Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Spetember 2011 - 02 November 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with International guidelines and GLP. All guideline validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Commission Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Guideline 2-1-19-1, Agricultural Production Bureau
Version / remarks:
25 November 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid
EC Number:
813-120-0
Cas Number:
1262967-45-2
Molecular formula:
C21H36O8 C26H46O8 C31H56O8 C36H66O8 C41H76O8
IUPAC Name:
Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid
Test material form:
liquid
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark and under nitrogen.
- Stability under test conditions: Assumed stable.
- Solubility and stability of the test substance in the solvent/vehicle: Diluted in acetone at a top concentration of 50 mg/mL (used to prepare plates at 5000 µg/plate)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): Diluted in acetone.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): N/A

OTHER SPECIFICS: No

Method

Species / strainopen allclose all
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:
S9 mix, prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver
Test concentrations with justification for top dose:
Seven concentrations up to 5000 µg/plate i.e. 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: test item was not soluble in DMSO and acetone was preferred over ethanol.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): Not reported

DURATION
- Preincubation period: N/A
- Exposure duration: 2-3 days
- Expression time (cells in growth medium): 2-3 days
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A

SELECTION AGENT (mutation assays): N/A

SPINDLE INHIBITOR (cytogenetic assays): N/A

STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: N/A

NUMBER OF CELLS EVALUATED: N/A

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): N/A

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: N/A

DETERMINATION OF CYTOTOXICITY
- Method: thinning/ absence of bacteria lawn

OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A

- OTHER: N/A
Rationale for test conditions:
The study was based on the in vitro technique described by Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:

1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values.
2. Any observed response was reproducible under the same treatment conditions.

The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.
Statistics:
The statistical significance of results was analysed using Dunnett’s test to aid in the evaluation of potential positive responses.

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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
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

Any other information on results incl. tables

Table 1       Test 1 (in the presence of S9 mix)

 

Strain

Concentration

(µg/plate)

Mean revertants per plate

SD

Fold increase relative to vehicle

Individual revertant colony counts

TA98

Solvent control

37.3

3.1

 

38, 40, 34

5

44.3

2.1

1.2

45, 46, 42

15

33.0

5.3

0.9

35, 27, 37

50

38.7

0.6

1.0

38, 39, 39

150

42.3

8.3

1.1

45, 49, 33

500

37.3

6.5

1.0

37, 31, 44

1500

42.0

3.6

1.1

43, 38, 45

5000

40.0

3.0

1.1

43, 40, 37

TA100

Solvent control

154.0

20.0

 

137, 149, 176

5

138.0

12.5

0.9

134, 152, 128

15

149.0

8.7

1.0

159, 144, 144

50

141.0

15.1

0.9

143, 125, 155

150

153.7

27.2

1.0

123, 163, 175

500

155.0

9.2

1.0

153, 165, 147

1500

151.3

7.8

1.0

145, 160, 149

5000

138.3

5.9

0.9

145, 136, 134

TA1535

Solvent control

21.7

1.5

 

23, 23, 22

5

18.7

1.2

0.9

20, 18, 18

15

19.3

1.2

0.9

20, 20, 18

50

25.0

1.7

1.2

24, 24, 27

150

23.3

1.2

1.1

22, 24, 24

500

20.3

4.9

0.9

26, 18, 17

1500

21.3

4.2

1.0

18, 20, 26

5000

18.7

2.3

0.9

20, 20, 16

TA1537

Solvent control

12.0

1.0

 

12, 13, 11

5

11.7

1.5

1.0

13, 10, 12

15

11.3

1.5

0.9

11, 13, 10

50

9.7

2.5

0.8

10, 7, 12

150

9.7

2.3

0.8

11, 11, 7

500

13.0

2.0

1.1

13, 15, 11

1500

12.7

2.9

1.1

11, 16, 11

5000

7.7

2.1

0.6

6, 10, 7

WP2 uvrA (pKM101)

Solvent control

139.0

7.9

 

142, 130, 145

5

157.7

21.9

1.1

145, 145, 183

15

156.7

9.1

1.1

147, 165, 158

50

134.7

31.0

1.0

165, 136, 103

150

158.7

10.0

1.1

149, 169, 158

500

157.7

16.3

1.1

165, 139, 169

1500

146.0

15.6

1.1

154, 128, 156

5000

128.0

15.6

0.9

110, 138, 136

TA98

2a

313.0

31.7

8.4

280, 343, 317

TA100

2b

902.0

138.7

5.9

787, 1056, 863

TA1535

2b

509.3

22.2

23.5

535, 497, 496

TA1537

50c

648.3

69.9

54.0

725, 632, 588

WP2 uvrA (pKM101)

