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Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay (Ames / OECD 471): negative
In vitro mammalian cell gene mutation assay (MLA / OECD 476): negative

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Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit
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
Additional strain / cell type characteristics:
other: All strains are deep rough and have a reduced capability to repair DNA-damage (except for TA 102). Strains TA 98, TA 100 and TA 102 contain the R-factor plasmid pkM101 enhancing an error prone DNA repair system.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from the livers of rats treated with phenobarbital/beta-naphthoflavone was achieved from Trinova Biochem GmbH and was cofactor supplemented.
Test concentrations with justification for top dose:
plate incorporation and preincubation test: 3.16, 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle used: distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix: Sodium azide: 10 µg/plate (TA 100, TA 1535), 4-NOPD: 10 µg/plate (TA 98)/40 µg/plate (TA1537), MMS: 1 µL/plate (TA 102); +S9 mix: 2-AA: 2.5 µg/plate (TA 98, TA 100, TA 1535, TA 1537) / 10 µg/plate (TA 102)
Details on test system and experimental conditions:
A preliminary plate incorporation experiment with the tester strains TA 98 and TA 100 was performed. The results were were in accordance with the acceptance criteria and are therfore reported as part of Experiment I.

METHOD OF APPLICATION:
Experiment I: in agar (plate incorporation);
Experiment II: preincubation

The initial plate incorporation test was conducted as follows:
Briefly, 0.1 mL test substance, 0.1 mL bacteria culture, 0.5 mL S9 mix (or buffer) and 2.0 mL soft agar were mixed and plated onto petri dishes with solid agar. After incubation at 37 °C for 48 h colonies were counted using an automatic counter. Each experiment was performed in triplicate.
The independent repeat was performed as preincubation test. Briefly, 0.1 mL test substance, 0.1 mL bacteria culture and 0.5 mL S9 mix (or buffer) were preincubated at 37 °C for 60 min. At the end of the preincubation period 2 mL of molten soft agar was added. After mixing, the suspension was plated, incubated for 48 h at 37 °C and colonies were counted using an automatic counter. Each experiment was performed in triplicate.

DETERMINATION OF CYTOTOXICITY
The reduction of background growth of bacteria on the plates as well as a reduction in the mutant count per plate to approximately 0.5 in comparison to control were used as marker for cytotoxicity.
Evaluation criteria:
The following criteria determined the acceptance of an assay:
1. The negative controls had to be within the expected range, as defined by the laboratories’ own historical data.
2. The positive controls had to show sufficient effects, as defined by the laboratories' experience
3. The bacteria strains TA 98, TA 100 and TA 102 demonstrate their typical response to ampicillin.
4. Corresponding background growth on negative control, solvent control and test plates is oberved
A clear and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 98, TA 100, and TA 102 this increase should be about twice that of negative controls. For TA 1535 and TA 1537 the increase should be about three times than that of solvent controls. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment.
Statistics:
Mean values and standard deviation were calculated.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
beginning at 1000 µg/plate in the plate incorporation experiment and 316 µg/plate in the preincubation experiment.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No precipitation was observed in any tester strain at any concentration.

Table 1: Test results of experiment 1 (plate incorporation).

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

TA102

TA98

TA1537

without S9-Mix

0

107 ± 7.5

6 ± 3.1

175 ± 10.6

25 ± 10.4

9 ± 2.5

3.16

128 ± 14.6

10 ± 6.7

196 ± 13.1

27 ± 6.4

14 ± 4.5

10.0

134 ± 4.2

7 ± 0.6

174 ± 13.4

21 ± 3.1

12 ± 3.1

31.6

134 ± 17.3

8 ± 1.7

185 ± 25.1

25 ± 2.5

9 ± 0.6

100

120 ± 15.0

8 ± 0.0

164 ± 15.1

28 ± 7.5

12 ± 2.5

316

95 ± 8.5

10 ± 3.2

182 ± 15.1

27 ± 9.7

8 ± 2.5

1000

74 ± 13.2 B

10 ± 4.2

215 ± 10.8

21± 3.1

9± 4.2

2500

38 ± 3.0 B

0± 0.0 B

179± 10.8

19± 6.5 B

6± 4.6 B

5000

0± 0.6 B

0± 0.0 B

148± 11.0

14± 2.5 B

0± 0.0 B

Positive controls:

