Propanoic acid, 3-[[bis(2-methylpropoxy)phosphinothioyl]thio]-2-methyl-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was found to cause clastogenicity and cytotoxicity in vitro (OECD 473, 1998), but no mutagenicity in bacteria (OECD 471, 1997).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 27, 1999- October 27, 1999

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:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

All Salmonella Typhimurium strains are histidine auxotrophic mutants. The E. coli strain is a tryptophan auxotrophic mutant.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: TA1537: his C 3076; rfa-; uvrB-; TA 98: his D 3052; rfa-; uvrB-; TAl535: his G 46; rfa-; uvrB-, TA 100: hisG46;rfa-;uvrB-. The uvrB derivative has a reduction in the activity of an excision repair system.

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: The uvrA derivative is deficient in the DNA repair process (excision repair damage).

Metabolic activation:

with and without

Metabolic activation system:

Mixture of co-factors with S9 fraction of liver from Wistar rats (HanIbm) induced with phenobarbital and beta-naphthoflavone.

Test concentrations with justification for top dose:

A preliminary toxicity test was carried out with concentrations of 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate suspended in dimethylsulfoxide. From the preliminary test, following concentrations: 0, 33, 100, 333, 1000, 2500, 5000 µg/plate, were used for assessment of mutagenicity, since only minor toxic effects were observed in one of the strains at the maximal concentration and 5000 µg/plate were chosen as maximal concentration. These concentrations were designated as Experiment I of the main mutagenicity test (Plate Incorporation Test).
Since results of Experiment I were negative a second mutagenicity test, designated as Experiment II, was undertaken with following concentrations:
0, 33, 100, 333, 1000, 2500, 5000 µg/plate (Pre-Incubation Test).

Vehicle / solvent:

- Vehicle: DMSO
The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria
No precipitation of the test item occurred up to the highest investigated dose.

Untreated negative controls:

yes

Remarks:

concurrent untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

(without metabolic activation)

Positive control substance:

sodium azide

Remarks:

TA 1535, TA 100; 10 µg/plate

Untreated negative controls:

yes

Remarks:

concurrent untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

(without metabolic activation)

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

TA 98, 10 µg/plate; TA 1537, 50 µg/plate

Untreated negative controls:

yes

Remarks:

concurrent untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

(without metabolic activation)

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli WP2 uvrA, 5 µL/plate

Untreated negative controls:

yes

Remarks:

concurrent untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

(with metabolic activation)

Positive control substance:

other: 2-aminoanthracene

Remarks:

All S. typhimurium, 2.5 µg/plate; E. coli WP2 uvrA, 10 µg/plate

Details on test system and experimental conditions:

The study consisted of a toxicity pre-screen test with and without metabolic activation (plate incorporation, reported as experiment I). Only minor toxic effects were observed. Experiment II was performed as pre-incubation test with and without metabolic activation.

METHOD OF APPLICATION:
Experiment I: plate incorporation test; Experiment II: preincubation test
In each experiment 0.1 mL of the test substance or the vehicle, 0.1 mL of a bacterial culture (in nutrient broth), 0.5 ml S9 mix (with metabolic activation) or S9 mix substitution buffer (without metabolic activation) in 2.0 mL of soft agar was used. In the pre-incubation assay soft agar was added after an incubation period of 1 h at 37 °C. The plates were incubated for about 48 hours at 37 °C in darkness. Each concentration and the controls were tested in triplicate.

DURATION
- Preincubation period: 1 h (Experiment II)
- Expression time: 48 h (Experiment I + II)

DETERMINATION OF CYTOTOXICITY
- Toxicity was assessed as clearing of the bacterial background lawn and/or as reduction of spontaneous revertants.

Evaluation criteria:

A test item was considered positive if either a dose related increase or a biologically relevant increase for at least one test concentration in the number of revertants was induced.
A test item was considered mutagenic if in the S. Typhimurium strains TA 98, TA 100, and E. coli WP2 uvrA the number of reversions was at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants was regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the above described enhancement factors or not.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

A decrease in the number of revertants was seen at concentrations of at least 2500 µg/plate (Experiment II, without S9 mix) and 1000 µg/plate (Experiment II, with S9 mix).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A decrease in the number of revertants was seen at concentrations of at least 2500 µg/plate (Experiment II, with and without S9 mix) and 5000 µg/plate (Experiment I, with S9 mix).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

After treatment with CG 37-1586 neither the number of histidine-prototrophic mutants nor the number of tryptophane-prototrophic mutants were increased in comparison to the negative control in any of the experiments. Plates incubated with test article showed normal background growth in either concentration with and without metabolic activation.

