Pentapotassium ({[(hydroxyphosphinato)methyl](phosphonatomethyl)nitroryl}methyl)phosphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacterial mutagencity: WoE read-across from ATMP-H, negative with and without metabolic activation in S. typhimurium TA 98; 100; 1535 and 1537 (similar to OECD Test Guideline 471) (Monsanto, 1981)

In vitro bacterial mutagencity: WoE read-across from ATMP-H, negative with and without metabolic activation in E. coli WP2 uvr A (OECD Test Guideline 471) (Envigo, 2018)

In vitro cytogenicity in mammalian cells: ATMP-N-oxide-xK was negative with and without metabolic activation in peripheral human lymphocytes (OECD Test Guideline 473) (Safepharm Laboratories, 1994)

In vitro mutagenicity in mammalian cells: read-across from ATMP-xNa, negative with metabolic activation in mouse lymphoma L5178Y cells (similar to OECD Test Guideline 476) (SRI, 1988)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994-02-22 to 1994-05-12

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:

1981

Deviations:

yes

Remarks:

experiment was not repeated to confirm negative result, exposure period without metabolic activation 20 hours

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: peripheral human

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 - induced rat liver S9

Test concentrations with justification for top dose:

393.75-1575 µg/ml (without metabolic activation), 787.5-3150 µg/ml (with metabolic activation). This is equivalent to an active acid content of approximately 250-1000 500-2000 µg/ml and 500-2000 µg/ml.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water. Hepes buffer was added to maintain pH balance.

- Justification for choice of solvent/vehicle: none given in report.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation: 500 µg/ml

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

without metabolic activation: 25 µg/ml

Details on test system and experimental conditions:

ACTIVATION: Aroclor induced rat liver S9 mix included NADP, G6P, KCl and MgCl₂ in Phosphate buffer, final concentration S9 mix in cultures was 1%

METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period:
- Exposure duration: 4 hours (with metabolic activation),
- Expression time (cells in growth medium): 16 hours (with metabolic activation),
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (2 h before harvest)
STAIN (for cytogenetic assays): Giesma

NUMBER OF REPLICATIONS: duplicate cultures for each dose level

NUMBER OF CELLS EVALUATED: 100 well-spread metaphases from each culture were scored for gaps, breaks or rearrangements. 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as mitotic index and percentage of vehicle control values.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; other: percentage of vehicle control values

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no

Evaluation criteria:

A positive response was recorded if the percentage of cells with aberrations markedly exceeded the concurrent vehicle control. For modest increases a dose-response relationship is generally required.

Statistics:

Fisher's exact test.

Species / strain:

lymphocytes: peripheral human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1575 µg/ml (without metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH was controlled with buffer
- Effects of osmolality: not recorded

COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle control cultures gave values of aberrations within the range of historical controls.

Results of Chromosome Aberration Assay

Test material: BRIQUEST 3010-25K

Harvest time: 20 hours

 

Treatment Group	Replicate Identification	No. of Cells Scored	Total Gaps	Chromatid		Chromosome		Others	Total Aberrations		Aberrant Cells		Mean Mitotic Index
				Breaks	Exchanges	Breaks	Exchanges	X	(+Gaps)	(-Gaps)	(+Gaps)	(-Gaps)
Without Metabolic Activation
Solvent Control	Total	200	3	1	0	0	0	0	4	7	4	1	8.05
393.75 (µg/ml)	Total	192	1	0	0	0	0	0	1	0	1	0	6.15
787.5 (µg/ml)	Total	200	3	0	0	0	1	0	4	1	4	1	4.73
1575 (µg/ml)	Total	200	1	0	0	1	0	0	2	1	2	1	3.25
Positive Control EMS 500 (µg/ml)	Total	200	30	41	0	14	0	0	85	55	57***	38***	4.58
With Metabolic Activation
Solvent Control	Total	200	3	2	0	0	0	0	5	2	5	2	5.05
787.5 (µg/ml)	Total	200	4	0	0	1	0	0	5	1	5	1	4.15
1575 (µg/ml)	Total	200	4	2	0	0	0	0	6	2	6	2	4.15
3150 (µg/ml)	Total	200	0	0	0	1	0	0	1	1	1	1	4.03
Positive Control CP 25 (µg/ml)	Total	200	24	13	1	4	0	1	42	18	30***	17***	1.70