Na-azide

MMS

4-NOPD

Concentration

(μg/plate)

10

1 µL/plate

10

40

Mean No. of colonies/plate

(average of 3 ± SD)

934 ± 22.0

758 ± 220.3

1782 ± 345.7

461 ± 23.8

119 ± 7.8

With S9-Mix

0

124 ± 13.8

7 ± 4.0

253 ± 5.3

26 ± 1.0

7 ± 3.5

3.16

120 ± 20.6

7 ± 7.1

272 ± 21.0

35 ± 5.2

11 ± 2.5

10.0

120 ± 8.1

10 ± 3.6

262 ± 2.5

35 ± 2.9

13 ± 4.5

31.6

113 ± 12.5

10 ± 3.2

262 ± 7.2

40 ± 7.2

10 ± 3.5

100

133 ± 16.7

7 ± 1.7

248 ± 19.7

35 ± 7.6

9 ± 1.0

316

121 ± 4.6

9 ± 1.0

277 ± 7.8

32 ± 3.5

12 ± 1.2

1000

117 ± 29.5

11 ± 1.7

274 ± 7.0

36 ± 5.3

13 ± 3.8

2500

100 ± 9.9

7 ± 1.5

233 ± 8.4

31 ± 3.1

6 ± 2.5

5000

61 ± 4.4 B

4 ± 1.0 B

238 ± 28.6

20 ± 5.5

9 ± 1.5

Positive controls:

2-AA

Concentrations

(μg/plate)

2.5

10

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

1581 ±

229.7

102 ±
9.0

516 ±
44.6

1959 ±

504.5

261 ±
14.9

MMS = Methylmethanesulfonate

4-NOPD = 4-Nitro-o-phenylene diamine

2-AA = 2-Aminoanthracene

B = Reduction of background lawn

Table 2: Test results of experiment 2 (pre-incubationtion).

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

TA102

TA98

TA1537

without S9-Mix

0

115± 23.1

10± 4.0

270± 12.2

22± 3.2

7± 1.5

3.16

129± 14.4

4± 2.1

241± 29.2

24± 1.2

10± 2.0

10.0

133± 9.0

9± 2.3

264± 29.4

31± 2.5

10± 6.0

31.6

119± 4.4

5± 1.0

278± 29.7

23± 2.6

12± 6.4

100

114± 15.9

8± 2.0

283± 23.1

26± 1.0

9± 3.2

316

100± 6.1

7± 1.2

262± 9.8

25± 2.1

8± 1.2 B

1000

58± 9.1 B

0± 0.0 B

280± 22.8

25 ± 0.6 B

0 ± 0.0 B

2500

0 ± 0.0 B

0 ± 0.0 B/N

155 ± 37.0

22 ± 6.1 B

0 ± 0.0 N

5000

0 ± 0.0 N

0 ± 0.0 N

125 ± 45.0 B

0 ± 0.0 B

0 ± 0.0 N

Positive controls:

Na-azide

MMS

4-NOPD

Concentration

(μg/plate)

10

1 µL/plate

10

40

Mean No. of colonies/plate

(average of 3 ± SD)

965± 120.2

1038± 227.1

2158± 67.5

389± 30.1

107± 7.0

With S9-Mix

0

108± 12.7

10± 2.5

292± 7.8

38± 4.5

9± 3.2

3.16

137± 30.7

5± 2.1

289± 15.9

39± 5.6

7± 2.1

10.0

139± 4.2

5± 2.5

323± 36.5

37± 2.0

4± 2.9

31.6

121± 14.0

7± 3.1

310± 21.2

32± 4.5

5± 4.9

100

134± 14.7

7± 1.5

297± 6.1

31± 6.0

7± 2.6

316

116± 7.6

8± 1.5

286± 24.0

31± 6.7

4 ± 1.2

1000

101± 15.5

7± 0.6

310± 7.4

30± 12.7

12± 3.5

2500

85± 11.8 B

4± 2.5 B

284± 15.0

22± 4.2 B

7± 3.5 B

5000

70± 4.6 B

0± 0.0 B

273± 10.7

21± 10.4 B

6± 3.6 B

Positive controls:

2-AA

Concentrations

(μg/plate)

