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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
The results from two Ames in vitro bacterial studies, an in vitro L5178Y TK+/- Mouse Lymphoma Assay and an in vitro chromosomal aberration study in cultured human lymphocytes were all negative for genetic toxicity.
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
Study period:
11 August 2009 and 2 September 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection 19th August 2008, Date of signature 4th March 2009
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium:
TA 1535, TA 100: hisG46
TA 1537: hisC3076
TA 98: hisD3052

Escherichia coli CM891 (WP2uvrA/pKM101):
contains ochre mutation; deficient in DNA repair system 9uvrA); contains the pKM101 plasmid
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 micrograms/plate.
Experiments 1 and 2: 50, 150, 500, 1500 and 5000 micrograms/plate.
Vehicle / solvent:
The chosen vehicle solvent was dimethyl sulfoxide. The test material was miscible in this vehicle.
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:
Plates without S9 mix

Migrated to IUCLID6: at 2 micrograms/plate, WP2uvrA-; 3 micrograms/plate, TA100; 5 micrograms/plate, TA1535
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Plates without S9 mix

Migrated to IUCLID6: at 80 micrograms/plate, TA1537
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Plates without S9 mix

Migrated to IUCLID6: at 0.2 micrograms/plate, TA98
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 1 microgram/plate, TA100; 2 microgram/plate, TA1535 and TA1537; and 10 microgram/plate for WP2uvrA-
Remarks:
Plates with S9 mix
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: at 5 microgram/plate, TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION:
A standard plate incorporation method was employed.

Preliminary Toxicity Test
A preliminary toxicity test was conducted over a series of 10 dose levels ranging from 0.15 to 5000 microgram/plate (and control). The test was performed by mixing 0.1 ml of 10-hour bacterial cultures of TA100 or WP2uvrA-, 2 ml of molten, trace histidine or tryptophan supplemented top agar, 0.1 ml of test material formulation and 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). After approximately 48 hours incubation at 37 deg C, the plates were assessed for the numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn. Manual counts had to be performed at 5000 microgram/plate due to excessive test material precipitation and the presence of an opaque film.

Test 1: (Without Pre-Incubation)
Tests were conducted in the presence or absence of S9. Five dose levels and control were tested up to and including 5,000 microg/plate. All testes were performed in triplicate.
- The test was performed by mixing in test tubes 0.1 ml of 10-hour bacterial cultures, 2 ml of molten, trace histidine or tryptophan supplemented top agar, 0.1 ml of test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each tube were mixed overlaid onto sterile plates of Vogel-Bonner Minimal agar (one tube/plate). The plates were incubated at 37 deg C for 48 hours. The frequency of revertant colonies was assessed using a Domino colony counter.

Test 2: (With Pre-Incubation)
A second assay was performed with pre-incubation at 27 deg C for 20 minutes before the addition of the agar overlay. The same 5 concentrations were used in this second test as in the first.

NUMBER OF REPLICATIONS:
3 replicates/strain

DETERMINATION OF CYTOTOXICITY
Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony counts or by the absence of a complete bacterial lawn.
Evaluation criteria:
The criteria for determining a positive response are a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one of more concentrations in at least one bacterial strain with or without metabolic activation. Statistical significance is not the only determining factor for a positive response and the biological relevance of the results should be considered first. A test material is considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
As reported in: Kirkland DJ, (Ed)(1989) Statistical Evaluation of Mutagenicity Test Data UKEMS sub-committee on Guidelines for Mutagenicity Testing. Report Part III - Cambridge University Press.
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Results Test 1 Without S9 Activation

Revertant colony counts (mean and SD 3 replicates)

Addition (micrograms/plate)

TA98

TA100

TA1535

TA1537

WP2uvrA-

0

18 +/- 2.1

87 +/- 11.2

19 +/- 9.0

9 +/- 4.4

20 +/- 2.5

50

18 +/- 4.0

90 +/- 14.7

20 +/- 4.9

10 +/- 0.6

27 +/- 6.7

150

20 +/- 2.1

86 +/- 13.2

16 +/- 1.7

9 +/- 2.0

21 +/- 6.7

500

22 +/- 5.8

88 +/- 3.2

17 +/- 2.6

9 +/- 1.0

21 +/- 4.7

1500

18 +/- 6.2

70 +/- 6.6

14 +/- 5.5

11 +/- 2.9

17 +/- 3.1

5000

18 +/- 6.1

85 +/- 8.6

12 +/- 4.4

5 +/- 0.0

16 +/- 0.0

2 -Nitroquinoline-N-oxide (0.2)

113 +/- 12.6

ENNG (3)

523 +/- 9.5

ENNG (5)

342 +/- 86.2

9-Aminoacridine (80)

1029 +/- 273.5

ENNG (2)

607 +/- 20.8

Table 2: Results Test 1 With S9 Activation

Revertant colony counts (mean and SD 3 replicates)

Addition (micrograms/plate)

TA98

TA100

TA1535

TA1537

WP2uvrA-

0

30 +/- 8.0

73 +/- 4.6

12 +/- 2.0

15 +/- 3.6

20 +/- 6.4

50

22 +/- 1.2

78 +/- 7.6

13 +/- 4.6

9 +/- 5.1

21 +/- 4.7

150

24 +/- 4.0

83 +/- 13.6

9 +/- 1.2

12 +/- 1.7

23 +/- 5.9

500

29 +/- 7.1

69 +/- 9.7

10 +/- 0.6

10 +/- 5.0

22 +/- 1.5

1500

27 +/- 3.8

62 +/- 7.9

10 +/- 3.1

11 +/- 1.2

23 +/- 3.5

5000

18 +/- 2.1

61 +/- 17.6

8 +/- 3.5

11 +/- 7.2

26 +/- 6.6

Benzo(a)pyrene (5)