X = > 10 aberrations per cell (not included in total aberrations)

*** = p < 0.001

Conclusions:

In an in vitro cytogenicity / chromosome aberration study in mammalian cells, ATMP-N-oxide-xK was tested according to OECD TG 473 and in compliance with GLP. No increase in the number of cells with aberrations was observed when peripheral human lymphocytes were tested up to cytotoxic concentrations. Exposure lasted for 4 hours in the presence of metabolic activation and for 20 hours in the absence of metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of the test.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

: not tested in the absence of metabolic activation

Principles of method if other than guideline:

Method: Clive et al

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

0-1.2 µl Dequest 2000/ml (calculated by previous reviewer as 0-0.78 µl active acid/ml). These concentrations were positive in the previous experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no information

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 2 days


SELECTION AGENT (mutation assays): 5ug/ml trifluorothymidine

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 6x10E06 (1st experiment); 9x10E06 (2nd experiment)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG)

Evaluation criteria:

Positive result: Dose-related increase in number of mutant colonies and mutant frequency at one or more concentrations of greater than or equal to twice control levels with an RTG of greater than 10%.

Statistics:

No statistical analysis of results was carried out.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Remarks:

Concentrations based on data from previous experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: test solution was neutralised to eliminate pH effects. Slight drop in pH seen at highest concentration but approximate fall in 0.08pH units only
- Precipitation: white precipitate reported at concentrations greater than 0.61µl/ml

RANGE-FINDING/SCREENING STUDIES: not conducted as this is a follow-up experiment

COMPARISON WITH HISTORICAL CONTROL DATA: no data

In the first experiment all treated cultures had higher RTGs than the control culture and a dose-related increase in mutant frequency was seen (see Table 1). The experiment was discounted because of the low control growth.

Table 1: Results of Mammalian Mutagenicity assay 1 with tester strain L5178Y in presence of metabolic activation

Concentration µl/ml	Mutant frequency x10E-06	RTG %
0*	23	100
0.49	22	195
0.51	33	134
0.77	48	149
0.96	59	133
1.2	58	121

In the second experiment no increases in mutant frequency and no cytotoxicity were seen. It had been proposed that a selective advantage of mutants in the presence of test substance was the reason for the increase in revertants observed in the previous experiment. No effects on levels of spontaneous mutants were seen when cells were plated immediately after treatment. This finding does not support the proposed reason for the previously observed positive result.

Table 2: Results of Mammalian Mutagenicity assay 2 with tester strain L5178Y in presence of metabolic activation

Concentration µl/ml	Mutant frequency x10E-06	RTG %
0*	101	100
0.61	91	100
0.77	112	74
0.96	98	92
1.2	100	88
1.5	112	103
Positive control	659	46

Conclusions:

In an in vitro gene mutation study in mammalian cells, conducted in a similar manner to OECD TG 476 and in compliance with GLP, a neutralized solution of ATMP acid did not increase the number of revertants in the presence of metabolic activation when tested up to the solubility limit. The test results indicates that the positive findings in the previous experiment with un-neutralised ATMP acid were an artefact of pH. It is concluded that ATMP acid is not mutagenic to mammalian cells under the conditions of this test.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07 November 2017 to 23 November 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Only one test strain of E. coli was used to detect mutations via cross-linking.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

Only one test strain of E. coli was used to detect mutations via cross-linking.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: On the day of the experiment, the test item ATMP-H was dissolved in deionized water. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was assumed to be stable for this period unless specified otherwise by the Sponsor.
- Preliminary purification step: The dose selection was adjusted to the purity of 33%.
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

To evaluate the toxicity of the test item, a pre-experiment was performed. Eight concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test). In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. Since no relevant toxic effects were observed, 5000 µg/plate were chosen as the maximal concentration.
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Remarks:

no treatment

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

ACTIVATION: 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.

