Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): non-mutagenic in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102, with and without metabolic activation

Chromosome aberration test (OECD 473): non-clastogenic in V79 cells with and without metabolic activation

RA from Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 80939-62-4)

HPRT test (OECD 476): non-mutagenic in V79 cells with and without metabolic activation

RA from Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 80939-62-4)

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:
11- 30 Jul 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Wistar rats induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw).
Test concentrations with justification for top dose:
Preliminary cytotoxicity test: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Experiment I: 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate with and without metabolic activation
Experiment II: 0.50, 1.58, 5.0, 15.8, 50, 158, 500 and 1580 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: the solvent was compatible with the survival of bacteria and S9 activity
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
yes
Remarks:
aqua dest.
Positive controls:
yes
Positive control substance:
other: -S9: sodium azide (10 µg/plate) for TA100 and TA1535; 4-nitro-o-phenylene-diamine (10 or 40 µg/plate) for TA98 and TA1537; methylmethanesulfonate (1 µL/plate) for TA102; +S9: 2-aminoanthracene (2.5 or 10 µg/plate) for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn or reduction in the number of revertants down to a mutation factor of ca. ≤ 0.5 compared to solvent control.
Evaluation criteria:
EVALUATION OF MUTAGENICITY
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values were used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occur and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with and without metabolic activation.
A biologically relevant increase is described as follows:
- if in the tester strains TA 98, TA 00 and TA 102 the number of reversions is at least twice as high than the reversion rate of the solvent control.
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Statistics:
A statistical evaluation of the results was not regarded as necessary since the biological relevance is the criterion for interpretation of results according to OECD 471.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. I: ≥ 316 µg/plate (-S9); ≥ 1000 µg/plate (+S9); Exp. II: ≥ 50 µg/plate (-S9), ≥ 500 µg/plate (+S9)
Vehicle controls validity:
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:
cytotoxicity
Remarks:
Exp. I: ≥ 100 µg/plate (-S9); ≥ 1000 µg/plate (+S9); Exp. II: ≥ 156 µg/plate (-S9), ≥ 1580 µg/plate (+S9)
Vehicle controls validity:
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:
cytotoxicity
Remarks:
Exp. I: ≥ 316 µg/plate (-S9); ≥ 1000 µg/plate (+S9); Exp. II: ≥ 158 µg/plate (-S9), ≥ 1580 µg/plate (+S9)
Vehicle controls validity:
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:
cytotoxicity
Remarks:
Exp. I: ≥ 316 µg/plate (-S9); ≥ 316 µg/plate (+S9); Exp. II: ≥ 50 µg/plate (-S9), ≥ 500 µg/plate (+S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. I: ≥ 316 µg/plate (-S9); ≥ 1000 µg/plate (+S9); Exp. II: ≥ 67 µg/plate (-S9), ≥ 1580 µg/plate (+S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: a preliminary cytotoxicity experiment in TA 98 and TA 100 was performed to determine appropriate concentrations for treatment in the main experiment. Cytotoxicity in TA 98 was observed at concentrations of ≥ 316 µg/plate in the absence of S9 mix and at concentrations of ≥ 1000 µg/plate in the presence of S9 mix. For TA 100, cytotoxic effects were observed at ≥ 100 µg/plate with and without S9 mix, respectively. In both strains, no bacterial background lawn was observed at ≥ 2500 µg/plate (with S9 mix) and at ≥ 5000 µg/plate (with and without S9 mix). Therefore, the highest concentration used in the main experiment was 2500 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: in experiment I in tester strain TA 102 (without metabolic activation), the mean value of revertant colony numbers of the negative control (aqua dest.) was below the lower limit of the historical control data for the negative control. Since the values fell only slightly below the lower limit of the control data, the data were considered as valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY: in experiment I, in the absence of S9 mix, cytotoxicity was observed at concentrations ≥ 100 µg/plate in tester strain TA 1537 and at ≥ 316 µg/plate in the remaining tester strains. In the presence of S9 mix, cytotoxicity was evident at concentrations ≥ 1000 µg/plate in all tester strains, except for TA 100, in which cytotoxicity was already seen at ≥ 316 µg/plate. In experiment II, cytotoxic effects were already visible at concentrations ≥ 50 µg/plate in TA 100, TA 1535 and TA 1537 in the absence of S9 mix. In TA 98 and TA 102, cytotoxicity was observed without S9 mix at concentrations ≥ 158 and 500 µg/plate, respectively. In the presence of S9 mix, tester strains TA 100 and TA 1535 showed cytotoxicity at ≥ 500 µg/plate, whereas in the remaining tester strains cytotoxicity was seen at ≥ 1580 µg/plate.

