Reaction mass of potassium sodium (2R,3R)-2-hydroxy-3-(phosphonatooxy)butanedioate and potassium sodium (2S,3S)-2-hydroxy-3-(phosphonatooxy)butanedioate

Genetic toxicity: in vitro

Administrative data

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:

15 Sep - 17 Nov 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): Reaction mass of tetra (potassium and sodium) (2R,3R)-2-hydroxy-3-(phosphonatooxy)succinate and potassium sodium (2S,3S)-2-hydroxy-3-(phosphonatooxy)succinate
- Physical state: white to yellowish powder of crystalline appearance
- Lot/batch No.: 010-2065
- Expiration date of the lot/batch: 16 Sep 2019
- Storage condition of test material: at temperatures < 40 °C, above fresh phosphorous pentoxide or in air tight sealed containers, reduce exposure to air to a minimum
- Other: very hygroscopic substance, pH = 9.45 (1% in solution)

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

31.6, 100, 316, 1000, 3160 and 5000 μg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: highly purified water
- Justification for choice of solvent/vehicle: the test substance dissolved completely in water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

highly purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Remarks:

-S9: sodium azide (10 µg/plate, TA100, TA1535); 2-nitrofluorene (10 µg/pl., TA98); 9-aminoacridine (100 µg/pl., TA1537); mitomycin C (10 µg/pl., TA102); +S9: benzo[a]pyrene (10 µg/pl., TA98, TA102, TA1537); 2-aminoanthracene (2 µg/pl., TA100, TA1535)

Details on test system and experimental conditions:

METHOD OF APPLICATION: experiment 1: in agar (plate incorporation); experiment 2: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3 replications in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test item is considered to show a positive response if:
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) at one or more concentrations compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient may be applied.
In general the biological relevance of the results should be considered first by comparison of the results with the historical control data. If the test item shows positive results, these have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates. A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the Ames test.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Species / strain:

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

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed.

RANGE-FINDING/SCREENING STUDIES: a range-finding study (plate incorporation method) was performed with test strain TA 100 at concentrations of 15.8 - 250,000 μg/plate, with and without metabolic activation. No signs of cytotoxicity were observed up to and including the highest dose level, which was the maximum technically feasible concentration.

COMPARISON WITH HISTORICAL CONTROL DATA:
Yes, the negative and positive controls fell within the historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxicity was observed in any strains at any dose level with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Experiment 1

EXPERIMENT 1 (plate incorporation test)
S9-mix	Without
Test item (µg/plate)	TA 98	TA 100	TA 102	TA 1535	TA 1537
NC	33.7 ± 1.2	170.0 ± 13.0	268.0 ± 14.2	21.3 ± 2.5	7.3 ± 0.6
31.6	29.3 ± 4.9	157.7 ± 4.9	272.7 ± 11.6	18.3 ± 3.1	5.3 ± 1.2
100	29.0 ± 1.0	145.3 ± 18.8	285.3 ± 10.4	22.7 ± 5.9	6.0 ± 0.0
316	33.7 ± 5.1	179.7 ± 11.9	276.7 ± 11.5	26.7 ± 2.1	6.0 ± 0.0
1000	31.7 ± 3.8	164.0 ± 19.1	280.7 ± 6.7	24.0 ± 2.6	5.0 ± 1.7
3160	30.7 ± 6.5	157.7 ± 14.0	246.7 ± 4.5	21.7 ± 1.2	5.7 ± 2.1
5000	21.0 ± 1.7	150.3 ± 5.5	252.0 ± 6.1	18.7 ± 7.2	5.7 ± 1.2
2-NF 10 µg/plate	179.7 ± 6.8	-	-	-	-
SA 10 µg/plate	-	1080.7 ± 53.1	-	161.0 ± 7.0	-
MMC 10 µg/plate	-	-	988.3 ± 13.1	-	-
9-AA 100 µg/plate	-	-	-	-	72.3 ± 5.7
S9-mix	With
Test item (µg/plate)	TA 98	TA 100	TA 102	TA 1535	TA 1537
NC	39.7 ± 3.5	154.3 ± 8.3	301.0 ± 25.2	21.7 ± 2.5	7.7 ± 0.6
31.6	35.3 ± 11.2	171.3 ± 22.0	300.3 ± 4.2	19.0 ± 5.6	4.0 ± 1.0
100	29.7 ± 5.0	168.7 ± 18.5	281.0 ± 22.5	22.3 ± 6.0	6.7 ± 0.6
316	31.3 ± 1.5	146.7 ± 23.1	284.3 ± 34.5	19.0 ± 3.5	5.7 ± 1.5
1000	37.0 ± 1.7	171.3 ± 5.9	288.3 ± 29.9	19.7 ± 3.5	4.7 ± 0.6
3160	34.3 ± 3.2	165.3 ± 10.2	260.0 ± 2.6	16.3 ± 5.9	7.0 ± 2.0
5000	29.3 ± 6.5	166.3 ± 6.7	263.7 ± 14.9	22.3 ± 1.5	8.3 ± 1.2
2AA 2 µg/plate	-	1125.0 ± 38.6	-	21.7 ± 2.5	-
B[a]P 10 µg/plate	183.3 ± 11.6	-	1035.3 ± 76.8	-	66.0 ± 4.4
NC = vehicle control, water 2-NF: 2-nitrofluorene SA: sodium azide 9AA: 9-aminocridine 2AA: 2-aminoanthracene B[a]P: benzo[a]pyrene MMC: mitomycin C  (for details see method description)
*, p<0.05