131 +/- 41.6

2 -Aminoanthracene (1)

1578 +/- 52.0

2 -Amionoanthracene (2)

268 +/- 33.5

2 -Aminoanthracene (2)

353 +/- 58.9

2 -Aminoanthracene (10)

311 +/- 15.0

Table 3: Results Test 2 Without S9 Activation (With Pre-Incubation)

Revertant colony counts (mean and SD 3 replicates)

Addition (micrograms/plate)

TA98

TA100

TA1535

TA1537

WP2uvrA-

0

23 +/- 2.5

110 +/- 3.2

16 +/- 4.0

9 +/- 1.5

19 +/- 1.0

50

22 +/- 3.5

107 +/- 7.8

14 +/- 2.6

11 +/- 1.5

19 +/- 3.1

150

16 +/- 2.0

105 +/- 13.9

14 +/- 3.5

12 +/- 2.3

19 +/- 1.2

500

20 +/- 4.6

108 +/- 10.7

17 +/- 4.6

12 +/- 2.0

18 +/- 3.1

1500

20 +/- 4.6

112 +/- 12.5

18 +/- 2.9

11 +/- 1.7

15 +/- 3.1

5000

19 +/- 5.0

106 +/- 3.8

13 +/- 1.5

9 +/- 0.6

20 +/- 1.0

2 -Nitroquinoline-N-oxide (0.2)

162 +/- 2.5

ENNG (3)

458 +/- 12.7

ENNG (5)

443 +/- 120.2

9-Aminoacridine (80)

694 +/- 281.5

ENNG (2)

757 +/- 84.7

Table 4: Results Test 2 With S9 Activation (With Pre-Incubation)

Revertant colony counts (mean and SD 3 replicates)

Addition (micrograms/plate)

TA98

TA100

TA1535

TA1537

WP2uvrA-

0

22 +/- 1.5

103 +/- 6.7

13 +/- 1.5

10 +/- 3.1

23 +/- 6.7

50

18 +/- 4.0

93 +/- 5.5

14 +/- 3.2

11 +/- 2.9

21 +/- 3.1

150

21 +/- 3.1

104 +/- 5.5

10 +/- 2.0

10 +/- 3.5

24 +/- 2.1

500

24 +/- 3.2

109 +/- 6.8

12 +/- 0.6

9 +/- 1.0

21 +/- 1.2

1500

22 +/- 3.2

100 +/- 9.5

11 +/- 2.1

10 +/- 1.5

19 +/- 2.6

5000

21 +/- 3.5

107 +/- 6.4

9 +/- 0.6

8 +/- 1.0

21 +/- 2.3

Benzo(a)pyrene (5)

168 +/- 27.1

2 -Aminoanthracene (1)

1630 +/- 71.7

2 -Amionoanthracene (2)

216 +/- 29.5

2 -Aminoanthracene (2)

375 +/- 28.4

2 -Aminoanthracene (10)

255 +/- 19.0

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test material was non-mutagenic under the conditions of this test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

A total of four (4) guideline or equivalent studies have been conducted with the candidate chemical. In a key study, it was negative for mutagenicity when tested at plate incorporation levels of up to 5000 micrograms/plate with Salmonella sp. TA 98, TA 100, TA 1535 and TA 1537 and in E. coli strain WP2uvrA/pkM101, either with or without rat liver S9 -mix. It was also negative for mutagenicity in a second supporting study in Salmonella sp. TA 97a, TA 98, TA 100 and TA 1535 when tested at plate incorporation levels up to 5000 micrograms/plate, either with or without rat liver S-9 mix. In a key L5178Y TK+/- Mouse Lymphoma Assay, the subject chemical did not induce any toxicologically significant increases in the frequency of mutations at the LK +/- locus either in the presence or absence of metabolic activation. In a key study with cultured human lymphocytes, the subject chemical induced a clear statistically significant increase in the frequency of cells with aberration in the absence of metabolic activation under conditions involving an initial 4 -hour treatment followed by a 20 -hour treatment-free period. This response was not repeated in a second experiment involving a 24 -hour exposure. The positive response was only seen at the maximum dose concentration of 3910 micrograms/mL, with no evidence of a true dose response. The response was due primarily to break-type aberrations which are unusual for a true clastogenic response. It was postulated that this response was not due to a true clastogenic response but resulted from apoptosis causing DNA fragmentation. The negative response from a Mouse Lymphoma Assay with the same material supports this contention. Therefore, the response seen in cultured human lymphocyte cells was considered of no toxicological significance.


Justification for selection of genetic toxicity endpoint
The AMES study conducted at Harlan Laboratories Ltd (report number 2600-0008) on the substance EHMC has been listed as the key study as opposed to the study with report number M07-5024 conducted at Consumer Product Testing Co., 70 New Dutch Lane, Fairfield, NJ 07004-2514 (USA).

Justification for classification or non-classification

The subject chemical was negative when tested in vitro in the Ames bacterial mutagenicity assay and in the mouse lymphoma L5178Y TK +/- mutagenicity assay. It also failed to produce a toxicologically significant positive response in the human lymphocyte chromosomal aberration test. Based on the results of these in vitro studies, the subject chemical would not be classified for Germ Cell Mutagenicity according to Directive 67/548/EEC, EU CLP (Regulation (EC) No. 1272/2008), and UN GHS.