2.5

10

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

789±
158.7

60 ±
15.0

628 ±
14.3

2943 ±
399.9

128 ±
29.8

MMS = Methylmethanesulfonate

4-NOPD = 4-Nitro-o-phenylene diamine

2-AA = 2-Aminoanthracene

B = Reduction of background lawn

N = No background lawn

Conclusions:
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
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidin kinase-locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
Co-factor supplemented S9 mix prepared from the livers of rats treated with phenobarbital/beta-naphthoflavone.
Test concentrations with justification for top dose:
Experiment I:
with S9 mix: 0.01, 0.05, 0.24, 0.28, 0.32, 0.36, 0.40, 0.44, 0.48 mM (incubation period: 4 h)
without S9 mix: 0.01, 0.02, 0.05, 0.10, 0.20, 0.24, 0.28, 0.32 mM (incubation period: 4 h)
Experiment II:
with S9 mix: 0.15, 0.35, 0.39, 0.43, 0.45, 0.47, 0.49, 0.53 mM (incubation period: 4 h)
without S9 mix: 0.0005, 0.001, 0.005, 0.01, 0.05, 0.10, 0.15, 0.20, 0.25 mM(incubation period: 24 h)
Vehicle / solvent:
Based on a solubility test RPMI (+5% horse serum) was used as solvent.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethanesulfonate (EMS): 200 + 300 µg/mL; Methylmethanesulfonate (MMS): 10 µg/mL; Benzo(a)pyrene (BaP): 2.5 µg/mL
Details on test system and experimental conditions:
Experiment I with and without S9 mix and Experiment II with S9 mix (incubation period: 4 h)
1 x 10E+7 cells were suspended in medium and exposed to the test item either in the presence or absence of metabolic activation in the mutation experiment for 4 h. Thereafter the test item was removed, the cells were washed and suspended in complete culture medium and grown for 2 days at 37 °C in humidified air. The cell density was determined each day and adjusted to 3 x 10E+5 cells/mL.

Experiment II without S9 mix (incubation period: 24 h)
5 x 10E+6 cells were suspended in medium and exposed to the test item in the absence of metabolic activation. After 24 h the test item was removed, cells were washed and suspended in complete culture medium and grown for 2 days at 37 °C in humidified air. The cell density was determined each day and adjusted to 3 x 10E+5 cells/mL.

After the expression period the cloning efficiency (CE) of the cells was determined by seeding a statistical number of 1.6 cells/well in two 96-well plates. The cells were incubated for at least 6 days at 37 °C in a humidified atmosphere. Additionally, cultures were seeded in selective medium. Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in selective medium with TFT. The plates were scored after an incubation period of approx. 14 days at 37 °C in humidified air.
The mutant frequency was calculated by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT. The mutant frequency is expressed as “mutants per 10E+6 viable cells”.

Suspension growth (SG) of the cell cultures reflects the number of times the cell number increases from the starting cell density.
The relative total growth (RTG) is the product of the relative suspension growth (RSG; calculated by comparing the SG of the dose groups with the SG of the control) and the relative cloning efficiency (RCE) for each culture: RTG = RSG x RCE /100.
The mutant frequencies obtained from the experiments were compared with the Global Evaluation Factor (GEF). The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation (GEF = 126 for the microwell method).
Evaluation criteria:
A mutation assay is considered acceptable if:
- At least 3/4 plates from the TFT resistance-testing portion of the experiment are scorable.
- The cloning efficiency of the negative and/or solvent controls is in the range 65% - 120%.
- The spontaneous mutant frequency in the negative and/or solvent controls is in the range 50-170 mutants per 10E+6 cells
- The cell number of the negative/solvent controls should undergo 8-32 fold increase during a 2 day growth period or 32-180 fold increase during a 3 day growth period (long-term treatment).
- The clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 10E+6 cells with at least 40% of the colonies being small colonies or with an induced small colony mutant frequency of at least 150 mutants per 10E+6 cells.
- The RTG is greater than 10%.

The test item is considered mutagenic if following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10E+6 cells.
- A dose-dependent increase in mutant frequency is detected.
Statistics:
The Poisson distribution was used to calculate the plating efficiencies for cells cloned without and with TFT selection. The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative /solvent controls.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No precipitation was observed at any concentration. The pH-value detected in medium containing the test item was within the physiological range.