DURATION
- Preincubation period: 60 minutes at 37°C
- Exposure duration: 48 hours at 37°C in the dark

SELECTION AGENT (mutation assays): selective agar

NUMBER OF REPLICATIONS: triplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: number of revertants
- Any supplementary information relevant to cytotoxicity: not specified

Rationale for test conditions:

To evaluate the toxicity of the test item, a pre-experiment was performed. Eight concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test). In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. Since no relevant toxic effects were observed 5000 µg/plate were chosen as maximal concentration.

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 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.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

E. coli WP2 uvr A

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not specified
- Effects of osmolality: not specified
- Evaporation from medium: not specified
- Water solubility: not specified
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES: 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 no relevant toxic effects were observed, 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.

HISTORICAL CONTROL DATA
- Positive historical control data: within the range of positive historical control data
- Negative historical control data: within the range of negative historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: number of revertants

Table 1: Summary of experiment 1

Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ± SD)
Without Activation		WP2 uvrA
Deionised water		48 ± 8
Untreated		48 ± 4
ATMP-H	3 µg	43 ± 5
	10 µg	48 ± 8
	33 µg	44 ± 8
	100 µg	48 ± 10
	333 µg	43 ± 9
	1000 µg	47 ± 11
	2500 µg	40 ± 7
	5000 µg	36 ± 5
MMS	2.0 µL	953 ± 87
With Activation
Deionised water		55 ± 3
Untreated		56 ± 11
ATMP-H	3 µg	51 ± 8
	10 µg	57 ± 8
	33 µg	51 ± 13
	100 µg	49 ± 15
	333 µg	51 ± 7
	1000 µg	55 ± 7
	2500 µg	39 ± 9
	5000 µg	47 ± 13
2-AA	10.0 µg	534 ± 112

Table 2: Summary of experiment 2

Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ± SD)
Without Activation			WP2 uvrA
Deionised water			32 ± 5
Untreated			34 ± 4
ATMP-H	33 µg		36 ± 5
	100 µg		35 ± 5
	333 µg		32 ± 6
	1000 µg		31 ± 2
	2500 µg		33 ± 4
	5000 µg		19 ± 2
MMS	2.0 µL		945 ± 40
With Activation
Deionised water			42 ± 2
Untreated			49 ± 12
ATMP-H	33 µg		34 ± 3
	100 µg		43 ± 2
	333 µg		37 ± 4
	1000 µg		36 ± 4
	2500 µg		35 ± 5
	5000 µg		28 ± 3
2-AA	10.0 µg		495 ± 16

MMS: methyl methane sulfonate

2-AA: 2-aminoanthracene

Conclusions:

In a valid bacterial reverse mutation assay, conducted according to OECD TG 471 and in compliance with GLP, ATMP-H has been tested using E. coli WP2 uvr A. No increase in the number of revertants was observed in any test strain, with or without metabolic activation when tested up to the limit concentration. Appropriate positive, negative and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The restrictions were that only four strains of bacteria were used.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

insufficient range of strains to meet requirements of current guideline

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced mouse and rat liver S9

Test concentrations with justification for top dose:

0.01, 0.04, 0.2, 1, 3,10 μl Dequest 2000/plate and 25 μl Dequest 2000 Dequest/plate for spot test. Expressed as ATMP: 0.0065, 0.026, 0.13, 0.65, 1.95, 6.5 μl/plate for plate incorporation; up to 9 μl for spot test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: none given

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 and TA 100 without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium nitrite

Remarks:

TA 1535 without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

TA 98 and TA 100 with activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA 1535 and TA 1537 with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); as impregnation on paper disk


DURATION
- Preincubation period: none
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

NUMBER OF REPLICATIONS: single applications (spot test), triplicate plates (plate incorporation)

DETERMINATION OF CYTOTOXICITY
- Method: other: reduction in microbial lawn

Evaluation criteria:

A positive response is indicated if three of more treatments are significantly greater than the solvent control with a significant dose-response.

