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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reverse gene mutation assay: Negative (non-mutagenic); OECD 471; May, K. (2011)

Mammalian cell cytogenicity: Negative (non-clastogenic); OECD 473; Pritchard, L. (2011)

Mammalian cell gene mutation: Negative (non-mutagenic); OECD 476; Yamakage, K. (2017)

Link to relevant study records

Referenceopen allclose all

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.
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
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
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.
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.
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

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

 

 

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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 July - 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 in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
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 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Health and Welfare. Evaluation and Licensing Division, Pharmaceutical and Medical Safety Bureau, Notification No. 1604
Version / remarks:
November 1999
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH (1996) Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals.
Version / remarks:
1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
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 500 mg/mL (used to prepare plates at 5000 µg/mL)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: At 5000 µg/mL a fluctuation in osmolality of more than 50 mOsm/kg was observed when compared to the solvent control. No fluctuation in pH of more than 1.0 unit was observed when compared with the solvent control.
Following serial dilution to lower concentrations of 4500 and 4000 μg/mL, a fluctuation in osmolality of more than 50 mOsm/kg was observed at 4500 μg/mL when compared with the solvent control. However, at 4000 μg/mL, no fluctuation in osmolality of more than 50 mOsm/kg was observed. No pH fluctuation of more than 1.0 unit was observed at either concentration when compared with the solvent control.
Concentrations with high ionic strength and osmolality may cause chromosomal aberrations (Galloway et al. 1987). Therefore, concentrations greater than 5000 μg/mL or 10 mM are not used in this test system.
In this case, the highest final concentration used for subsequent testing was 4000 μg/mL.

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
Species / strain / cell type:
lymphocytes: Human blood was collected aseptically from two healthy, non-smoking male donors,
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Human blood was collected aseptically from two healthy, non-smoking male donors.
- Suitability of cells: Not reported
- Cell cycle length, doubling time or proliferation index: Not reported
- Sex, age and number of blood donors if applicable: Not reported
- Whether whole blood or separated lymphocytes were used if applicable: Not reported
- Number of passages if applicable: Not reported
- Methods for maintenance in cell culture if applicable: Blood was pooled and diluted with RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM glutamine. Aliquots (0.4 mL blood : 4.5 mL medium : 0.1 mL phytohaemagglutinin) of the cell suspension were placed in sterile universal containers and incubated at 37 °C in a 5 % CO2 atmosphere for approximately 48 hours. The cultures were gently shaken daily to resuspend the cells.
- Modal number of chromosomes: Not reported
- Normal (negative control) cell cycle time: Not reported

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: See above
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not reported
- Periodically checked for karyotype stability: Not reported
- Periodically 'cleansed' against high spontaneous background: Not reported
Metabolic activation:
with and without
Metabolic activation system:
S9 mix; obtained 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:
40.31, 67.18, 111.97, 186.62, 311.04, 518.4, 864, 1440, 2400 and 4000 μg/mL.

At 5000 μg/mL, a fluctuation in osmolality of more than 50 mOsm/kg was observed when compared to the solvent control. Following serial dilution to lower concentrations of 4500 and 4000 μg/mL, a fluctuation in osmolality of more than 50 mOsm/kg was observed at 4500 μg/mL when compared with the solvent control. However, at 4000 μg/mL, no fluctuation in osmolality of more than 50 mOsm/kg was observed.

Concentrations with high ionic strength and osmolality may cause chromosomal aberrations (Galloway et al. 1987). Therefore, concentrations greater than 5000 μg/mL or 10 mM are not used in this test system. In this case, the highest final concentration used for subsequent testing was 4000 μg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid was found to be miscible in acetone at 500 mg/mL (1M)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Not reported

DURATION
- Preincubation period: N/A
- Exposure duration: 3 h
- Expression time (cells in growth medium): 18 h
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): 10 mins

SELECTION AGENT (mutation assays): N/A

SPINDLE INHIBITOR (cytogenetic assays): Colcemid® (at 0.1 µg/mL)

