kg Cyclophosphamide

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

At least one intact femur was prepared from each sacrificed animal (not pretreated with a spindle inhibitor). A suitable instrument was used to sever the pelvic bones and lower leg.
The femur was separated from muscular tissue. The lower-leg stump, including the knee and all attached soft parts, was separated in the distal epiphyseal cartilage by a gentle pull at the distal end. The proximal end of the femur was opened at its extreme end with a suitable instrument, e.g. fine scissors, making visible a small opening in the bone-marrow channel. A suitable tube was filled with sufficient fetal calf serum. A small amount of serum was drawn from the tube into a
suitable syringe with a thin cannula. The cannula was pushed into the open end of the marrow
cavity. The femur was then completely immersed in the calf serum and pressed against the wall of the tube, to prevent its slipping off. The contents were then flushed several times and the bone
marrow was passed into the serum as a fine suspension. Finally, the flushing might be repeated from the other end, after it had been opened. The tube containing the serum and bone marrow was centrifuged in a suitable centrifuge at approximately 1000 rpm for five minutes.
The supernatant was removed with a suitable pipette (e.g. Pasteur pipette), leaving only a small remainder. The sediment was mixed to produce a homogeneous suspension. One drop of the viscous suspension was placed on a well cleaned slide and spread with a suitable object, to allow
proper evaluation of the smear. The labeled slides were dried overnight. If fresh smears needed to be stained, they needed to be dried with heat for a short period.
The Staining of Smears:
The smears were stained automatically with an Ames Hema-Tek Slide Stainer from the Miles Company. The slides were then "destained" with methanol, rinsed with deionized water, and left to dry.
The Covering of Smears:
Following this treatment, the smears were transferred to a holder. A cuvette was filled with xylene, into which the holder was immersed for approximately ten minutes. The slides were removed singly (e.g. with tweezers) to be covered. A small amount of covering agent was taken from a bottle with a suitable object (e.g. glass rod) and applied to the coated side of the slide. A cover glass was then placed in position without trapping bubbles. The slides were not evaluated until the covering agent had dried.

Evaluation criteria:

Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the a nucleated erythrocytes. They can be distinguished from artifacts by varying the focus.

If the ratio for a single animal amounts to distinctly more than 3000 normochromatic erythrocytes per 1000 polychromatic ones, or if such a ratio seems likely without other animals in the group showing similar effects, then the case may be regarded as pathological and unrelated to treatment, and the animal may be omitted from the evaluation. A test was considered positive if, at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control. test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of negative controls.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

A negative result is reported for a mouse bone marrow micronucleus assay with propineb, using single doses of 500 and 700 mg/kg bw (sampling at 16, 24 and 48 hours).

There is evidence of target tissue exposure (altered NCE:PCE ratio) and clear evidence of systemic toxicity, including mortality at 700 mg/kg bw. Furthermore, target tissue exposure can be assumed due to the dosing method used.

Conclusions:

No relevant indications of a clastogenic effect of propineb were found after a single intraperitoneal treatment with 500 and 700 mg/kg bw.

Executive summary:

The micronucleus test was employed to investigate propineb in male and female mice for a possible clastogenic effect on the chromosomes of bone-marrow erythroblasts in two independent experiments. The known clastogen and cytostatic agent, cyclophosphamide, served as positive control. The treated animals received a single intraperitoneal administration of either a propineb product (containing 70% propineb) or cyclophosphamide.

The femoral marrow of groups treated with propineb was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours. The doses of propineb were 500 mg/kg bw in the first trial and 700 mg/kg bw in the second experiment. For the positive control, cyclophosphamide, 20 mg/kg body weight were used for both studies. The animals treated with propineb showed symptoms of toxicity after administration. Two of forty animals died before the end of the test due to the acute intraperitoneal toxicity of 700 mg/kg bw propineb. There was an altered ratio between polychromatic and normochromatic erythrocytes. No relevant indications of a clastogenic effect of propineb were found after a single intraperitoneal treatment with 500 and 700 mg/kg.

Cyclophosphamide, the positive control, had a clear clastogenic effect, as is shown by the biologically relevant increase in polychromatic erythrocytes with micronuclei. The ratio of polychromatic to normochromatic erythrocytes was not altered.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

A complete battery of mutagenicity studies has been conducted with propineb.
A new Ames test was performed showing no bacterial mutagenicity potential for the test substance. Results from an in vitro mammalian mutagenicity study showed no concern for mutagenicity. An in vitro chromosome aberration test showed effects on chromosome aberration at cytotoxic doses. However, concern for propineb clastogenic and/or cytogenetic potential was dismissed by in vivo bone marrow micronucleus tests in mice following intraperitoneal and oral dosing with propineb. In conclusion, propineb is not genotoxic.

Justification for classification or non-classification

The available data on genetic toxicity does not meet the criteria for classification according to Regulation (EC) 1272/2008, and is therefore conclusive but not sufficient for classification.