Statistics:

Analysis of log10 revertants per plate used Bartlett's test for homogeneity of variance and comparison of treatments with controls used within-levels pooled variance and a one-sided t-test. Where values were found to be significant, a Grubb's test was applied and significance of dose-response was evaluated by a t-test.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1-3ul/plate for plate incorporation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the main study toxicity was seen as follows: +S9; 10 μl, 3 μl (TA98, TA1535); 10 μl, 3 μl, 1 μl (TA100, TA1537) -S9: 10 μl, 3 μl (all strains)

Remarks on result:

other: No mutagenic potential

No increase in revertants in any strain/metabolic activation condition. Full details of results presented in report. Positive and negative values acceptable.

Conclusions:

In an in vitro gene mutation study in bacteria with the test substance ATMP acid (ATMP-H), conducted according to OECD TG 471 and in compliance with GLP, a lack of mutagenic potential occurred. Four Salmonella strains (S. typhimurium TA 98; 100; 1535; 1537) were tested in the absence and presence of rat and mouse liver S9-mix up to the toxicity limit (1-3μl/plate). It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

n vivo micronucleus study: read-across from ATMP-xNa, negative (OECD Test Guideline 474) (Covance, 1998)

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

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:

12/96 final draft

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: Crl: CD-1 (ICR) BR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Labs Raleigh NC
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 30.6 - 33.9 grams (males); 23.6-28.4 grams (females)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: 5 animals per polycarbonate cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26 °C
- Humidity (%): 30-70%
- Air changes (per hr): at least 10 per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Amount of vehicle (if gavage or dermal): 10 ml/kg

Duration of treatment / exposure:

2 days

Frequency of treatment:

24, 48h

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Range finding experiments: 3 male and 3 female per dose; 6 males per dose in main experiment.

Control animals:

yes, concurrent vehicle

Positive control(s):

- cyclophosphamide;
- Justification for choice of positive control(s): none given
- Route of administration: oral gavage
- Doses / concentrations: 80 mg/kg

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on two range-finding studies.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 500 mg/kg bw, 1000 mg/kg bw and positive control harvested at 24 hours; 2000 mg/kg bw and vehicle control harvested at 24 and 48 hours.


DETAILS OF SLIDE PREPARATION: Giesma stain

METHOD OF ANALYSIS: scored for micronuclei and PCE NCE ratio

Evaluation criteria:

Criteria for positive: Statistically significant increase in micronucleated PCEs or at least one dose level and a statistically significant dose-related response.

Statistics:

ANOVA followed by Dunnett's t-test when ANOVA is positive

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

up to limit concentration

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

No clinical toxicity and no cytotoxicity to the bone marrow was observed in any treated animals. No increase in the frequency of micronucleated erythrocytes occurred in any test substance dose.

Table 1: Mean Results of in vivo micronucleus test in mouse bone marrow

-	Solvent Control (water)		Positive Control (Cyclophosphamide)	Low dose 500 mg/kg	Mid dose 1000 mg/kg	High dose 2000 mg/kg
Sampling time (h)	24	48	24	24	48	24	48
Number of cells analysed	2000	2000	2000	2000	2000	2000	2000
Micronucleated cells per animal %	0.07	0.07	2.73	0.04	0.09	0.02	0.06
Ratio PCE/NCE	0.52	0.47	0.50	0.59	0.44	0.41	0.60

Conclusions:

In an in vivo mammalian erythrocyte micronucleus study, conducted according to OECD 174 and in compliance with GLP, ATMP sodium salt did not increase the frequency of micronucleated erythrocytes. It is concluded that ATMP-xNa is negative for the induction of micronuclei under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies:

Reliable information is available on the cytogenicity (in vitro) of ATMP-N-oxide-5K. For endpoints where reliable studies were not available for ATMP-N-oxide-5K, key studies for ATMP-H or ATMP-xNa were read across. Read-across between acids and salts is considered to be scientifically justified as the counterions are not significant in genotoxicity and have been assessed in depth in the public literature. The results of all the studies on ATMP-N-oxide-5K were negative.