None of the experimental results met the criteria for considering the test substance mutagenic, except for the highest dose level in experiment I with metabolic activation. There a significantly increased number of mutants at 0.48 mM and a dose dependency were seen. In addition the Global Evaluation Factor of 126 was exceeded by the induced mutant frequency. This effect was only seen in the highly cytotoxic range (RTG below 10%) and was not seen in the verification experiment (experiment II with metabolic activation). Therefore this effect was considered to be of no biological relevance.

Table 1: Experiment I - 4 h exposure – With and without Metabolic Activation

Test group

Concentration
[mM]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants/1E+06 surviving cells

Induced mutant frequency

 

+S9 mix

Control 1

0

82.9

100

73.7

-

Control 2

0

70.6

92.8

Test Item

0.01

73.7

90.7

89.2

5.9

0.05

79.3

107.8

66.8

-16.5

0.24

71.7

97.2

93.8

10.6

0.28

80.5

93.1

98.1

14.9

0.32

72.7

78.0

88.4

5.1

0.36

80.5

60.1

70.2

-13.0

0.40

72.7

40.4

104.3

21.0

0.44

93.5

27.3

141.3 *

58.0

0.48

82.9

9.4

217.2 *

134.0

Benzo[a]pyrene

2.5

58.8

48.3

829.8 *

746.5

 

-S9 mix

Control 1

0

92.1

100.0

76.3

/

Control 2

0

84.1

83.3

/

Test Item

0.01

98.0

107.4

85.9

6.1

 

0.02

85.4

101.2

73.3

-6.5

 

0.05

82.9

109.0

82.8

2.9

 

0.10

96.5

110.7

87.0

7.2

 

0.20

110.1

79.6

70.8

-9.0

 

0.24

102.9

42.3

93.5

13.7

 

0.28

132.6

31.0

137.4*

57.6

 

0.32

114.0

23.6

126.4*

46.5

EMS

300

77.0

63.5

713.3*

633.5

MMS

10

79.3

64.3

447.5*

367.7

*: p <0.05

EMS   Ethyl methane sulphonate,       MMS Methyl methane sulphonate

Induced mutant frequency = mutant frequency of Sample – mean value of mutant frequency in corresponding controls

 

Table 2: Experiment II - 4 h Exposure - With Metabolic Activation

Test group

Concentration
[mM]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants/1E+06 surviving cells

Induced mutant frequency

Control 1

0

68.7

100.0

115.5

/

Control 2

0

86.6

75.6

/

Test Item

0.15

93.5

121.1

89.6

-5.9

0.35

67.7

77.0

130.3

34.7

0.39

92.1

82.2

94.5

-1.0

0.43

93.5

60.7

118.5

22.9

0.45

116.0

60.8

102.5

7.0

0.47

108.2

36.1

148.6*

53.0

0.49

89.3

18.9

169.4*

73.9

0.53

89.3

11.1

220.6*

125.1

Benzo[a]pyrene

2.5

68.7

68.3

691.7*

596.1

*: p <0.05

Induced mutant frequency = mutant frequency of Sample – mean value of mutant frequency in corresponding controls

 

Table 3: Experiment II - 24 h exposure - Without Metabolic Activation

Test group

Concentration
[mM]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants/1E+06 surviving cells

Induced mutant frequency

Control 1

0

93.5

100.0

75.3

/ 

Control 2

0

95.0

108.4

/ 

Test Item

0.0005

118.1

124.7

77.4

-14.4

0.001

118.1

130.7

82.6

-9.2

0.005

89.3

98.1

74.9

-16.9

0.01

106.4

98.3

72.7

-19.1

0.05

127.4

94.1

69.3

-22.5

0.10

125.0

84.9

69.5

-22.4

0.15

89.3

43.7

92.0

0.2

0.20

92.1

26.0

113.6

21.8

0.25

80.5

7.9

188.6*

96.8

EMS

200

38.9

12.2

3002.5*

2910.7

MMS

10

34.2

10.1

2321.3*

2229.5

*: p <0.05

EMS   Ethyl methane sulphonate;      MMS  Methyl methane sulphonate

Induced mutant frequency = mutant frequency of Sample – mean value of mutant frequency in corresponding controls