Table 3. Test results of experiment I (plate incorporation)

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (mutation factor) (n=3 ± SD)

EXPERIMENT I

S9-Mix

Without

 

Concentration (per plate)

TA 98

TA 100

TA 1535

TA 1537

TA 102

NC

23 ± 2 (0.7)

129 ± 8 (1.2)

11 ± 4 (0.7)

14 ± 2 (1.0)

161 ± 11 (0.9)

SC

30 ± 5 (1.0)

103 ± 6 (1.0)

16 ± 6 (1.0)

14 ± 6 (1.0)

181 ± 8 (1.0)

Test item

 

 

 

 

 

3.16 µg

30 ± 2 (1.0)

129 ± 7 (1.2)

12 ± 2 (0.8)

11 ± 4 (0.8)

193 ± 21 (1.1)

10.0 µg

26 ± 5 (0.9)

124 ± 12 (1.2)

10 ± 3 (0.6)

9 ± 2 (0.6)

224 ± 12 (1.2)

31.6 µg

31 ± 2 (1.0)

119 ± 17 (1.2)

19 ± 8 (1.2)

10 ± 1 (0.7)

184 ± 20 (1.0)

100 µg

22 ± 5 (0.7)

100 ± 37 (1.0)

24 ± 7 (1.5)

11 ± 5B (0.8)

169 ± 72 (0.9)

316 µg

16 ± 6B (0.5)

66 ± 16B (0.6)

36 ± 16B (2.2)

24 ± 6B (1.7)

68 ± 72B (0.4)

1000 µg

7 ± 3B (0.2)

36 ± 54B (0.3)

1 ± 2B (0.1)

6 ± 10B (0.4)

0 ± 0B (0.0)

2500 µg

0 ± 0N (0.0)

0 ± 0N (0.0)

0 ± 0N (0.0)

0 ± 0N (0.0)

0 ± 0N (0.0)

PC

 

 

 

 

 

NaN3 (10 µg)

-

948 ± 123 (9.2)

931 ± 14 (58.2)

-

-

2AA (2.5 µg)

-

-

-

-

-

2-AA (10 µg)

-

-

-

-

-

4NOPD (10 µg)

492 ± 33 (16.2)

-

-

-

-

4NOPD (40 µg)

-

-

-

137 ± 4 (9.8)

-

MMS (1 µL)

-

-

-

-

1074 ± 146 (5.9)

S9-Mix

 

With

Concentration (per plate)

TA 98

TA 100

TA 1535

TA 1537

TA 102

NC

33 ± 7 (1.0)

147 ± 6 (1.0)

13 ± 2 (0.9)

7 ± 2 (1.0)

238 ± 15 (1.0)

SC

34 ± 4 (1.0)

145 ± 17 (1.0)

14 ± 1 (1.0)

7 ± 3 (1.0)

237 ± 18 (1.0)

Test material

 

 

 

 

 

3.16 µg

37 ± 3 (1.1)

122 ± 28 (0.8)

15 ± 6 (1.0)

9 ± 1 (1.2)

229 ± 20 (1.0)

10.0 µg

39 ± 3 (1.1)

134 ± 28 (0.9)

14 ± 4 (1.0)

8 ± 3 (1.1)

215 ± 15 (0.9)

31.6 µg

34 ± 6 (1.0)

132 ± 13 (0.9)

18 ± 2 (1.3)

6 ± 2 (0.9)

244 ± 44 (1.0)

100 µg

40 ± 8 (1.2)