 

An in vitro gene mutation study in bacteria with the test substance ATMP-H, conducted according to a protocol similar to OECD Test Guideline 471 and in compliance with GLP, showed a lack of mutagenic potential. Four Salmonella strains (S. typhimurium TA 98; 100; 1535 and 1537) were tested in the absence and presence of rat and mouse liver S9-mix up to the toxicity limit (1-3μl/plate). Appropriate vehicle and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test (Monsanto, 1981).

 

In a valid bacterial reverse mutation assay, conducted according to OECD Test Guideline 471 and in compliance with GLP, ATMP-H was tested using E. coli WP2 uvr A. No increase in the number of revertants was observed in any test strain, with or without metabolic activation when tested up to the limit concentration. Appropriate positive, negative and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test (Envigo, 2018). 

In a low-reliability supporting in vitro bacterial gene mutation study, conducted according to OECD Test Guideline 471 and in compliance with GLP, ATMP-N-oxide-5K was tested in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538. No evidence of induction of revertants was observed. Cytotoxicity was not recorded, and the substance was tested up to a concentration equivalent to approximately 1350 µg/plate. It is concluded that the substance is negative for mutagenicity to bacteria under the conditions of the test (Instituto di Ricerche Biomediche, 1992).

 

In an additional low-reliability supporting in vitro bacterial mutagenicity study, conducted in a similar manner to OECD Test Guideline 471 and in compliance with GLP, ATMP-N-oxide-5K was tested in Salmonella typhimurium strains TA98 and TA100. No test substance related increase in the number of revertants was observed in the presence and absence of metabolic activation and no toxicity to bacteria was observed. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test (Safepharm Laboratories, 1994).

In an in vitro cytogenicity / chromosome aberration study in mammalian cells, conducted according to OECD Test Guideline 473 and in compliance with GLP, ATMP-N-oxide-5K did not cause any increase in the number of cells with aberrations when peripheral human lymphocytes were tested up to cytotoxic concentrations. Exposure lasted for 4 hours in the presence of metabolic activation and for 20 hours in the absence of metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of the test (Safepharm Laboratories, 1994).

In a supporting in vitro chromosome aberration test, conducted according to OECD Test Guideline 473 and in compliance with GLP, ATMP-xNa did not induce chromosome aberrations in Chinese hamster ovary (CHO) cells in the presence and absence of metabolic activation. In absence of metabolic activation, no cytotoxicity was observed with 3-hour treatment with <500 µg/ml. With continuous treatment, cytotoxicity was induced. The top dose of 250 µg/ml had a relative mitotic index of 60%. The higher dose of 500 µg/ml had a relative mitotic index of <10%. It is concluded that ATMP-xNa does not cause chromosomal aberrations under the conditions of the test (Covance, 1998).

 

In an in vitro gene mutation study in mammalian cells, conducted according to a protocol similar to OECD Test Guideline 476 and in compliance with GLP, no increase in the number of revertants was observed in the presence of metabolic activation when ATMP-xNa was tested up to the solubility limit. It is concluded that ATMP-xNa is not mutagenic to mammalian cells under the conditions of this test (SRI, 1988).

 

In vivo study:

In an in vivo mammalian erythrocyte micronucleus study, conducted according to OECD Test Guideline 474 and in compliance with GLP, ATMP-xNa did not increase the frequency of micronucleated erythrocytes. It is concluded that ATMP-xNa is negative for the induction of micronuclei under the conditions of this test (Covance, 1998).

Justification for classification or non-classification

The available data indicate that when tested in vitro, ATMP-N-oxide-5K did not cause chromosomal aberrations when tested in vitro in the presence or absence of metabolic activation. The information on mutagenicity comes from tests on ATMP acid, which did not cause an increase in revertants in bacterial cells. This is supported by the reliability 4 data for ATMP-N-oxide-5K. A neutralised solution of ATMP acid did not cause mutations in mammalian cells. On the basis of this data, it is considered that classification for mutagenicity is not required according to Regulation (EC) No 1272/2008.