Summary of Results

without S9 mix

Concentration	TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
µg/plate	I	II	I	II	I	II	I	II	I	II
Negative control	20	12	10	25	15	20	98	81	44	46
Solvent control	12	12	14	23	15	19	93	88	40	55
Positive control	1297	615	69	89	440	270	Il49	879	820	273
33	14	8	15	26	12	18	93	82	34	54
100	15	6	15	27	12	17	93	87	37	53
333	16	8	13	16	12	17	101	91	36	54
1000	17	8	13	18	12	15	94	87	30	48
2500	18	3	13	10	10	16	92	86	35	34
5000	6	3	8	3	12	11	66	79	38	35

with S9 mix

Concentration	TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
µg/plate	I	II	I	II	I	II	I	II	I	II
Negative control	13	11	7	25	16	23	115	92	49	52
Solvent control	9	10	11	18	15	16	101	94	49	44
Positive control	105	80	56	81	448	694	896	683	204	182
33	9	8	12	22	11	19	98	92	46	71
100	8	7	12	27	16	12	95	87	45	54
333	12	6	9	28	18	16	97	91	45	42
1000	13	3	10	13	10	12	106	93	37	41
2500	10	I	10	4	9	10	93	83	39	36
5000	8	4	4	2	7	8	80	78	30	35

I Plate incorporation test

II Preincubation test

Conclusions:

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, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 27, 1999 - May 09, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1998

Deviations:

yes

Remarks:

Guideline recommends to score 200 well-spread metaphases. 100 well-spread metaphases were scored in the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable, since chromosome aberrations were assessed.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix was obtained by adding cofactor-solution to S9 fraction prepared from livers of beta-naphtoflavone (orally) and phenobarbital (i.p.) treated male Wistar rats.

Test concentrations with justification for top dose:

A range finding growth inhibition test was performed. On basis of the results, concentrations of test article in experiment 1 (with and without S9 mix) ranged from 50 to 250 µg/mL. Due to results of this experiment, concentrations in a second experiment ranged from 12.5 to 125 µg/mL (without S9 mix) and from 100 to 225 µg/mL (with S9 mix). For details see additional information on materials and methods.

Vehicle / solvent:

- Vehicle: DMSO in a final concentration of 0.5 % in cell culture medium .

Untreated negative controls:

yes

Remarks:

concurrent negative (culture medium)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

4.8 mM and 8.0 mM; without metabolic activation

Untreated negative controls:

yes

Remarks:

concurrent negative (culture medium)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

2.5 µM; with metabolic activation

Details on test system and experimental conditions:

Experiment I
Exposure period 4 hours.
Culture medium was replaced with test article in serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with 10 % FCS (v/v). For the treatment with metabolic activation 50 µL/mL S9 mix per mL culture medium were added. Concurrent negative, solvent and positive controls were performed. After 4 h the cultures were washed twice with Saline G and then the cells were cultured in complete medium for the remaining culture time (14 h and 24 h, respectively).

Experiment II
Exposure period 4 hours.
Culture medium was replaced with test article in serum-free medium. For the treatment with metabolic activation 50 µl/mL S9 mix per ml culture medium were added. Concurrent negative, solvent and positive controls were performed. After 4 h the cultures were washed twice with Saline G and then the cells were cultured in complete medium for the remaining culture time (14 h and 24 h, respectively).
Exposure period 18 and 28 hours.
These incubation periods were performed without metabolic activation. The culture medium was replaced with complete test article in culture medium (10 % FCS).The medium was not changed until preparation of the cells.

Preparation of the cultures
Two hours prior harvesting 0.2 µg/mL culture medium colcemid was added to the cultures. Cells were treated with hypotonic solution (0.4 % KCI) for 20 min and fixed with methanol:glacial acetic acid (3:1), thereafter. After preparation the cells were stained with Giemsa.
Two cultures per group were set up in parallel (without colcemid-treatment). The cell number within 10 defined fields per slide was assessed to determine the growth inhibitory effects of the test article.

The number of polyploid cells was determined of each test group in a sample of 500 cells per culture. The mitotic index was determined in a sample of 1000 cells per culture of each test group. 100 well-spread metaphase plates per culture were scored for cytogenetic damage on coded slides.

Evaluation criteria:

Analysis of Metaphase Cells
Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis for chromosomal aberrations like: Breaks, fragments, deletions, exchanges and chromosome disintegrations. Gaps were recorded but not included in the calculation of the aberration rates.

To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells was determined.