STAIN (for cytogenetic assays): 10% Giemsa, prepared in buffered water (pH 6.8)

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid® to each culture at a final concentration of 0.1 μg/mL. After 2 hours incubation, each cell suspension was transferred to a centrifuge tube and centrifuged for 5 minutes at 500 g. The cell pellets were treated with a hypotonic solution (0.075M KCl), pre-warmed at 37 °C. After a 10 minute period of incubation at 37 °C, the suspensions were centrifuged at 500 g for 5 minutes and the cell pellets fixed by addition of freshly prepared cold fixative (3 parts methanol : 1 part glacial acetic acid). The fixative was replaced until it was clear. The pellets were resuspended, then centrifuged at 500 g for 5 minutes and finally resuspended in a small volume of fresh fixative. A few drops of the cell suspensions were dropped onto pre-cleaned microscope slides and allowed to air dry. The slides were then stained in 10 % Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and mounted in DPX. The remainder of the cell pellets in fixative were stored at approximately 4 °C until slide analysis was completed.

NUMBER OF CELLS EVALUATED: The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures, or cultures where there were no signs of cytotoxicity.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): One hundred metaphase figures were examined from each culture. Chromosome aberrations were scored according to the classification of the ISCN (1985).

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy: Polyploid cells were noted when seen using a low power objective and examined at a magnification of x1000 using an oil immersion objective.
- Determination of endoreplication: Endoreduplicated cells were noted when seen using a low power objective and examined at a magnification of x1000 using an oil immersion objective.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A

- OTHER: In a second test a 21 hour continuous treatment was used in the absence of S9 mix. In the presence of S9 mix, a 3 hour treatment was used, as in the first test. However, to modify study parameters the final concentration of S9 mix was increased from 2% v/v to 5% v/v.
Evaluation criteria:
An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range.

The test substance is considered to cause a positive response if the following conditions are met:
Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
The increases exceed the solvent control range of this laboratory, taken at the 99% confidence limit.
The increases are reproducible between replicate cultures.
The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
Evidence of a concentration-related response is considered to support the conclusion.

A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.
Statistics:
The number of aberrant metaphase cells in each test substance group was compared with the solvent control value using the one-tailed Fisher exact test (Fisher 1973).
A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups. If this is significant at the 1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.
D20s (the minimum concentration (mg/mL) at which aberrations were found in 20 % of metaphases) were estimated using logistic regression on a log(concentration) scale, allowing the number of control aberrations to be non-zero (Armitage et al., 2002).
Key result
Species / strain:
lymphocytes: human
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No fluctuation in pH of more than 1.0 unit was observed when compared with the solvent control.
- Effects of osmolality: At 5000 μg/mL, a fluctuation in osmolality of more than 50 mOsm/kg was observed when compared to the solvent control. Following serial dilution to lower concentrations of 4500 and 4000 μg/mL, a fluctuation in osmolality of more than 50 mOsm/kg was observed at 4500 μg/mL when compared with the solvent control. However, at 4000 μg/mL, no fluctuation in osmolality of more than 50 mOsm/kg was observed.

Concentrations with high ionic strength and osmolality may cause chromosomal aberrations (Galloway et al. 1987). Therefore, concentrations greater than 5000 μg/mL or 10 mM are not used in this test system. In this case, the highest final concentration used for subsequent testing was 4000 μg/mL.
- Evaporation from medium: No
- Water solubility: Not soluble. Acetone used as a vehicle
- Precipitation: Not observed
- Definition of acceptable cells for analysis: not reported
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: In the absence of S9 mix following 3 hour treatment,the test item caused a reduction in the mitotic index to 68 % of the solvent control value at 4000 μg/mL. The concentrations selected for metaphase analysis were 40.31, 864 and 4000 μg/mL. In the presence of S9 mix (2 % v/v final concentration) following 3 hour treatment, the test item caused a reduction in the mitotic index to 86 % of the solvent control value at 4000 μg/mL. The concentrations selected for metaphase analysis were 186.62, 864 and 4000 μg/mL. In both the absence and the presence of S9 mix, the test item caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the solvent control.


CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: N/A

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Within laboratory historical control range (99 % CI).
- Negative (solvent/vehicle) historical control data: All mean values for the solvent control (acetone), and alltest item treatment concentrations were within laboratory historical control range, when taken at the 99% confidence limit.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Mitotic indices of cultured human lymphocytes treated with Test item were compared with solvent control values.
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]: No

Table 1       First test summary of results

 

Exposure period

(h)

S9 mix

(v/v)

Test concentration

(µg/mL)

Cells with aberrations (excluding gaps)

Cells with aberrations (including gaps)

Relative mitotic index

(%)

Polyploidy mean incidence

(%)

Individual values

(%)

Mean

(%)

Individual values

(%)

Mean

(%)

3

-

Solvent control

2.0

2.0

2.0

4.0

2.0

3.0

100

0.5

40.31

0.0

2.0

1.0

0.0

3.0

1.5

93

0.0

864

0.0

1.0

0.5

0.0

2.0

1.0

85

1.5

4000

1.0

1.0

1.0

1.0

1.0

1.0

68

0.0

0.2a

14.0

13.0

13.5***

15.0

15.0

15.0***

-

0.0

 

3

+

(2 %)

Solvent control

0.0

0.0

0.0

0.0

0.0

0.0

100

0.0

186.62

1.0

0.0

0.5

1.0

2.0

1.5

106

1.0

864

3.0

1.0

2.0

3.0

3.0

3.0

83

0.5

4000

0.0

1.0

0.5

0.0

1.0

0.5

86

0.5

5b

19.0

17.0

18.0***

21.0

18.0

19.5***

-

0.5

amitomycin C

bcyclophosphamide

*** p <0.001 (one-tailed Fisher’s exact test)

 

Table 2       Second test summary of results

 

Exposure period

(h)

S9 mix

(v/v)

Test concentration

(µg/mL)

Cells with aberrations (excluding gaps)

Cells with aberrations (including gaps)

Relative mitotic index

(%)

Polyploidy mean incidence

(%)

Individual values

(%)

Mean

(%)

Individual values

(%)

Mean

(%)

3

-

Solvent control

1.0

3.0

2.0

5.0

4.0

4.5

100

1.5

3000

1.0

3.0

2.0

3.0

4.0

3.5

101

1.0

3500

3.0

2.0

2.5

4.0

4.0

4.0

90

0.5

4000

2.0

2.0

2.0

2.0

3.0

2.5

112

0.0

0.1a

14.0

20.0

17.0***

16.0

22.0

19.0***

-

1.0

 

3

+

(5 %)

Solvent control

0.0

0.0

0.0

1.0

0.0

0.5

100

1.0

2000

0.0

1.0

0.5

0.0

1.0

0.5

104

2.0

3000

0.0

0.0

0.0

0.0

0.0

0.0

110

0.5

4000

0.0

0.0

0.0

0.0

0.0

0.0

100

0.0

5b

10.0

9.0

9.5***

10.0

10.0

10.0***

-

0.0

amitomycin C

bcyclophosphamide

*** p <0.001 (one-tailed Fisher’s exact test)

 

 

 

Conclusions:
It is concluded that the test substance Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.
Executive summary:

OECD 473 (2011) - In a mammalian cell cytogenetics assay (in vitro chromosome aberration, OECD 473), primary lymphocyte cultures were exposed to Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid at concentrations of 40.31, 67.18, 111.97, 186.62, 311.04, 518.4, 864, 1440, 2400 and 4000 µg/mL for 3 h with and without metabolic activation. Additionally, a continuous exposure of 24 h was tested with and without metabolic activation at test item concentrations of 25, 100, 250, 500, 100, 1500, 200, 2500, 3000, 3500 and 4000 µg/mL and 100, 250, 500, 1000, 2000, 3000 and 4000 µg/mL, respectively. The S9 mix concentration in the second test was raised from 2 to 5 % v/v as a modification parameter. 