 

Table 2: Experiment 2

EXPERIMENT 2 (preincubation test)
S9-mix	Without
Test item (µg/plate)	TA 98	TA 100	TA 102	TA 1535	TA 1537
NC	28.3 ± 2.1	124.0 ± 7.9	270.7 ± 26.3	19.0 ± 1.0	7.7 ± 1.2
31.6	25.0 ± 2.0	126.3 ± 6.8	288.7 ± 12.4	16.0 ± 1.7	6.3 ± 1.5
100	26.7 ± 7.2	115.7 ± 4.5	282.0 ± 9.8	16.0 ± 1.0	5.3 ± 1.2
316	24.7 ± 0.6	125.0 ± 13.2	287.0 ± 20.1	16.7 ± 3.8	6.7 ± 2.5
1000	25.0 ± 2.0	112.3 ± 2.5	263.0 ± 15.7	15.0 ± 3.0	5.7 ± 2.1
3160	30.3 ± 3.2	125.3 ± 9.1	268.0 ± 3.6	16.0 ± 2.6	6.0 ± 0.0
5000	27.3 ± 2.5	117.7 ± 2.5	253.7 ± 0.6	19.3 ± 2.5	5.3 ± 1.5
2-NF 10 µg/plate	174.7 ± 1.5	-	-	-	-
SA 10 µg/plate	-	833.0 ± 3.6	-	140.7 ± 0.6	-
MMC 10 µg/plate	-	-	1074.7 ± 115.0	-	-
9-AA 100 µg/plate	-	-	-	-	45.0 ± 3.6
S9-mix	With
Test item (µg/plate)	TA 98	TA 100	TA 102	TA 1535	TA 1537
NC	28.3 ± 2.3	133.7 ± 17.0	286.3 ± 23.4	14.3 ± 0.6	8.7 ± 1.2
31.6	32.0 ± 3.5	124.0 ± 0.0	292.0 ± 3.0	16.0 ± 2.6	7.7 ± 4.2
100	29.3 ± 1.5	121.3 ± 9.3	273.3 ± 1.5	20.3 ± 1.5	5.0 ± 1.0
316	33.3 ± 6.0	142.3 ± 1.5	267.3 ± 14.4	17.7 ± 4.5	4.3 ± 1.2
1000	35.0 ± 1.0	152.7 ± 17.0	256.7 ± 2.9	21.0 ± 3.5	6.0 ± 2.6
3160	35.7 ± 4.5	147.3 ± 29.1	265.7 ± 11.6	17.7 ± 1.2	5.3 ± 1.2
5000	32.3 ± 2.5	121.7 ± 11.2	256.0 ± 1.0	17.7 ± 5.7	5.0 ± 2.6
2AA 2 µg/plate	-	827.3 ± 7.6	-	134.7 ± 3.1	-
B[a]P 10 µg/plate	173.3 ± 0.6	-	1004.0 ± 125.1	-	53.3 ± 5.0
NC = vehicle control, water 2-NF: 2-nitrofluorene SA: sodium azide 9AA: 9-aminocridine 2AA: 2-aminoanthracene B[a]P: benzo[a]pyrene MMC: mitomycin C  (for details see method description)
*, p<0.05

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative