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Toxicological information

Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please see justification for read across for genetic toxicity attached to the read across object in chapter 7.6.2 record "Read across object-MN.000"
Cross-referenceopen allclose all
Reason / purpose:
read-across source
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
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
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254
Test concentrations with justification for top dose:
4 to 10,000 microgram/plate. 5,000 microgram/plate was chosen as the highest dose in the second experiment.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Remarks:
Without metabolic activation
Positive control substance:
other: NA-azide (TA 100, TA 1535), 9-aminoacridine (TA 1537), 2-Nitroflourene (TA 98, TA 1538), N-Metnyl-N-nitro-N-nitrosoguanidine (WP2uvrA).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Remarks:
With metabolic activation
Positive control substance:
other: Benzo[a]pyrene: TA 98, TA 100, TA 1535, TA 1537, TA 1538. WP2uvrA, 2-Aminoanthracene: TA 98, TA 100, TA 1535, TA 1537, TA 1538, WP2uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: Top agar is prepared for the Salmonella strains by mixing 100 ml agar (0.6 % agar, 0.5 ~ NaCl) with 10 m1 of a 0.5 % histidine-biotin solution. With E. coli histidine is replaced by tryptophan (2.5 ml, 0.5 mM). The following ingredients are added (in order) to 2 ml of molten top agar at 45 degrees C:
- 0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
- 0.1 ml test compound solution
- 0.5 ml S-9 Mix (if required) or buffer
After mixing, the liquid is poured into a petridish with minimal agar (1.5 % agar, Vogel-Bonner E medium With 2 % glucose).


DURATION
- Incubation period: 46 to 72h @ 37 degrees C in the dark. Colonies (his+ revertants) are counted .
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or presence of 5-9 Mix. No dose dependent effect was obtained.

Surviving colonies per plate – experiment one

Without metabolic activation

ug/plate

TA100

TA1535

TA1537

TA1538

TA98

WP2uvrA

0

200

15

10

14

22

62

4

226

19

11

12

26

65

20

217

20

12

13

24

63

100

211

18

8

16

25

67

500

206

15

8

18

25

62

2500

207

18

9

14

31

59

10000

209

21

12

17

26

67

+ve control

627

465

154

500

373

357

Sterility contr

0

0

0

0

0

0

 

With metabolic activation

ug/plate

TA100

TA1535

TA1537

TA1538

TA98

WP2uvrA

0

175

16

9

20

34

67

4

167

11

10

19

29

74

20

192

10

11

21

26

74

100

191

16

7

21

33

75

500

211

11

8

19

39

64

2500

228

17

13

22

37

72

10000

205

17

8

20

43

72

2-aminoanthr

668

177

97

436

426

233

Benzo[a]pyr.

614

20

144

192

648

92

S9 mix

0

0

0

0

0

0

Sterility contr

0

0

0

0

0

0

 

Surviving colonies per plate – experiment two

Without metabolic activation

ug/plate

TA100

TA1535

TA1537

TA1538

TA98

WP2uvrA

0

198

16

9

16

27

53

4

185

16

9

11

26

57

20

189

17

9

10

30

43

100

204

18

10

13

23

60

500

193

20

7

11

25

55

2500

215

13

12

18

28

61

5000

224

20

12

13

31

66

+ve control

622

398

157

533

392

380

Sterility contr

0

0

0

0

0

0

 

With metabolic activation

ug/plate

TA100

TA1535

TA1537

TA1538

TA98

WP2uvrA

0

208

14

8

19

29

70

4

212

12

11

17

31

48

20

210

13

9

13

30

54

100

209

13

10

22

33

51

500

201

12

9

18

30

57

2500

200

12

10

20

40

67

5000

208

13

10

19

31

70

2-aminoanthr

736

159

90

431

415

281

Benzo[a]pyr.

633

26

136

151

677

73

S9 mix

0

0

0

0

0

0

Sterility contr

0

0

0

0

0

0

 

Conclusions:
Interpretation of results (migrated information):
negative

Methyltriglykol is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at tne dose levels investigated.
Executive summary:

In a bacterial reverse mutation (Ames) assay using the S. typhimurium strains TA 1535, TA 1537, TA1538, TA 98 and TA 100 plus the E coli strain WP2 uvrA, no mutagenic activity was observed either with our without metabolic activation up to the maximum tested dose of 10mg/plate.

Reason / purpose:
read-across source
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
6 December 2010 to 27 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): ethylene glycol monopropyl ether
- Physical state: clear colorless liquid
- Analytical purity: 99.820 wt%
- Impurities (identity and amount): 0.012 wt%
- Purity test date: 15 November 2010 (COA)
- Lot/batch No.: TXEG
- Receipt date: 25 November 2010
- Expiration date of the lot/batch: 25 November 2011
- Storage condition of test material: room temperature in the dark
- Other: The integrity of supplied data relating to the identity, purity and stability of the test item was the responsibility of the Sponsor.
Target gene:
Salmonella typhimurium:
TA 1535: his G 46; rfa-; uvrB-
TA 98: his D 3052; rfa-; uvrB-; R-factor
TA 1535: his G 46; rfa-; uvrB-
TA 100: his G 46; rfa-; uvrB-; R-factor

Escherichia coli (WP2uvrA/pKM101): trp-; uvrA-
contains the pKM101 plasmid
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced SD rat liver S9 fraction
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 microg/plate.
Main Experiments: 50, 150, 500, 1500 and 5000 microg/plate.
Vehicle / solvent:
Sterile distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
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
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
A standard plate incorporation method was employed.