 

4000 µg/mL was determined as the highest concentration that did not induce unacceptable fluctuations in osmolarity in a screening test. This concentration was assigned as the top concentration in both the first and secondary tests.

 

Both the solvent control and positive controls induced the appropriate responses and were with laboratory historical ranges with 99 % CI.

 

There was no evidence (or a concentration related positive response) of chromosome aberration induction over background values at the highest tested concentration.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline In vitro Mammalian Chromosome Aberration Test (OECD 473) in human lymphocyte cells.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 March - 09 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with International guidleines and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
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
Target gene:
Hprt
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: V79 cells originated from Chinese hamster lung cells obtained from Health Science Reserch Resources Bank.
- Cell cycle length, doubling time or proliferation index: Doubling time = 13 hours
- Sex, age and number of blood donors if applicable: N/A
- Whether whole blood or separated lymphocytes were used if applicable: Not reported
- Number of passages if applicable: 6 for preliminary testing and 10 for main test.
- Methods for maintenance in cell culture if applicable: Cultured in Eagle's Minimum Medium (MEM) supplemented with 10 vol % fetal calf serum in a hhumidified CO2 incubator (5 % CO2, 37 ºC).
- Modal number of chromosomes: 22
- Normal (negative control) cell cycle time: 13 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No
- Periodically checked for karyotype stability: No
- Periodically 'cleansed' against high spontaneous background: No
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Prelim: 0.0078 - 0.25 mg/mL
Main: 0.031, 0.063, 0.13 and 0.25 mg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Soluble at 25 mg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension
- Cell density at seeding (if applicable): 300 x 10E4

DURATION
- Preincubation period: 4 h
- Exposure duration: 4 and 24 h
- Expression time (cells in growth medium): 48 h
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days

STAIN (for cytogenetic assays): Crystal violet (0.1 % w/v)

NUMBER OF REPLICATIONS: 2
Evaluation criteria:
Positive result if;
1. Mean MF's in treatment groups are 3 times higher than control values.
2. The mean MF's in the treatment groups are over ranges of negative (all data) in the historical control data.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Table 1       Mutation frequency on Hprt gene in V79 cells treated with test item for 4 hours without S9 mix (mean values)

 

Concentration

(mg/mL)

Cell confluency

Cytotoxicity

Mutation

Cell confluency

OD %/ dish

% of control

Relative cell numbers

Cytotoxicity

(colonies/well)

Relative cloning efficiency

(%)

Relative survival

(%)

Mutant

colonies/ well

Relative cloning efficiency

(%)

Mean number of resistant colonies

Mutation frequency

(x106)

Solvent control

98.8

100.0

100.0

119.2

100.0

100.0

106.5

100.0

0.6

5.8

0.031

99.2

100.3

102.3

119.5

100.3

102.6

93.5

87.7

0.6

6.0

0.063

99.5

100.7

104.6

111.2

93.3

97.6

94.7

89.3

0.3

3.4

0.13

98.8

100.0

105.7

111.2

93.3

98.5

98.5

92.7

0.7

6.6

0.25

98.5

99.7

107.8

103.0

86.4

93.1

88.7

83.5

0.5

5.0

1a

94.0

95.1

107.9

84.8

71.2

76.8

80.2

75.5

94.6

1179.2

apositive control (ethyl methanesulfonate)

 

Table 2       Mutation frequency on Hprt gene in V79 cells treated with test item for 4 hours with S9 mix (mean values)

 

Concentration

(mg/mL)

Cell confluency

Cytotoxicity

Mutation

Cell confluency

OD %/ dish

% of control

Relative cell numbers

Cytotoxicity

(colonies/well)

Relative cloning efficiency

(%)

Relative survival

(%)

Mutant

colonies/ well

Relative cloning efficiency

(%)

Mean number of resistant colonies

Mutation frequency

(x106)