2d

2259.0

200.3

16.3

2280, 2448, 2049

a2-nitrofluorene

bsodium azide

c9-aminoacridine

d4-nitroquinoline-1-oxide

 

Table 2       Test 1 (in the absence of S9 mix)

 

Strain

Concentration

(µg/plate)

Mean revertants per plate

SD

Fold increase relative to vehicle

Individual revertant colony counts

TA98

Solvent control

52.3

1.2

 

53, 51, 53

5

58.3

8.7

1.1

68, 51, 56

15

52.3

3.1

1.0

53, 49, 55

50

45.7

6.4

0.9

42, 53, 42

150

47.3

2.3

0.9

46, 50, 46

500

49.7

4.2

0.9

53, 45, 51

1500

41.0

7.5

0.8

40, 49, 34

5000

41.0

6.1

0.8

44, 34, 45

TA100

Solvent control

164.7

10.1

 

170, 153, 171

5

162.0

17.6

1.0

155, 182, 149

15

154.3

5.0

0.9

149, 155, 159

50

156.0

13.0

0.9

171, 148, 149

150

159.3

18.5

1.0

138, 171, 169

500

148.7

8.1

0.9

145, 158, 143

1500

146.3

8.4

0.9

156, 141, 142

5000

148.3

12.7

0.9

163, 141, 141

TA1535

Solvent control

19.3

2.3

 

18, 18, 22

5

22.0

3.5

1.1

20, 26, 20

15

22.7

0.6

1.2

23, 22, 23

50

17.3

1.2

0.9

18, 18, 16

150

14.0

1.7

0.7

13, 13, 16

500

17.7

2.9

0.9

21, 16, 16

1500

17.7

2.1

0.9

16, 20, 17

5000

15.0

2.0

0.8

17, 13, 15

TA1537

Solvent control

38.0

4.6

 

42, 39, 33

5

33.0

6.0

0.9

27, 39, 33

15

32.0

7.2

0.8

38, 24, 34

50

31.7

5.5

0.8

26, 32, 37

150

29.7

3.2

0.8

26, 32, 31

500

22.7

2.3

0.6

24, 24, 20

1500

24.0

1.7

0.6

23, 26, 23

5000

27.0

3.5

0.7

29, 29, 23

WP2 uvrA (pKM101)

Solvent control

159.7

10.5

 

160, 149, 170

5

154.7

19.1

1.0

175, 137, 152

15

146.3

14.0

0.9

160, 132, 147

50

158.0

11.3

1.0

145, 165, 164

150

142.3

19.6

0.9

137, 164, 126

500

139.3

11.9

0.9

143, 149, 126

1500

142.7

15.1

0.9

160, 136, 132

5000

133.0

5.0

0.8

138, 128, 133

TA98

5a

219.7

29.2

4.2

236, 186, 237

TA100

5b

2259.7

405.0

13.7

1858, 2668, 2253

TA1535

5b

302.0

4.6

15.6

297, 306, 303

TA1537

5a

241.7

17.0

6.4

241, 259, 225

WP2 uvrA (pKM101)

10b

1332.7

61.1

8.3

1398, 1323, 1277

abenzo(a)pyrene

b2-aminoanthracene

 

Table 3       Test 2 (in the presence of S9 mix)

 

Strain

Concentration

(µg/plate)

Mean revertants per plate

SD

Fold increase relative to vehicle

Individual revertant colony counts

TA98

Solvent control

36.7

4.6

 

42, 34, 34

50

35.7

2.9

1.0

39, 34, 34

150

36.7

8.5

1.0

45, 37, 28

500

38.3

10.8

1.0

46, 43, 26

1500

41.3

5.7

1.1

46, 43, 35

5000

38.7

5.0

1.1

44, 38, 34

TA100

Solvent control

123.3

16.3

 

120, 109, 141

50

138.7

16.2

1.1

120, 148, 148

150

129.0

6.1

1.0

136, 125, 126

500

117.7

8.1

1.0

109, 119, 125

1500

118.7

6.5

1.0

112, 125, 120

5000

122.7

3.1

1.0

126, 122, 120

TA1535

Solvent control

22.7

0.6

 

22, 23, 23

50

26.0

2.0

1.1

24, 26, 28

150

23.0

1.0

1.0

23, 22, 24

500

22.3

7.0

1.0

15, 23, 29

1500

20.7

1.2

0.9

20, 22, 20

5000

20.7

1.2

0.9

20, 20, 20

TA1537

Solvent control

16.7

5.0

 