Preliminary Toxicity Test:
In order to select appropriate dose levels for use in the main test, a preliminary test were conducted in the presence or absence of S9. Ten dose levels and controls were tested up to and including 5,000 microg/plate. The assay was conducted by mixing 0.1 ml the bacterial culture (TA100 or WP2uvrA-), and 0.1 ml of the vehicle or test chemical mixture, 0.5 mL of S9-mix or phosphate buffer and 2.0 ml of molten agar supplemented with trace histidine or tryptophan 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 numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Experiment 1:
Five concentrations of the test material were assayed with or without S9-mix against each tester strain using the above described direct plate incorporation method. An additional dose level and expanded dose range were selected in order to achieve both four non-toxic doses and the toxic limit of the test material.

Experiment 2:
A second experiment was performed at the same concentrations as in Experiment 1 with fresh bacterial cultures, test material and control solutions. Preincubation was employed. Thus, measured aliquots (0.1 mL) of each bacterial culture were dispensed into sets of test tubes followed by 0.5 mL of S9-mix or phosphate buffer and 0.1 mL of vehicle or test material formulation and incubated for 20 minutes at 37 deg C with shaking at approximately 130 rpm prior to addition of 2 mL of molten trace histidine or tryptophan supplemented top agar. The contents of the tubes were mixed and poured onto the surface of Vogel-Bonner Minimal agar plates. This procedure was repeated for each bacterial strain either with or without S9.

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:
Acceptance Criteria:

The following criteria must be met for acceptance:
- All tester strain cultures must exhibit a characteristic number of spontaneous revertants per plate in vehicle and untreated controls.
- The appropriate characteristics of each tester strain must be confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor.
- All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.
- All positive control chemicals should induce marked increases in the frequency of revertant colonies, both with and without metabolic activation.
- The test should include a minimum of four non-toxic dose levels.
- There should be no evidence of excessive contamination.

Evaluation Criteria:

A dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain either with or without metabolic activation. Biological relevance of the response is to be considered first, as recommended by the UKEMS sub-committee on Guidelines for Mutagenicity Testing (1989). Statistical methods can be used as an aid to evaluation but may not be the only determining factor for a positive response. Fold increase greater than 2x the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response.

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Mean values with standard deviations were reported.
Species / strain:
S. typhimurium TA 1535
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:
S. typhimurium TA 1537
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:
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:
not applicable
Positive controls validity:
valid
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:
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
Conclusions:
Interpretation of results: negative with metabolic activation; negative without metabolic activation.
This substance dose not produce gene mutation in bacteria.
Executive summary:
In a guideline and GLP reverse mutation assay of the substance 2 -propoxyethanol in bacteria strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvr A pKM 101 there was no evidence of mutation with and without metabolic activation up to the maximum dose of 5mg/plate which did not cause significant cell toxicity.
Reason / purpose:
read-across source
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report which meets basic scientific principles. Only four bacterial strains tested.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
, Only four bacterial strains tested.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
his-
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Liver S-9 mix from Aroclor 1254 induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
20 - 5000 ug/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see details on test system and conditions below
Details on test system and experimental conditions:
Test substance No . : 88/745
METHOD OF APPLICATION: Standard plate test (SPT) and plate incorporation test (PIT)


NUMBER OF REPLICATIONS: 3


OTHER: Positive controls
with S-9 mix: 10 µg 2-aminoanthracene (dissolved in DMSO) for the strains TA 100, TA 98, TA 1537 and TA 1535)
without S-9 mix: 5 µg N-methyl-N -nitro-N-nitrosoguanidine (MNNG) (dissolved in DMSO ) for the strains TA 100 and TA 1535
10 µg 4-nitro-o-phenylendiamine (dissolved in DMSO) for the strain TA 98
100 µg 9-aminoacridine chloride monohydrate (dissolved in DMSO) for the strain TA 1537.
Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

An increase in the number of his+ revertants higher than 1.3 relative to the control was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

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

Diethylenglycolmonomethylether was not mutagenic in the Ames test.
Executive summary:

In a bacterial reverse mutation (Ames) assay, 2 -(2 -methoxyethoxy)ethanol gave a negative response for reverse mutation with and without metabolic activation in the Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537.

Reason / purpose:
read-across: supporting information
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See read across justification covering all genotoxicity end points see attached justification below.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

Data source

Materials and methods

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
no data

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 97
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
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
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
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium 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
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
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
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
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
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
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
Positive controls validity:
valid
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
Positive controls validity:
valid

Applicant's summary and conclusion