Solvent control

100.3

100.0

100.0

109.7

100.0

100.0

104.8

100.0

0.2

1.7

0.031

98.8

98.5

106.7

102.8

93.8

100.0

107.3

103.5

1.0

9.4

0.063

99.3

99.0

103.8

105.5

96.2

99.8

114.0

109.4

0.4

3.5

0.13

98.3

98.0

103.0

118.2

107.8

111.1

107.0

102.5

0.5

4.5

0.25

97.7

97.3

101.9

110.0

100.4

102.3

118.0

115.3

0.4

3.3

1a

96.8

96.5

103.8

98.5

89.9

93.3

102.8

100.9

41.0

107.0

apositive control (N-nitrosodimethylamine)

Conclusions:
Under the conditions of this test Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid did not induce gene mutation in the Hprt locus in V79 cells.
Executive summary:

OECD 476 (2017) - In a mammalian cell gene mutation assay (HPRT locus of V79 cells), Chinese hamster lung cells cultured in vitro were exposed to Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid at concentrations of 0.031, 0.063, 0.13 and 0.25 µg/mL in the absence and presence of mammalian metabolic activation (S9-mix) for an exposure period of 4 hours.

 

The test item was tested up to solubility limits which were defined in a screening test. There was no evidence of cytotoxicity or induced mutant colonies over background levels at any of the concentrations tested.

 

The positive controls induced the appropriate response.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

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

Genetic toxicity in vivo

Description of key information

Mammalian somatic cell: Negative (non-clastogenic); OECD 474; Barfield, W. (2011)

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18th July - 24 Aug 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
OJ L 142/240.
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
EPA 712-C-98-226.
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
November 24, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL

- Source and lot/batch No.of test material: JA01YX10
- Expiration date of the lot/batch: April 2012
- Purity test date: 95.8%



RADIOLABELLING INFORMATION (if applicable): N/A




STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL

- Storage condition of test material: Room temperature (ca 20°C), in the dark under nitrogen.
- Stability under test conditions: Not stated

- Solubility and stability of the test substance in the solvent/vehicle: Not stated

- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not stated



TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Treatment of test material prior to testing: Suspensions of the test substance were prepared in Corn oil obtained from Sigma batch number MKBF6012V.
- Preliminary purification step (if any): N/A

- Final dilution of a dissolved solid, stock liquid or gel: 10 mL/kg/day
- Final preparation of a solid: N/A



FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:

TEST ANIMALS
 - Source: Charles River UK Limited, Margate, Kent, England, animals
- Age at study initiation: Males and females ca 35 days old. Males and females ca 40 days
- Weight at study initiation: Males weighed between 30.0 g to 30.4 g. Females weighed between 22.4 g to 25.5 g
- Assigned to test groups randomly: yes

- Fasting period before study: Not stated

- Housing: Each group was kept, with the sexes separated, in cages and maintained in a controlled environment
- Diet (e.g. ad libitum): Nestlets
- Water (e.g. ad libitum): tap water ad libitum.
- Acclimation period: 5 days


 ENVIRONMENTAL CONDITIONS

- Temperature (°C): 19 to 23 °C
- Humidity (%): 40 to 70 %
- Air changes (per hr): Not stated

- Photoperiod (hrs dark / hrs light): artificial light for 12 hours per day

IN-LIFE DATES: From: To: 27 July 2011 to 25 August 2011

Route of administration:
oral: gavage
Vehicle:
Corn oil obtained from Sigma batch number MKBF6012V
Details on exposure:
Preliminary toxicity test:
A dose of 2000 mg/kg/day (200 mg/mL) was administered to 4 animals (2/sex) on two consecutive occasions approximately 24 hours apart followed by 48 hours observation.

The micronucleus test:
Dose levels of 500, 1000 and 2000 mg/kg/day were used once to 5 groups of animals once followed by 24hour observation
Duration of treatment / exposure:
24 hours
Frequency of treatment:
Once
Post exposure period:
Preliminary toxicity test:
48 hours observation.