137 ± 19 (0.9)

14 ± 5 (1.0)

5 ± 1 (0.7)

263 ± 36 (1.1)

316 µg

28 ± 3 (0.8)

89 ± 28B (0.6)

19 ± 6 (1.3)

7 ± 5 (1.0)

194 ± 33 (0.8)

1000 µg

24 ± 6B (0.7)

46 ± 18B (0.3)

17 ± 6B (1.2)

2 ± 1B (0.3)

84 ± 4 (0.4)

2500 µg

1 ± 1B (0.0)

3 ± 5B (0.0)

0 ± 0B/N (0.0)

0 ± 0N (0.0)

0 ± 0B (0.0)

PC

 

 

 

 

 

NaN3 (10 µg)

-

-

-

-

-

2AA (2.5 µg)

3089 ± 278 (90.8)

1517 ± 214 (10.5)

125 ± 17 (8.7)

273 ± 32 (39.0)

-

2-AA (10 µg)

-

-

-

-

497 ± 32 (2.1)

4NOPD (10 µg)

-

-

-

-

-

4NOPD (40 µg)

-

-

-

-

-

MMS (1 µL)

-

-

-

-

-

NC = Negative control (aqua dest.), SC = Solvent control (ethanol); PC = Positive control substances; SD = standard deviation, B = background lawn reduced, N = no background lawn;

4NOPD = 4-nitro-o-phenylene-diamine, 2AA = 2-aminoanthracene, NaN3 = sodium azide, MMS = methylmethane-sulfonate;

Mutation factor = mean revertants (test item)/ mean revertants (solvent control)

 

Table 4. Test results of experiment II (plate incorporation)

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (mutation factor) (n=3 ± SD)

EXPERIMENT II

S9-Mix

Without

 

Concentration (per plate)

TA 98

TA 100

TA 1535

TA 1537

TA 102

NC

20 ± 6 (0.7)

127 ± 12 (1.1)

6 ± 2 (1.1)

6 ± 4 (0.8)

184 ± 0 (0.9)

SC

27 ± 8 (1.0)

113 ± 5 (1.0)

5 ± 2 (1.0)

7 ± 1 (1.0)

213 ± 8 (1.0)

Test item

 

 

 

 

 

0.5 µg

22 ± 5 (0.8)

132 ± 16 (1.2)

9 ± 3 (1.7)

6 ± 1 (0.9)

190 ± 12 (0.9)

1.58 µg

21 ± 1 (0.8)

113 ± 5 (1.0)

4 ± 1 (0.8)

7 ± 1 (1.0)

191 ± 31 (0.9)

5.0 µg

20 ± 8 (0.7)

136 ± 35 (1.2)

4 ± 2 (0.8)

7 ± 1 (1.0)

198 ± 48 (0.9)

15.8 µg

24 ± 2 (0.9)

107 ± 9 (1.0)

8 ± 3 (1.5)

6 ± 4 (0.8)

184 ± 6 (0.9)

50 µg

21 ± 7 (0.8)

88 ± 9B (0.8)

3 ± 1B (0.6)

4 ± 1 (0.5)

186 ± 8 (0.9)

158 µg

22 ± 4B (0.8)

76 ± 22B (0.7)

3 ± 3B (0.5)

6 ± 2B (0.8)

158 ± 12 (0.7)

500 µg

19 ± 2B (0.7)

2 ± 4B (0.0)

0 ± 0B (0.0)

0 ± 0B/N (0.0)

67 ± 13B (0.3)

1580 µg

0 ± 0N (0.0)

0 ± 0N (0.0)

0 ± 0N (0.0)

0 ± 0N (0.0)

13 ± 23B/N (0.1)

PC

 

 

 

 

 

NaN3 (10 µg)

-

771 ± 59 (6.8)

744 ± 149 (139.6)

-

-

2AA (2.5 µg)

-

-

-

-

-

2-AA (10 µg)

-

-

-

-

-

4NOPD (10 µg)

284 ± 11 (10.4)

-

-

-

-

4NOPD (40 µg)