22, 16, 12

50

15.3

2.1

0.9

16, 13, 17

150

19.7

1.5

1.2

18, 21, 20

500

14.0

1.7

0.8

13, 16, 13

1500

18.3

1.5

1.1

17, 18, 20

5000

14.7

1.5

0.9

15, 16, 13

WP2 uvrA (pKM101)

Solvent control

145.7

16.4

 

152, 127, 158

50

156.3

21.6

1.1

141, 181, 147

150

152.7

9.1

1.0

161, 154, 143

500

152.0

6.2

1.0

159, 150, 147

1500

140.0

8.7

1.0

145, 145, 130

5000

145.3

13.6

1.0

150, 156, 130

TA98

2a

404.7

29.7

11.0

438, 395, 381

TA100

2b

1015.3

103.6

8.2

1125, 1002, 919

TA1535

2b

701.3

24.0

30.9

729, 689, 686

TA1537

50c

501.7

20.6

30.1

500, 523, 482

WP2 uvrA (pKM101)

2d

1816.7

181.5

12.5

1782, 2013, 1655

a2-nitrofluorene

bsodium azide

c9-aminoacridine

d4-nitroquinoline-1-oxide

 

Table 4       Test 2 (in the absence of S9 mix)

 

Strain

Concentration

(µg/plate)

Mean revertants per plate

SD

Fold increase relative to vehicle

Individual revertant colony counts

TA98

Solvent control

47.0

6.2

 

42, 54, 45

50

51.0

9.5

1.1

62, 46, 45

150

43.7

1.5

0.9

45, 44, 42

500

51.7

11.2

1.1

64, 42, 49

1500

46.3

10.0

1.0

50, 35, 54

5000

44.7

4.6

1.0

50, 42, 42

TA100

Solvent control

133.7

21.0

 

150, 110, 141

50

136.0

4.4

1.0

131, 139, 138

150

141.7

12.3

1.1

152, 145, 128

500

148.3

9.9

1.1

155, 137, 153

1500

140.7

13.6

1.1

128, 139, 155

5000

133.7

6.8

1.0

139, 136, 126

TA1535

Solvent control

25.0

7.2

 

17, 31, 29

50

20.7

5.0

0.8

26, 20, 16

150

21.3

4.6

0.9

16, 24, 24

500

22.0

5.3

0.9

28, 18, 20

1500

19.0

2.6

0.8

17, 22, 18

5000

19.3

2.9

0.8

21, 21, 16

TA1537

Solvent control

30.0

6.9

 

26, 26, 38

50

28.3

4.0

0.9

26, 33, 26

150

28.3

2.3

0.9

27, 27, 31

500

28.3

3.2

0.9

32, 27, 26

1500

31.3

6.8

1.0

39, 29, 26

5000

27.0

1.7

0.9

26, 29, 26

WP2 uvrA (pKM101)

Solvent control

156.0

14.4

 

172, 152, 144

50

165.0

11.3

1.1

159, 158, 178

150

158.3

15.5

1.0

158, 174, 143

500

154.7

4.5

1.0

155, 159, 150

1500

157.7

4.6

1.0

155, 163, 155

5000

152.7

4.2

1.0

156, 154, 148

TA98

5a

380.7

36.9

8.1

409, 394, 339

TA100

5b

2087.7

206.8

15.6

1864, 2272, 2127

TA1535

5b

243.7

68.6

9.7

169, 304, 258

TA1537

5a

236.0

14.5

7.9

222, 251, 235

WP2 uvrA (pKM101)

10b

1179.3

60.6

7.6

1117, 1238, 1183

abenzo(a)pyrene

b2-aminoanthracene

 

 

Applicant's summary and conclusion

Conclusions:
The test item was considered to be non-mutagenic under the conditions of the test.
Executive summary:

OECD 471 (2011) - In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 (S. typhimurium) and WP2uvrA (pKM101) (Escherichia coli) were exposed to Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid in acetone at concentrations of 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in a screening experiment followed by concentrations of 50, 150, 1500 and 5000 µg/plate in a secondary experiment. Both tests were conducted in the presence and absence of S9 mix. The S9 mix content was increased from 10 % v/v to 20 % v/v in the secondary test.

 

The test item was tested up to the standard limit concentration recommended in the regulatory guidelines i.e. 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains.

 

There was no evidence of cytotoxicity or induced mutant colonies (over background levels) in any of the test item treated colonies in either of the two tests.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.