The micronucleus test:
24hour observation
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Test item
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Test item
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
Test item
Dose / conc.:
12 mg/kg bw/day (nominal)
Remarks:
Mitomycin C
No. of animals per sex per dose:
6 animals per group and 5 for positive control
Control animals:
yes
Positive control(s):
Mitomycin C
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):Femurs were cleaned of all excess tissue and blood and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal were flushed out and pooled in a total volume of 3 mL of filtered foetal calf serum by aspiration.

FIXATION AND SLIDE STAINING:
1 Fixed for a minimum of 10 minutes in methanol and allowed to air-dry
2 Rinsed in purified water
3 Stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes
4 Washed in purified water for 5 minutes
5 Rinsed in cold tap water for 2 minutes
6 Stored at room temperature until required
7 Immediately prior to scoring, slides are wet mounted with coverslips using purified water


DETAILS OF SLIDE PREPARATION:
The resulting cell suspensions were centrifuged at 1000 rpm (150 × g) for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing in the conventional manner on glass microscope slides

METHOD OF ANALYSIS:
Fluorescence microscopy and 2000 polychromatic erythrocytes per animal were examined for the presence of micronuclei.

OTHER:
The proportion of polychromatic erythrocytes was assessed by examination of a total of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes was recorded.
Evaluation criteria:
The following criteria were applied for assessment of assay acceptability:
1. Each treated and control group should include at least 5 analysable animals.
2. Vehicle control values for micronucleated polychromatic erythrocytes must be consistent
with the laboratory historical vehicle control data.
3. Positive controls must show clear unequivocal positive responses.
Statistics:
The data were received in an Excel document and analysed using SAS 9.1.3 (SAS Institute Inc., 2002) (Jonckheere's and Wilcoxon tests) and StatXact 3 (Cytel 1995) (Linear-by-Linear and Permutation tests).
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No mortalities were observed throughout the duration of the micronucleus test. No clinical signs of toxicity were noted for the vehicle control, positive control and Test item group animals over the duration of the test. Some small incidences of bodyweight loss in all dose groups, including controls, were recorded.

Table 3       Summary of results and statistical analysis

Sampling time after 2nddose

Treatment

Concentration (mg/mL)

Proportion of

PCE (%) #

Incidence MPCE (mean) #

24 Hours

 

Vehicle

-

51.4

1.3

Test item

500

47.0

1.7

Test item

1000

50.0

1.8

Test item

2000

53.2

1.8

Mitomycin Ca

12

51.3

53.6**

Vehicle = Corn Oil

PCE = Polychromatic erythrocytes

MPCE = Number of micronucleated polychromatic erythrocytes observed per 2000 polychromatic erythrocytes examined

a = Positive control dosed once only approximately 24 hours prior to termination at a dose volume of 20 mL/kg/day.

# Occasional apparent errors of ± 1% may occur due to rounding of values for presentation in the table

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities):

** p < 0.01 (significant), otherwise p > 0.01 (not significant)

Table 4       Results for individual animals - 24 hour sampling time.

Treatment (mg/kg/day)

AnimalNO

Proportion PCE (%)

MPCE

 

PCE

 

NCE

 

MNCE

 

Vehicle

401

402

403

404

405

406

 

50.6

53.2

54.5

49.2

48.3

52.7

1

2

3

0

1

1

515

535

548

500

495

537

503

471

457

516

529

482

0

0

0

0

0

0

Test item (500)

411

451

452

414

415

416

41.5

45.7

53.6

47.9

45.8

47.7

1

1

0

1

5

2

417

457

540

484

467

490

588

544

467

526

553

537

0

0

0

0

0

0

Test item (1000)

421

422

423

424

425

426

51.1

50.4

49.7

50.1

55.8

43.0

1

2

2

1

2

3

514

514

506

510

564

435

492

505

512

507

446

576

0

0

0

0

0

0

Test item (2000)

431

432

433

434

435

436

59.6

59.4

44.5

49.8

55.1

50.6

3

2

3

1

1

1

600

602

452

519

572

513

407

411

563

524

467

501

0

0

0

0

0

0

Mitomycin Ca(12)

441

442

443

444

445

51.5

50.8

52.5

48.6

51.2

58

60

35

40

75

540

508

528

488

516

470

492

477

516

491

0

0

0

0

0

 

Vehicle = Corn Oil

PCE = Polychromatic erythrocytes

MPCE = Number of micronucleated polychromatic erythrocytes observed per 2000 polychromatic erythrocytes examined

NCE = Total number of normochromatic erythrocytes examined for micronuclei

MNCE: Number of micronucleated normochromatic erythrocytes observed

a = Positive control dosed once only approximately 24 hours prior to termination at a dose volume of 20 mL/kg/day.

Table 5       Body weight - Preliminary Toxicity Test

Treatment (mg/kg/day)

 

Animal Number

 

Bodyweights (g)

 

Day after arrival

 

Day 1

Day 2

Day 3

Individual

 

Mean

 

Individual

 

Mean

 

Individual

 

Mean

 

Individual

 

Mean

 

Test item (2000)

      

M -21

30.0

30.2

34.3

 

34.5

33.6#

33.8

35.3

34.7

M-22

30.4

34.6

 

34.0#

34.1

F -27

22.4

24.0

24.2

 

24.6

23.6#

24.4

23.7

24.7

F- 28

25.5

25.0

25.2

 

25.7

# = Denotes weight loss from previous weighing

M Male

F Female

Table 6       Body weight - Micronucleus Test

Treatment (mg/kg/day)

 

Animal Number

 

Bodyweights (g)

 

Day after arrival

 

Day 1

Day 2

Day 3

Individual

 

Mean

 

Individual

 

Mean

 

Individual

 

Mean

 

Individual

 

Mean

 

Vehicle

401

402

403

404

405

406

29.8

32.0

29.6

29.5

30.7

31.4

30.5

31.8

32.2

34.1

32.7

31.8

33.6

32.7

31.8

34.0

33.4#

33.2

31.9

33.1#

 

32.9

31.9

34.7

32.7#

32.7#

31.6#

33.8

 

 

32.9

Test item (500)

 

411

451

452

414

415

416

30.9

31.1

31.3

29.9

28.0

29.5

30.1

33.0

33.2

34.7

31.3

31.0

30.6

32.3

34.5

33.1#

34.8

31.6

31.5

31.3

 

32.8

35.3

33.5

35.0

32.6

32.5

31.8

33.5

Test item (1000)

 

421

422

423

424

425

426

30.5

28.8

33.2

31.8

31.2

30.1

30.9

32.8

31.6

35.6

34.4

34.4

32.4

33.6

32.8

31.9

35.2#

35.0

34.4#

32.6

 

 

33.7

33.0

32.4

35.8

35.0

34.2#

32.7

 

33.9

Test item (2000)

431

432

433

434

435

436

31.1

30.3

31.2

32.1

30.6

29.6

30.8

33.3

33.2

33.7

35.1

32.2

32.0

33.3

33.0#

33.0#

33.7

35.2

31.8#

32.3

 

 

 

33.2

32.5#

33.5

34.0

35.1#

32.4

32.7

 

 

33.4

Mitomycin Ca(12)

 

441

442

443

444

445

30.4

30.5

31.1

32.0

30.5

30.9

-

-

-

-

-

-

31.2

31.7

33.5

32.7

35.5

32.9

31.8

31.5#

33.9

32.8

35.8

33.2

# = Denotes weight loss from previous weighing

M Male

F Female

Conclusions:
Under the condition of the study, it is concluded that Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid is not clastogenic.
Executive summary:

OECD 474 (2011) - In a study designed to assess the potential induction of micronuclei by Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid in bone marrow cells of CD1 mice, the animal were treated with the test item by oral garage on two occasions approximately 24hr apart.

The preliminary toxicity test demonstrated that a dose of 2000 mg/kg/day, (the standard limit dose for the micronucleus test) administered on two consecutive occasions approximately 24 hours apart was tolerated.  Therefore the main study was conducted at dose levels of 500, 1000 and 2000 mg/kg/day using a dose volume of 10 mL/kg/day.  

The negative control group received the vehicle corn oil (dose volume of 10 mL/kg/day) and the positive control group received Mitomycin C at 12 mg/kg using a dose volume of 20 mL/kg.

Bone marrow smears were obtained from all test groups 24 hours after administration.  One smear from each animal was examined for the presence of micronuclei in 2000 polychromatic erythrocytes. The proportion of polychromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal.

The test item did not induce any statistically significant increases in the frequency of micronucleated polychromatic erythrocytes or decreases in the proportion of polychromatic erythrocytes were observed in all tested concentrations.  Both control control groups produced results within historical control data.

It is concluded that under the condition of the study, the test it is not considered clastogenic.

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

Mode of Action Analysis / Human Relevance Framework

Not applicable as there were no adverse effects observed.

Additional information

Mutagenicity (in vitro)

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.

OECD 473 (2011) - In a mammalian cell cytogenetics assay (in vitro chromosome aberration, OECD 473), primary lymphocyte cultures were exposed to Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid at concentrations of 40.31, 67.18, 111.97, 186.62, 311.04, 518.4, 864, 1440, 2400 and 4000 µg/mL for 3 h with and without metabolic activation. Additionally, a continuous exposure of 24 h was tested with and without metabolic activation at test item concentrations of 25, 100, 250, 500, 100, 1500, 200, 2500, 3000, 3500 and 4000 µg/mL and 100, 250, 500, 1000, 2000, 3000 and 4000 µg/mL, respectively. The S9 mix concentration in the second test was raised from 2 to 5 % v/v as a modification parameter. 

 

4000 µg/mL was determined as the highest concentration that did not induce unacceptable fluctuations in osmolarity in a screening test. This concentration was assigned as the top concentration in both the first and secondary tests.

 

Both the solvent control and positive controls induced the appropriate responses and were with laboratory historical ranges with 99 % CI.

 

There was no evidence (or a concentration related positive response) of chromosome aberration induction over background values at the highest tested concentration.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline In vitro Mammalian Chromosome Aberration Test (OECD 473) in human lymphocyte cells.

OECD 476 (2017) - In a mammalian cell gene mutation assay (HPRT locus of V79 cells), Chinese hamster lung cells cultured in vitro were exposed to Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid at concentrations of 0.031, 0.063, 0.13 and 0.25 µg/mL in the absence and presence of mammalian metabolic activation (S9-mix) for an exposure period of 4 hours.

 

The test item was tested up to solubility limits which were defined in a screening test. There was no evidence of cytotoxicity or induced mutant colonies over background levels at any of the concentrations tested.

 

The positive controls induced the appropriate response.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

Mutagenicity (in vivo)

OECD 474 (2011) - In a study designed to assess the potential induction of micronuclei by Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid in bone marrow cells of CD1 mice, the animal were treated with the test item by oral garage on two occasions approximately 24 hr apart.

The preliminary toxicity test demonstrated that a dose of 2000 mg/kg/day, (the standard limit dose for the micronucleus test) administered on two consecutive occasions approximately 24 hours apart was tolerated.  Therefore the main study was conducted at dose levels of 500, 1000 and 2000 mg/kg/day using a dose volume of 10 mL/kg/day.  

The negative control group received the vehicle corn oil (dose volume of 10 mL/kg/day) and the positive control group received Mitomycin C at 12 mg/kg using a dose volume of 20 mL/kg.

Bone marrow smears were obtained from all test groups 24 hours after administration.  One smear from each animal was examined for the presence of micronuclei in 2000 polychromatic erythrocytes. The proportion of polychromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal.

The test item did not induce any statistically significant increases in the frequency of micronucleated polychromatic erythrocytes or decreases in the proportion of polychromatic erythrocytes were observed in all tested concentrations.  Both control control groups produced results within historical control data.

It is concluded that under the condition of the study, the test it is not considered clastogenic.

Justification for classification or non-classification

The substance does not meet the criteria for classification in accordance with Regulation (EC) No 1272/2008 (CLP).