The chromosome aberration assay is considered acceptable if the number of structural aberrations found in the negative and solvent controls falls within the range of historical laboratory control data. Additionally, the number of chromosome aberration of the positive control needs to be significant different from the control group. Therefore, a test article is classified as mutagenic, if the number of induced structural chromosome aberrations are not in the range of historical control data and a significant increase of the number of structural chromosome aberrations is observed.

Statistics:

Statistical significance was confirmed by means of the Fischer's exact test.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

CG 37-1586 caused a dose dependent increase in chromosome aberrations, statistically significant at and above a concentration of 100 µg/mL (2.5 % aberrant cells). This effect was within the historical laboratory control (0–4 % aberrant cells).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

After 18 h exposure to 100 µg/mL CG 37-1586 the mitotic index and the cell numbers were decreased in comparison to the control (56 and 49%, respectively).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

After the 28 h interval (4 h exposure) the aberration frequency showed a dose dependent increase, being significant at concentrations of 200 µg/mL or higher (13.5 % or higher).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

After the 28 h interval (4 h exposure) cytotoxicity was increased at and above concentrations of 150 µg/mL (41 % or higher).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CHROMOSOME ABERRATION
EXPERIMENT I
In the presence of S9 mix (18 h time interval) a statistically significant increase of chromosome aberrations (5 % without gaps) was seen at a test concentration of 200 µg/mL versus 0.5 % in solvent control. The frequency obtained was slightly beyond the historical control values. Since the result was considered to be inconclusive Experiment II was undertaken.

EXPERIMENT II
After the 28 h interval (4 h exposure, with S9 mix) a dose dependent increase of chromosome aberrations was seen with 175, 200, 225 µg/mL (2.5, 13.5, 36.5 % aberrant cells, without gaps, versus 0.0 % in solvent control). Additionally, the increase of cells with aberrations in the two highest groups was statistically significant (7 and 23 %).
Continuous exposure (without S9 mix) for 28 h was evaluated for chromosome aberration at a test concentration of 50 µg/mL only (see table in additional remarks above). Chromosome aberration frequency was in the range of the solvent control (0.5 % without gaps vs. 1 %).
Continuous exposure (without S9 mix) for 18 h was evaluated for chromosome aberration at a test concentration of 25, 50, 75, 100 µg/mL. At the highest concentration of 100 µg/mL the chromosome aberration frequency (without gaps) was 2.5 % versus 0 % for the solvent control. This value was statistically significant.

CYTOTOXICITY
EXPERIMENT I
In the absence of S9 mix (18 h time interval) the mitotic index was 83.5, 93.3 and 60.3 % at 50, 100 and 150 µg/mL, respectively; Mitotic index in the solvent control was 100 %.
In the presence of S9 mix (18 h time interval) the mitotic index was 93.7, 93.7 and 87.1 % at 50, 100 and 200 µg/mL, respectively; Mitotic index in the solvent control was 100 %.

EXPERIMENT II
After the 28 h interval (4 h exposure, with S9 mix) the mitotic index was 68.6, 99.6, 86, 41.3, 38 % at 100, 150, 175, 200 and 225 µg/ml, respectively.
Following continuous exposure (without S9 mix) for 28 h the mitotic index was 95.9 % at 50 µg/ml; Mitotic index in the solvent control was 100 %.
Following continuous exposure (without S9 mix) for 18 h the mitotic index was 152.9, 90.4, 58.6 and 56.1 % at 25, 50, 75, 100 µg/ml, respectively; Mitotic index in the solvent control was 100 %.

REMARK
Increased aberration frequencies were observed only in the presence of reduced cell numbers and/or mitotic indices. It was stated in the report that therefore, a non-genotoxic DNA damaging mechanism can not be excluded.

