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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From February 05, 2018 to February 26, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Amides, C18-unsatd., N-hydroxyethyl
Molecular formula:
No molecular formula available for this UVCB.
IUPAC Name:
Amides, C18-unsatd., N-hydroxyethyl
Test material form:
solid: bulk
Specific details on test material used for the study:
- No correction for purity required
- The test material is a UVCB

Method

Target gene:
- Salmonella typhimurium
- Tryptophan locus of Escherichia coli strain
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
The preliminary toxicity assay conducted at dose levels of 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate in water The maximum dose of 5000 µg per plate was achieved using a concentration of 5.00 mg/mL and a 1000 µL plating aliquot. The test article in water formed clear solutions from 0.00667 to 0.333 mg/mL and workable suspensions from 0.667 to 5.00 mg/mL. No toxicity was observed. Precipitate was observed beginning at 667 µg per plate in the absence of S9 activation and beginning at 1000 µg per plate in the presence of S9 activation. Dose responsive increases in revertant counts were observed with tester strain TA98 in the presence of S9 activation.

Based upon the results of the preliminary toxicity assay, the dose levels selected for the mutagenicity assay were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. No toxicity was observed. Precipitate was observed beginning at 1500 µg per plate with all conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.
Vehicle / solvent:
Water was the vehicle of choice based on compatibility with the target cells. The test article formed workable suspensions in water at concentrations of approximately 10 to 25 mg/mL in the solubility test conducted.
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
Details on test system and experimental conditions:
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

Overnight cultures were prepared by inoculating from the appropriate frozen permanent stock into a vessel, containing 30 to 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175 rpm and incubating at 37±2°C for approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.
Evaluation criteria:
The following criteria must be met for the mutagenicity assay to be considered valid:
All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene.
Based on historical control data (95% control limits), all tester strain cultures must exhibit characteristic numbers of spontaneous revertants per plate with the vehicle controls. The mean revertants per plate must be within the following ranges (inclusive).

95% Control Limits (99% Upper Limit)
TA98 TA100 TA1535 TA1537 WP2 uvrA
-S9 6-26 (31) 66-114 (126) 3-23 (28) 1-13 (16) 9-41 (49)
+S9 9-37 (44) 68-128 (143) 3-23 (28) 3-15 (18) 12-44 (52)
With Study Director justification, values including the 99% control limit and above are acceptable.


To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x109 cells/mL.
The mean of each positive control must exhibit at least a 3.0 fold increase in the number of revertants over the mean value of the respective vehicle control and exceed the corresponding acceptable vehicle control range cited above.
A minimum of three non toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn (background code 3, 4 or 5).

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not valid
Untreated negative controls validity:
valid
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
Vehicle controls validity:
not valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.
Remarks on result:
other:
Remarks:
Precipitate was observed beginning at 1500 µg per plate with all conditions

Any other information on results incl. tables

Historical Negative and Positive Control Values

2015

Revertants per plate

Strain

Control

Activation

None

Rat Liver

Mean

SD

Min

Max

95% CL

Mean

SD

Min

Max

95% CL

TA98
(2015)

Neg

16

5

6

43

6-26

23

7

5

53

9-37

Pos

190

191

42

2468

 

329

176

51

1786

 

TA100
(2015)

Neg

90

12

62

233

66-114

98

15

63

157

68-128

Pos

697

172

239

1767

 

671

284

138

2692

 

TA1535
(2015)

Neg

13

5

2

35

3-23

13

5

3

33

3-23

Pos

624

196

50

2509

 

137

110

24

1060

 

TA1537
(2015)

Neg

7

3

1

20

1-13

9

3

2

23

3-15

Pos

392

292

24

2887

 

73

53

19

574

 

WP2uvrA
(2015)

Neg

25

8

7

73

9-41

28

8

10

96

12-44

Pos

336

112

89

1026

 

352

117

78

1409

 

SD=standard deviation; Min=minimum value; Max=maximum value; 95% CL = Mean ±2 SD (but not less than zero); Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control

Applicant's summary and conclusion

Conclusions:
Under the study conditions, results indicate the target substance was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.
Executive summary:

A study was conducted to determine the mutagenic potential pf the test substance by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system according to OECD Guideline 471. Water was used as the vehicle.

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate. No toxicity was observed. Precipitate was observed beginning at 667 µg per plate in the absence of S9 activation and beginning at 1000 µg per plate in the presence of S9 activation. Dose responsive increases in revertant counts were observed with tester strain TA98 in the presence of S9 activation. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 µg per plate.

In the mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. No toxicity was observed. Precipitate was observed beginning at 1500 µg per plate with all conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Under the study conditions, results indicate the target substance was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.