-

-

-

72 ± 16 (9.9)

-

MMS (1 µL)

-

-

-

-

1225 ± 36 (5.7)

S9-Mix

 

With

Concentration (per plate)

TA 98

TA 100

TA 1535

TA 1537

TA 102

NC

24 ± 2 (0.8)

147 ± 30 (1.1)

7 ± 3 (1.2)

6 ± 1 (0.9)

263 ± 8 (1.1)

SC

30 ± 3 (1.0)

134 ± 23 (1.0)

6 ± 2 (1.0)

7 ± 5 (1.0)

243 ± 36 (1.0)

Test material

 

 

 

 

 

0.5 µg

45 ± 8 (1.5)

142 ± 15 (1.1)

10 ± 5 (1.8)

11 ± 5 (1.7)

236 ± 16 (1.0)

1.58 µg

27 ± 7 (0.9)

155 ± 5 (1.2)

10 ± 5 (1.8)

8 ± 4 (1.2)

273 ± 39 (1.1)

5.0 µg

25 ± 6 (0.8)

151 ± 20 (1.1)

7 ± 2 (1.2)

6 ± 3 (0.9)

261 ± 10 (1.1)

15.8 µg

31 ± 1 (1.0)

138 ± 9 (1.0)

7 ± 2 (1.2)

8 ± 4 (1.2)

236 ± 29 (1.0)

50 µg

34 ± 5 (1.2)

129 ± 5 (1.0)

4 ± 3 (0.8)

7 ± 5 (1.1)

272 ± 45 (1.1)

158 µg

42 ± 4 (1.4)

115 ± 20 (0.9)

8 ± 2 (1.4)

6 ± 5 (0.9)

269 ± 22 (1.1)

500 µg

30 ± 6 (1.0)

78 ± 8B (0.6)

7 ± 3B (1.2)

7 ± 5 (1.1)

206 ± 10 (0.9)

1580 µg

1 ± 1B (0.0)

0 ± 0B/N (0.0)

0 ± 0B (0.0)

1 ± 1B (0.1)

70 ± 10B (0.3)

PC

 

 

 

 

 

NaN3 (10 µg)

-

-

-

-

-

2AA (2.5 µg)

2393 ± 641 (80.7)

1902 ± 282 (14.2)

91 ± 20 (16.0)

290 ± 139 (43.5)

-

2-AA (10 µg)

-

-

-

-

534 ± 32 (2.2)

4NOPD (10 µg)

-

-

-

-

-

4NOPD (40 µg)

-

-

-

-

-

MMS (1 µL)

-

-

-

-

-

NC = Negative control (aqua dest.), SC = Solvent control (ethanol); PC = Positive control substances; SD = standard deviation, B = background lawn reduced, N = no background lawn;

4NOPD = 4-nitro-o-phenylene-diamine, 2AA = 2-aminoanthracene, NaN3 = sodium azide, MMS = methylmethane-sulfonate;

Mutation factor = mean revertants (test item)/ mean revertants (solvent control)

 

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Remarks:
Harlan Cytotest Cell Research GmbH (Harlan CCR), In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
Type of assay:
other: in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
The V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing each batch is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
Experiment I
4 h without metabolic activation: 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, 100 and 200 µg/mL; top dose: 12.5, 25 and 50 µg/mL
4 h with metabolic activation: 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, 100 and 200 µg/mL; top dose: 25, 50 and 100 µg/mL

Experiment II
18 h without metabolic activation: 0.6, 1.2, 2.3, 4.7, 9.4, 18.8, 37.5, 75 and 150 µg/mL; top dose: 0.6, 1.2 and 2.3 µg/mL
18 h with metabolic activation: 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, 100 and 200 µg/mL; top dose: 6.3, 12.5 and 25 µg/mL
28 h without metabolic activation: 0.6, 1.2, 2.3, 4.7, 9.4, 18.8, 37.5, 75 and 150 µg/mL; top dose: 1.2, 2.3 and 4.7 µg/mL

The cytogenetic evaluation of higher concentrations in the respective intervals (with and without S9 mix) was impossible due to strong test item induced cytotoxic effects (reduced cell numbers and/or low metaphase numbers, partially paralleled by poor metaphase quality).
Vehicle / solvent:
On the day of the experiment (immediately before treatment), the test item was dissolved in culture medium (MEM-Medium). The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures. The osmolarity and pH-value were determined in the solvent control and the maximum concentration without metabolic activation. The test item was dissolved completely in alkalysed medium and afterwards the pH was adjusted to physiological values with 2N HCL in the stock solutions in Experiment I and II.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
- Pretests for dose selection: A pre-test on cell growth inhibition was performed in order to determine the toxicity of the test item, the solubility during exposure and changes in osmolarity and pH value at experimental conditions. In agreement with the OECD guideline no. 473 the test item was applied up to a maximum concentration of 200.0 μg/mL. At the selected dose no influence on solubility or osmolarity was detected.
- Dose selection: The highest concentration used in the cytogenetic experiments was chosen considering the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced cell numbers or mitotic indices below 50% of control, whichever is the lowest concentration, and/or the occurrence of precipitation. In case of non-toxicity the maximum concentration should be 5 mg/mL, 5 μL/mL or 10 mM, whichever is the lowest, if formulation in an appropriate solvent is possible. With respect of the results obtained in the HPRT test (1452400) 5000 μg/mL of Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates was applied as top concentration for treatment of the cultures. Test item concentrations between 0.8 and 200.0 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. No precipitation of the test item was observed after 4 hours treatment time. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I. In Experiment I cytotoxicity was observed after treatment with 100.0 μg/mL in the absence and presence of S9 mix. Therefore, 150.0 μg/mL was chosen as top dose for Experiment II in the absence of S9 mix an d 200.0 μg/mL in the presence of S9 mix.

EXPERIMENTAL PROCEDURE
- Preparation of test cultures: Colcemid was added to the culture medium (0.2 μg/mL) 2.5 hours prior to cell harvest. The cells were treated, 2.5 hours later, on the slides in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively). After preparation the cells were stained with Giemsa and labelled with a computer-generated random code to prevent scorer bias.
- Treatment of test cultures: Exposure period 4 hours: The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 μL S9 mix per mL culture medium were added. After 4 hours the cultures were washed twice with "Saline G" (pH 7.2) containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose * H2O, 192 mg/L Na2HPO4 * 2 H2O and 150 mg/L KH2PO4. The cells were then cultured in complete medium containing 10 % (v/v) FBS for the remaining culture time of 14 respectively 24 hours.
Exposure period 18 and 28 hours: The culture medium of exponentially growing cell cultures was replaced with complete medium containing 10 % (v/v) FBS including the test item without S9 mix. The medium was not changed until preparation of the cells. Concurrent solvent and positive controls were performed in the absence and presence of metabolic activation. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

EVALUATION
- Evaluation of cell numbers: The evaluation of cytotoxicity indicated by reduced cell numbers was made after the preparation of the cultures on spread slides. The cell numbers were determined microscopically by counting 10 defined fields per coded slide. The cell number of the treatment groups is given in percentage compared to the respective solvent control.
- Cell count: Evaluation of the cultures was performed according to the OECD guideline using NIKON microscopes with 100x objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphases per culture were evaluated for cytogenetic damage on coded slides, except for the positive control in Experiment II without S9 mix and 18 hours treatment, where only 50 metaphases were evaluated. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype). Additionally the number of endomitotic cells evaluated at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases).
Evaluation criteria:
A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory historical control data and
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of the laboratory historical control data and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of the laboratory historical control data
Statistics:
Statistical significance was confirmed by means of the Fisher’s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
No visible precipitation of the test item in the culture medium was observed. No relevant influence on osmolarity or pH value was observed.

RANGE-FINDING/SCREENING STUDIES:
A pre-test on cell growth inhibition was performed in order to determine the toxicity of the test item, the solubility during exposure and changes in osmolarity and pH value at experimental conditions. The test item was applied up to a maximum concentration of 200.0 μg/mL. At the selected dose no influence on solubility or osmolarity was detected.

COMPARISON WITH HISTORICAL CONTROL DATA:
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 2.5 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.0 - 1.5 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data. No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.5 - 3.9 %) as compared to the rates of the solvent controls (1.7 - 3.9 %). No biologically relevant increase in the rate of endomitotic metaphases was found after treatment with the test item (0.0 - 0.1 %) as compared to the rates of the solvent controls (0.0 - 0.1 %).
In both experiments, either EMS (600.0 or 1000.0 μg/mL) or CPA (2.0 or 2.4 μg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment I in the absence of S9 mix and in Experiment II in the absence of S9 mix with 18 hours continuous treatment concentrations showing clear cytotoxicity were not scorable for cytogenetic damage.
A very steep dose-cytotoxicity course was observed in these two experimental parts. In Experiment I and II in the presence of S9 mix and in Experiment II in the absence of S9 mix with 28 hours continuous treatment clear cytotoxicity indicated as reduced mitotic index was observed at the highest evaluated concentration.
Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Remarks:
Harlan Cytotest Cell Research GmbH (Harlan CCR), In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
Type of assay:
other: mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Cytokinesis block (if used):
The V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I:
4 h without metabolic activation: 2.5, 5, 10, 20, 40, 60 and 80 µg/mL; top dose: 2.5, 5, 10, 20 and 40 µg/mL
4 h with metabolic activation: 1.3, 2.5, 5, 10, 20, 30 and 40 µg/mL; top dose: 5, 10, 20, 30 and 40 µg/mL

Experiment II:
24 h without metabolic activation: 0.31, 0.63, 1.3, 2.5, 5, 7.5 and 10 µg/mL; top dose: 0.31, 0.63, 1.3, 2.5 and 5 µg/mL
4 h with metabolic activation: 1.3, 2.5, 5, 10, 20, 30 and 40 µg/mL; top dose: 5, 10, 20, 30 and 40 µg/mL

In experiment I and II the cultures at the two lowest concentrations with metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines. The cultures at the two highest concentrations without metabolic activation were not continued due to exceedingly strong cytotoxic effects.
Vehicle / solvent:
On the day of the experiment (immediately before treatment), the test item was suspended (pre-experiment) or dissolved (main experiments) in culture medium. The osmolarity and pH-value was determined in the solvent control and in the highest concentration of the pre-experiment without metabolic activation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
- Expression/fixation time: Three or four days after treatment 1.5x10^6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5x10^5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 7-10 days.
The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: The study was performed in two independent experiments, using identical experimental procedures.

NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).
Evaluation criteria:
Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
The numbers of mutant colonies per 10^6 cells found in the solvent controls falls within the laboratory historical control data.
The positive control substances should produce a significant increase in mutant colony frequencies.
The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.
The data of this study comply with the above mentioned criteria.

Evaluation of Results
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory's historical control data range, a concentration-related increase of the mutations within this range has to be discussed.
The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS GENOTOXICITY:
No relevant and reproducible increase in mutant colony numbers/10E06 cells was observed in the main experiments up to the maximum concentration with and without metabolic activation. The threshold of three times the mutation frequency of the corresponding solvent control was exceeded in the first culture of the first experiment at 40.0 µg/mL without metabolic activation. This increase was judged as biologically irrelevant as the mutation frequency did not exceed the historical range of solvent controls and the parallel culture showed no comparable increase under identical conditions.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.4 up to 17.7 mutants per 10E06 cells; the range of the groups treated with the test item was from 3.2 up to 47.7 mutants per 10E06 cells.
EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

