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Genetic toxicity in vitro

Description of key information

The Ames Test for HEBMP-H is negative with and without metabolic activation.

Two studies in vitro (CHO HPRT and chromosome aberration) are available for sodium salt of test item (HEBMP-xNa) and both studies are negative.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2000
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
METI, MHLW and MAFF
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon (Salmonella strains); tryptophan operon (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
50-5000 µg active acid/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water- Justification for choice of solvent/vehicle: no information in study report
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without metabolic activation Migrated to IUCLID6: 3 µg/plate TA 100, 5 µg/plate TA 1535, 2 µg/plate WP2uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation Migrated to IUCLID6: 80 µg/plate TA 1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation Migrated to IUCLID6: 2 µg/plate TA 98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 1 µg/plate TA 100, 2 µg/plate TA 1535 and TA 1537, 10 µg/plate E. coli WP2uvrA
Remarks:
with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation Migrated to IUCLID6: 5 µg/plate TA 98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)DURATION- Preincubation period: none- Exposure duration: 48 hours- Expression time (cells in growth medium): 48 hoursSELECTION AGENT (mutation assays): histidine/tryptophan deficient agarNUMBER OF REPLICATIONS: Plated in triplicate, experiment repeatedDETERMINATION OF CYTOTOXICITY- Method: other: condition of background lawnMETABOLIC ACTIVATION: S9 mix included glucose-6-phosphate, NADP and NADPH as co-factors. 0.5 ml of 10% S9 solution was added to 0.1 ml test material, 0.1 ml of bacterial culture and 2 ml of top agar - final concentration approx 2% S9.
Evaluation criteria:
A test material is considered positive if it produces a reproducible, dose-related and statistically significant increase in the revertant count of at least one strain of bacteria.
Statistics:
Dunnett's method of linear regression
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No test-specific confounding factors were reported, and no precipitation or toxicity were observed.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1 Cytotoxicity assay, revertants per plate

+/- S9

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA 100

68

67

82

76

75

71

67

80

65

56

60

+

89

89

106

103

96

120

80

129

87

87

86*

-

WP” uvrA

20

24

22

15

20

16

20

22

17

28

23

+

24

21

30

37

30

30

21

13

41

28

39

* Sparse bacterial background lawn

Table 2 Experiment 1, plate incorporation, revertants per plate (mean of 3 plates)

Dose (µg/plate)

TA 100

TA1535

E. coli WP2 uvrA

TA 98

TA1537

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

Negative control**

112

-

33

-

17

-

29

-

12

-

0*

99

102

33

16

29

23

20

33

13

17

50

93

99

33

16

21

24

14

30

11

16

150

96

94

41

17

23

25

21

23

14

14

500

114

97

28

15

22

25

20

34

12

15

1500

109

99

32

14

21

28

18

33

12

15

5000

95

75

31

16

24

26

19

24

12

15

Positive control

442

868

211

314

816

1002

166

27117

1177

466

* solvent control with water

** spontaneous reversion rate

Table 2 Experiment 2, plate incorporation, revertants per plate (mean of 3 plates)

Dose (µg/plate)

TA 100

TA1535

E. coli WP2 uvrA

TA 98

TA1537

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

Negative control**

120

-

27

-

25

-

25

-

12

-

0*

108

123

31

14

27

34

24

39

12

14

50

122

124

29

11

19

28

22

35

13

17

150

127

124

32

21

27

28

22

38

17

15

500

117

120

36

17

31

35

25

39

12

19

1500

120

121

34

19

27

31

17

31

15

14

5000

113

109

33

22

28

34

22

45

16

17

Positive control

363

1232

251

 

615

1146

138

247

982

330

* solvent control with water

** spontaneous reversion rate

Conclusions:
Ames Test for HEBMP-H was negative with and without metabolic activation.
Executive summary:

HEBMP-H has been tested for mutagenicity to bacteria in a reliable assay conducted according to OECD 471 and under GLP. No increase in the number of revertants per plate was found with and without activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and E.col WP2 uvrA in either the initial or the repeat plate incorporation assay. Appropriate positive, negative (spontaneous reversion) and solvent controls were included and gave expected results. It is concluded that the test substance is negative for the induction of mutation in bacteria under the conditions of the test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Data are available for bacterial mutagenicity for HEBMP-H. No further data are available for the registered substance, however, data are available for HEBMP-xNa for in vitro cytogenicity and mutagenicity to mammalian cells.

In dilute aqueous conditions of defined pH, such as the ones in which in vitro studies are conducted, a salt will behave no differently to the parent acid, at identical concentrations of the particular speciated form present, and will be fully dissociated to yield HEBMP-H and salt. Hence genetic toxicity properties for HEBMP-xNa (in contact with water or in aqueous media) can be directly read across to the parent acid and vice versa. In the present context the effect of the alkaline metal counter-ion (sodium) will not be significant and has been extensively discussed in the public literature. In biological systems and the environment, polyvalent metal ions will be present, and the phosphonate ions show very strong affinity to them.

HEBMP-H has been tested for mutagenicity to bacteria in a reliable assay conducted according to OECD 471 and under GLP (SafePharm, 2003). No increase in the number of revertants per plate was found with and without activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and E.col WP2 uvrA in either the initial or the repeat plate incorporation assay. Appropriate positive, negative (spontaneous reversion) and solvent controls were included and gave expected results. It is concluded that the test substance is negative for the induction of mutation in bacteria under the conditions of the test.

Information on potential for induction of chromosome aberrations in vitro is available from a reliable study in which HEBMP-xNa was tested. The study was conducted according to OECD 473 and in compliance with GLP (Bowles A., 2013). The test substance did not induce statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. Appropriate positive and solvent controls were included and gave expected results. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro under the conditions of the test.

HEBMP-xNa has been tested in a reliable study conducted according to OECD 476 and in compliance with GLP (Morris A., 2012). The test substance did not induce any significant or dose-related increases in mutant frequency in either the presence or absence of metabolic activation in either of the two experiments. Appropriate positive and solvent controls were included and gave expected results. The test item was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test. In vivo testing is not required as the results of the in vitro studies were all negative.

Justification for selection of genetic toxicity endpoint
The selected studies are the only available studies for the registered substance or the parent acid. They were conducted according to appropriate OECD guidelines and in compliance with GLP.


Short description of key information:
In vitro:
Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without activation in Salmonella typhimurium strains TA98 and TA100 (OECD 471) (SafePharm, 2003).
Cytogenicity in mammalian cells: negative with and without activation in peripheral human lymphocytes (OECD 473) (Bowles A., 2013).
Mutagenicity in mammalian cells: negative with and without activation at HPRT locus in Chinese hamster ovary cells (OECD 476) (Morris A., 2012).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available in vitro genetic toxicity data, HEBMP-H is not classified for mutagenicity according to the CLP Regulation (EC n.1272/2008).