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

Summary of Results

						Aberrant cells in %
Exp.	Preparation interval	Test Item Concentration in µg/ml	Polyploid cells in %	Cell number in % of control	Mitotic index in % of control	incl. gaps	excl. gaps*	exchanges
Exposure period 4 h without S9 mix
I	18 h	negative control	3.7	n.t.	100.0	2.5	1.0	0.5
		solvent control^1	6.5	100.0	100.0	1.5	1.5	0.0
		positive control^3	5.0	n.t.	81.0	16.5	16.0^S	14.0
		50.0	5.2	81.0	84.0	3.0	0.5	0.5
		100.0	4.0	69.0	93.0	3.0	1.5	0.0
		150.0	5.8	46.0	60.0	1.0	0.5	0.5
Exposure period 18 h without S9 mix
II	18 h	negative control	5.2	n.t.	100.0	1.5	1.5	0.5
		solvent control^1	4.4	100.0	100.0	0.0	0.0	0.0
		positive control^2	4.7	n.t.	67.0	26.5	24.0^S	13.5
		25.0	5.2	101.0	153.0	1.0	0.0	0.0
		50.0	3.0	104.0	90.0	0.5	0.5	0.0
		75.0	7.5	67.0	59.0	1.5	1.5	0.0
		100.0	2.6	49.0	56.0	4.0	2.5^S	0.0
Exposure period 28 h without S9 mix
II	28h	negative control	3.9	n.t.	100.0	2.5	1.5	0.5
		solvent control^1	5.9	100.0	100.0	1.5	1.0	0.0
		positive control^2	3.0	n.t.	67.0	30.5	29.0^S	14.5
		50.0	5.5	86.0	96.0	1.0	0.5	0.0
Exposure period 4 h with S9 mix
I	18 h	negative control	2.7	n.t.	100.0	1.0	0.0	0.0
		solvent control^1	4.3	100.0	100.0	1.0	0.5	0.0
		positive control^4	4.6	n.t.	78.0	12.0	12.0^S	8.0
		50.0	3.0	100.0	94.0	1.5	1.0	0.0
		100.0	4.2	90.0	94.0	1.0	0.5	0.0
		200.0	4.2	56.0	87.0	7.0	5.0^S	2.5
II	28 h	negative control	3.4	n.t.	100.0	2.5	1.5	0.0
		solvent control^1	3.2	100.0	100.0	1.5	0.0	0.0
		positive control^4	4.5	n.t.	82.0	13.0	9.5^S	3.5
		100.0	4.7	86.0	69.0	0.5	0.5	0.0
		150.0	2.6	59.0	100.0	1.0	0.5	0.0
		175.0	5.1	58.0	86.0	2.5	2.5	0.5
		200.0	6.6	31.0	41.0	14.5	13.5^S	7.0
		225.0	8.6	28.0	38.0	37.0	36.5^S	23.0

* inclusive cells carrying exchanges

n.t. not tested

^S aberration frequency statistically significant higher than corresponding solvent control values

¹ DMSO 0.5 %

² EMS 600 pg/mL

³ EMS 1000 pg/mL

⁴ CPA 0.7 Mg/mL

Conclusions:

Under the experimental conditions reported, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells in vitro. Therefore, the test item is considered to be clastogenic in this chromosome aberration test in the presence of S9 mix.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

No genotoxicity was observed in the micronucleus test upon oral application of a toxic dose to mice (OECD 474, 1997).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 10, 2000 - June 13, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study conducted according to GLP. Deviations from the protocol were stated (example given: relative humidity under which the experiment was conducted ranged between 18-70 % and not between 30-70 % as described in the protocol) which however, did not affect the validity of the experiment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(1997)

Deviations:

yes

Remarks:

Deviations from the protocol were stated (example given: relative humidity under which the experiment was conducted ranged between 18-70 % and not between 30-70 % as described in the protocol) which however, did not affect the validity of the experiment.

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd. Biotechnology & Animal Breeding Division CH-4414 Füllinsdorf
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: 33 +/- 2.7 g (males), 28.4 +/- 1.8 g (females)
- Assigned to test groups randomly: yes
- Housing: individually in Makrolon type-I cages
- Diet (ad libitum): pelleted standard diet, ALTROMIN 1324
- Fasting period before study: 18 h
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 4
- Humidity (%): 18 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle: corn oil
- Concentration of test material in vehicle: In a pretest 2000 mg/kg bw, 1800 mg/kg bw in main study
- Application volume: 10 mL/kg bw

Duration of treatment / exposure:

After single oral administration the following dose levels of the test item were investigated:
24 h preparation interval: 450, 900 and 1800 mg/kg bw
48 h preparation interval: 1800 mg/kg bw

Frequency of treatment:

Single oral administration

Post exposure period:

MAIN STUDY:
450, 900 and 1800 mg/kg bw: 24 h
1800 mg/kg bw: 48 h

Dose / conc.:

450 mg/kg bw/day (actual dose received)

Remarks:

observation period 24 h

Dose / conc.:

900 mg/kg bw/day (actual dose received)

Remarks:

observation period 24 h

Dose / conc.:

1 800 mg/kg bw/day (actual dose received)

Remarks:

observation period 24 h and 48 h

No. of animals per sex per dose:

Six animals per sex per dose (5 per sex were evaluated)

Control animals:

yes, concurrent no treatment

Positive control(s):

- Positive Control: 40 mg/kg bw Cyclophosphamide
- Volume administered: 10 mL/kg bw
- Route of administration: single oral application

Tissues and cell types examined:

Bone marrow was harvested from the femurs. 2000 PCE per animal were used to determine the incidence of micronucleated PCE. Normochromatic erythrocytes (NCE) as well as polychromatic erythrocytes (PCE) were counted in the same sample for determination of the PCE/NCE-ratio.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pre-test with 2000 mg/kg bw a female died 24 h after application of CG 37-1586 per gavage. In a second pre-test with 4 animals (2 males, 2 females) no mortality was observed within 48 h after administration of 1800 mg/kg bw test article. Therefore, 1800 mg/kg bw CG 37-1586 was selected as maximum tolerated dose level.

DETAILS OF SLIDE PREPARATION:
Bone marrow was harvested from the shafts of femurs. Preparations were air-dried and stained in May-Grünwald/Giemsa's. Cover slips were mounted in Eukitt.

Evaluation criteria:

In this report, a test article was classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant and biologically relevant positive response.

A test item producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and biologically relevant positive response is considered non-mutagenic.

Statistics:

The significance of difference was assessed by the non-parametric Mann-Whitney test.

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

PCEs with micronuclei 0.03, 0.05, 0.035 % after 24 h at 450, 900 and 1800 mg/kg bw, respectively versus 0.05 % in control. After 48 h at 1800 mg/kg bw the percentage was 0.015 %. Positive control (24 h) showed 1.67 % micronucleated PCE.

Toxicity:

yes

Remarks:

The number of NCE increased slightly at the dose level of 1800 mg/kg bw (2027 NCE/2000 PCE after 24 h; 2117 NCE/2000 PCE after 48 h) in comparison to vehicle control (1732 NCE/2000 PCE).

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

In a pre-test with 2000 mg/kg bw a female died 24 h after application of the test article per gavage. In a second pre-test with 4 animals (2 males, 2 females) no mortality was observed within 48 h after administration of 1800 mg/kg bw test article. Therefore, 1800 mg/kg bw test material was selected as maximum tolerated dose level. However, animals at this dose level showed clinical signs of toxicity (reduction of spontaneous activity, eyelid closure, apathy, abdominal position). During main study one female died in the highest dose level within 24 h. It was replaced with a satellite animal so that the overall number of animals was sufficient.

No statistically significant increase in the frequency of micronuclei at any preparation interval and dose level after administration of the test item was seen. The mean values of micronuclei observed after treatment with the test item were below or as high as the value of the vehicle control group and within the historical control data range.

The percentage of micronucleated cells in the high dose group at the 48 h sampling time was below the historical data range. However, the authors stated that such values have been obtained in previous studies and do not affect the validity of the study.

 

The positive control (cyclophosphamide) showed a statistically significant increase of induced micronucleus frequency.

Summary of Micronucleus Test Results

Test group	Dose mg/kg bw	Sampling time (h)	PCEs with micronuclei (%)	Range*	PCE/NCE
Vehicle	0	24	0.050	0 - 3	2000 / 1732
Test item	450	24	0.030	0 - 2	2000 / 1638
Test item	900	24	0.050	0 -3	2000 / 1917
Test item	1800	24	0.035	0 - 2	2000 / 2027
Cyclo phosphamide	40	24	1.670	7 - 52	2000 / 1909
Test item	1800	48	0.015	0 - 2	2000 / 2117

* Number of micronucleated PCEs

Conclusions:

Under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo

The test item belongs to the group of dithiophosphoric acid esters, the most prominent member of which being malathione (Cas no.121-75-5). The test item caused no mutagenicity in bacteria (2000). In the chromosome aberration test in vitro, aberration frequency of cells increased in the presence of induced rat liver S9 mix (2000). Cytotoxicity was observed both in the presence and absence of metabolic activation. No clastogenicity was observed upon oral application of a toxic dose to mice (2000). The oral route of application is acceptable since the substance is stable in regard to hydrolysis and caused toxic effects on the bone marrow cells. This genotoxicity profile is similar to that of technical grade malathion. Malathion is related to the ester by-product of the commercial product, whereas the main product itself is related to the non toxic acid metabolite of malathion. From the metabolism data on malathion it is speculated that the clastogenicity is caused by the ester by-product rather than the parent acid compound. Malathion undergoes CYP-catalysed oxidative desulfuration whereas the acid metabolite appears to be eliminated without further oxidative metabolism. The genotoxicity and carcinogenicity of malathione has been studied in detail. Malathione was tested for carcinogenicity in rats and mice (NCI/NTP Carcinogenesis Technical Report Series, National Cancer Institute/National Toxicology Program, U.S. Department of Health and Human Services, TR-192 Y79 and TR-24 Y78) and it was found to be non carcinogenic upon dietary application.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008, Annex I,XX.,3.5. Germ cell mutagenicity. As a result the substance is not considered to be classified under Regulation (EC) No. 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218.