TEST-SPECIFIC CONFOUNDING FACTORS
No precipitation or phase separation occurred up to the maximum concentration with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
The first pre-test, performed in the concentration range from 19.5 to 5000 µg/mL, was not analysable in the absence of metabolic activation following 24 hours treatment due to exceedingly severe cytotoxicity down to the lowest concentration. Therefore, this part of the pre-test was repeated in a concentration range from 0.16 and 20.0 µg/mL without metabolic activation (24 hours treatment).
In the pre-tests relevant toxic effects were observed at 78.1 µg/mL and above in the absence of metabolic activation and at 39.1 µg/mL and above in the presence of metabolic activation after 4 hours treatment. Following 24 hours treatment without metabolic activation strong toxic effects occurred at 10.0 µg/mL and above. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. No precipitation or phase separation occurred up to the maximum concentration after 4 hours and 24 hours treatment with and without metabolic activation. No relevant shift of osmolarity of the medium even in the stock solution of the test item was noted in the first pre-experiment. The pH at the highest concentration was adjusted to neutral with 2 N sodium hydroxide. Based on the results of the pre-experiments, the individual concentrations of the main experiments were selected. The concentration series was generally spaced by a factor of 2. Narrower spacing at high concentrations was used to cover the cytotoxic range more closely.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in the first experiment without metabolic activation at 20.0 µg/mL and above. In the second experiment without metabolic activation cytotoxic effects as described above were noted at 5.0 µg/mL and above. The recommended cytotoxic range of approximately 10-20% relative cloning g efficiency I or relative cell density was covered. In the presence of metabolic activation strong toxic effects in one of the parallel cultures were observed in both main experiments at 40.0 µg/mL. Although one of the parallel cultures showed only moderate or no cytotoxicity, the relative cloning efficiency I of the other parallel culture was reduced down to 0.0% indicating that the cytotoxic concentration range was reached.
Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for Read-across

For Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates (CAS 96690-34-5) a bacterial mutagenicity test is available. In addition, read-across from an appropriate substance Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 80939-32-4) is considered in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. in order to fulfill the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VII-VIII, 8.4. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in IUCLID Section 13.

Gene mutation in bacteria

The bacterial gene mutation assay (Ames test) with Amines, C12-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 96690-34-5) was performed in compliance with OECD guideline 471 and GLP (BSL Bioservice, 2012). In a preliminary cytotoxicity test in Salmonella typhimurium strains TA 98 and TA 100, cytotoxicity was observed in TA 98 at concentrations of ≥ 316 µg/plate in the absence of S9 mix and at concentrations of ≥ 1000 µg/plate in the presence of S9 mix. For TA 100, cytotoxic effects were observed at ≥ 100 µg/plate with and without S9 mix, respectively. Since no cytotoxicity was observed at ≥ 2500 µg/plate (only with S9 mix) and at ≥ 5000 µg/plate (with and without S9 mix), the highest concentration used in the main experiment was 2500 µg/plate. The main experiment was performed in two independent experiments with and without metabolic activation in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 according to the plate incorporation method. In the first experiment bacterial strains were exposed to concentrations ranging from 3.16 to 2500 µg/plate for 48 h. In the absence of S9 mix, cytotoxicity was observed at concentrations ≥ 100 µg/plate in tester strain TA 1537 and at ≥ 316 µg/plate in the remaining tester strains. In the presence of S9 mix, cytotoxicity was evident at concentrations ≥ 1000 µg/plate in all tester strains, except for TA 100, in which cytotoxicity was already seen at ≥ 316 µg/plate. Based on the observed cytotoxic effects, tester strains were treated with lower concentrations (0.5-1500 µg/plate) in the second experiment using the same exposure period.In the repeat experiment, cytotoxic effects were already visible at concentrations ≥ 50 µg/plate in TA 100, TA 1535 and TA 1537 in the absence of S9 mix. In TA 98 and TA 102, cytotoxicity was observed without S9 mix at concentrations ≥ 158 and 500 µg/plate, respectively. In the presence of S9 mix, tester strains TA 100 and TA 1535 showed cytotoxicity at ≥ 500 µg/plate, whereas in the remaining tester strains cytotoxicity was seen at ≥ 1580 µg/plate.

The mean number of revertant colonies per plate was not increased in any tester strain and at any test concentration with or without metabolic activation in both experiments. The mean numbers of spontaneous revertants in the negative (solvent) controls and the mean number of revertants in the positive controls of the strains used were all within the normal historical ranges, and thus fulfilled the validity criteria of the assay. Based on these results, the test substance was considered to be non-mutagenic in the selected strains of S. typhimurium under the conditions of this Ames test.