Conclusions:
negative
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Reason / purpose:
read-across source
Target gene:
his-operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: All strains are deep rough and have a reduced capability to repair DNA-damage (except for TA 102). Strains TA 98, TA 100 and TA 102 contain the R-factor plasmid pkM101 enhancing an error prone DNA repair system.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from the livers of rats treated with phenobarbital/beta-naphthoflavone was achieved from Trinova Biochem GmbH and was cofactor supplemented.
Test concentrations with justification for top dose:
plate incorporation and preincubation test: 3.16, 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle used: distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix: Sodium azide: 10 µg/plate (TA 100, TA 1535), 4-NOPD: 10 µg/plate (TA 98)/40 µg/plate (TA1537), MMS: 1 µL/plate (TA 102); +S9 mix: 2-AA: 2.5 µg/plate (TA 98, TA 100, TA 1535, TA 1537) / 10 µg/plate (TA 102)
Details on test system and experimental conditions:
A preliminary plate incorporation experiment with the tester strains TA 98 and TA 100 was performed. The results were were in accordance with the acceptance criteria and are therfore reported as part of Experiment I.

METHOD OF APPLICATION:
Experiment I: in agar (plate incorporation);
Experiment II: preincubation

The initial plate incorporation test was conducted as follows:
Briefly, 0.1 mL test substance, 0.1 mL bacteria culture, 0.5 mL S9 mix (or buffer) and 2.0 mL soft agar were mixed and plated onto petri dishes with solid agar. After incubation at 37 °C for 48 h colonies were counted using an automatic counter. Each experiment was performed in triplicate.
The independent repeat was performed as preincubation test. Briefly, 0.1 mL test substance, 0.1 mL bacteria culture and 0.5 mL S9 mix (or buffer) were preincubated at 37 °C for 60 min. At the end of the preincubation period 2 mL of molten soft agar was added. After mixing, the suspension was plated, incubated for 48 h at 37 °C and colonies were counted using an automatic counter. Each experiment was performed in triplicate.

DETERMINATION OF CYTOTOXICITY
The reduction of background growth of bacteria on the plates as well as a reduction in the mutant count per plate to approximately 0.5 in comparison to control were used as marker for cytotoxicity.
Evaluation criteria:
The following criteria determined the acceptance of an assay:
1. The negative controls had to be within the expected range, as defined by the laboratories’ own historical data.
2. The positive controls had to show sufficient effects, as defined by the laboratories' experience
3. The bacteria strains TA 98, TA 100 and TA 102 demonstrate their typical response to ampicillin.
4. Corresponding background growth on negative control, solvent control and test plates is oberved
A clear and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 98, TA 100, and TA 102 this increase should be about twice that of negative controls. For TA 1535 and TA 1537 the increase should be about three times than that of solvent controls. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment.
Statistics:
Mean values and standard deviation were calculated.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
beginning at 1000 µg/plate in the plate incorporation experiment and 316 µg/plate in the preincubation experiment.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No precipitation was observed in any tester strain at any concentration.

Table 1: Test results of experiment 1 (plate incorporation).

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

TA102

TA98

TA1537

without S9-Mix

0

107 ± 7.5

6 ± 3.1

175 ± 10.6

25 ± 10.4

9 ± 2.5

3.16

128 ± 14.6

10 ± 6.7

196 ± 13.1

27 ± 6.4

14 ± 4.5

10.0

134 ± 4.2

7 ± 0.6

174 ± 13.4

21 ± 3.1

12 ± 3.1

31.6

134 ± 17.3

8 ± 1.7

185 ± 25.1

25 ± 2.5

9 ± 0.6

100

120 ± 15.0

8 ± 0.0

164 ± 15.1

28 ± 7.5

12 ± 2.5

316

95 ± 8.5

10 ± 3.2

182 ± 15.1

27 ± 9.7

8 ± 2.5

1000

74 ± 13.2 B

10 ± 4.2

215 ± 10.8

21± 3.1

9± 4.2

2500

38 ± 3.0 B

0± 0.0 B

179± 10.8

19± 6.5 B

6± 4.6 B

5000

0± 0.6 B

0± 0.0 B

148± 11.0

14± 2.5 B

0± 0.0 B

Positive controls:

Na-azide

MMS

4-NOPD

Concentration

(μg/plate)