Cytogenicity in mammalian cells

An in vitro mammalian chromosome aberration test was performed with the source substance Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 80939-32-4) in Chinese hamster lung fibroblasts (V79) according to OECD guideline 473 and GLP, in the presence and absence of metabolic activation (Harlan, 2013). In the first experiment cells were exposed for 4 h to test substance concentrations of 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, 100 and 200 µg/mL with and without metabolic activation (prepared from the livers of rats, treated with phenobarbital and β-naphthoflavone). In the second experiment cells were exposed for 18 hours to 0.6, 1.2, 2.3, 4.7, 9.4, 18.8, 37.5, 75 and 150 µg/mL without metabolic activation and to 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, 100 and 200 µg/mL with metabolic activation. In addition, cells were exposed for 28 h to 0.6, 1.2, 2.3, 4.7, 9.4, 18.8, 37.5, 75 and 150 µg/mL without metabolic activation. Ethylmethanesulphonate and cyclophosphamide were used as positive control substances and showed distinct increases in cells with structural chromosome aberrations with and without metabolic activation, respectively. Precipitation of the test substance was not observed. In Experiment I in the absence of S9 mix and in Experiment II in the absence of S9 mix with 18 hours continuous treatment concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. A very steep dose-cytotoxicity course was observed in these two experimental parts. In Experiment I and II in the presence of S9 mix and in Experiment II in the absence of S9 mix with 28 hours continuous treatment clear cytotoxicity indicated as reduced mitotic index was observed at the highest evaluated concentration (48.5, 43.4 and 47.1 %, respectively).

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.5 - 3.9 %) as compared to the rates of the solvent controls (1.7 - 3.9 %).

No biologically relevant increase in the rate of endomitotic metaphases was found after treatment with the test item (0.0 - 0.1 %) as compared to the rates of the solvent controls (0.0 - 0.1 %). No visible precipitation of the test item in the culture medium was observed. Based on the results of the conducted study, the test substance was not considered to exhibit clastogenic properties in V79 cells with and without metabolic activation.

 

Gene mutation in mammalian cells

An in vitro mammalian cell gene mutation assay according to OECD guideline 476 and under GLP conditions was performed with the source substance Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 80939-32-4) in Chinese hamster lung fibroblasts (V79) (Harlan, 2013). In experiment I, the cells were treated with the test substance for 4 h at 1.3, 2.5, 5, 10, 20, 30 and 40 µg/mL and 2.5, 5, 10, 20, 40, 60 and 80 µg/mL with and without metabolic activation, respectively. In experiment II, the cells were treated for 24 h at 0.31, 0.63, 1.3, 2.5, 5, 7.5 and 10 µg/mL without metabolic activation, and for 4 h at 1.3, 2.5, 5, 10, 20, 30 and 40 µg/mL with metabolic activation. In the pre-tests relevant toxic effects were observed at 78.1 µg/mL and above in the absence of metabolic activation and at 39.1 µg/mL and above in the presence of metabolic activation after 4 hours treatment. Following 24 hours treatment without metabolic activation strong toxic effects occurred at 10.0 µg/mL and above. No relevant and reproducible increase in mutant colony numbers/10E+06 cells was observed in the main experiments up to the maximum concentration with and without metabolic activation. The threshold of three times the mutation frequency of the corresponding solvent control was exceeded in the first culture of the first experiment at 40.0 µg/mL without metabolic activation. This increase was judged as biologically irrelevant as the mutation frequency did not exceed the historical range of solvent controls and the parallel culture showed no comparable increase under identical conditions. In addition, a linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups. Therefore, no biological significant increase in the mutation frequency at the HPRT locus was observed after treatment either in the absence or in the presence of S9-mix and the test substance was not mutagenic in Chinese hamster lung fibroblasts (V79) under the experimental conditions described.

Justification for classification or non-classification

Based on the analogue approach, the available data on gene mutation in bacterial and mammalian cells, and on in vitro cytogenicity do not meet the classification criteria according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.