10

1 µL/plate

10

40

Mean No. of colonies/plate

(average of 3 ± SD)

934 ± 22.0

758 ± 220.3

1782 ± 345.7

461 ± 23.8

119 ± 7.8

With S9-Mix

0

124 ± 13.8

7 ± 4.0

253 ± 5.3

26 ± 1.0

7 ± 3.5

3.16

120 ± 20.6

7 ± 7.1

272 ± 21.0

35 ± 5.2

11 ± 2.5

10.0

120 ± 8.1

10 ± 3.6

262 ± 2.5

35 ± 2.9

13 ± 4.5

31.6

113 ± 12.5

10 ± 3.2

262 ± 7.2

40 ± 7.2

10 ± 3.5

100

133 ± 16.7

7 ± 1.7

248 ± 19.7

35 ± 7.6

9 ± 1.0

316

121 ± 4.6

9 ± 1.0

277 ± 7.8

32 ± 3.5

12 ± 1.2

1000

117 ± 29.5

11 ± 1.7

274 ± 7.0

36 ± 5.3

13 ± 3.8

2500

100 ± 9.9

7 ± 1.5

233 ± 8.4

31 ± 3.1

6 ± 2.5

5000

61 ± 4.4 B

4 ± 1.0 B

238 ± 28.6

20 ± 5.5

9 ± 1.5

Positive controls:

2-AA

Concentrations

(μg/plate)

2.5

10

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

1581 ±

229.7

102 ±
9.0

516 ±
44.6

1959 ±

504.5

261 ±
14.9

MMS = Methylmethanesulfonate

4-NOPD = 4-Nitro-o-phenylene diamine

2-AA = 2-Aminoanthracene

B = Reduction of background lawn

Table 2: Test results of experiment 2 (pre-incubationtion).

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

TA102

TA98

TA1537

without S9-Mix

0

115± 23.1

10± 4.0

270± 12.2

22± 3.2

7± 1.5

3.16

129± 14.4

4± 2.1

241± 29.2

24± 1.2

10± 2.0

10.0

133± 9.0

9± 2.3

264± 29.4

31± 2.5

10± 6.0

31.6

119± 4.4

5± 1.0

278± 29.7

23± 2.6

12± 6.4

100

114± 15.9

8± 2.0

283± 23.1

26± 1.0

9± 3.2

316

100± 6.1

7± 1.2

262± 9.8

25± 2.1

8± 1.2 B

1000

58± 9.1 B

0± 0.0 B

280± 22.8

25 ± 0.6 B

0 ± 0.0 B

2500

0 ± 0.0 B

0 ± 0.0 B/N

155 ± 37.0

22 ± 6.1 B

0 ± 0.0 N

5000

0 ± 0.0 N

0 ± 0.0 N

125 ± 45.0 B

0 ± 0.0 B

0 ± 0.0 N

Positive controls:

Na-azide

MMS

4-NOPD

Concentration

(μg/plate)

10

1 µL/plate

10

40

Mean No. of colonies/plate

(average of 3 ± SD)

965± 120.2

1038± 227.1

2158± 67.5

389± 30.1

107± 7.0

With S9-Mix

0

108± 12.7

10± 2.5

292± 7.8

38± 4.5

9± 3.2

3.16

137± 30.7

5± 2.1

289± 15.9

39± 5.6

7± 2.1

10.0

139± 4.2

5± 2.5

323± 36.5

37± 2.0

4± 2.9

31.6

121± 14.0

7± 3.1

310± 21.2

32± 4.5

5± 4.9

100

134± 14.7

7± 1.5

297± 6.1

31± 6.0

7± 2.6

316

116± 7.6

8± 1.5

286± 24.0

31± 6.7

4 ± 1.2

1000

101± 15.5

7± 0.6

310± 7.4

30± 12.7

12± 3.5

2500

85± 11.8 B

4± 2.5 B

284± 15.0

22± 4.2 B

7± 3.5 B

5000

70± 4.6 B

0± 0.0 B

273± 10.7

21± 10.4 B

6± 3.6 B

Positive controls:

2-AA

Concentrations

(μg/plate)

2.5

10

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

789±
158.7

60 ±
15.0

628 ±
14.3

2943 ±
399.9

128 ±
29.8

MMS = Methylmethanesulfonate

4-NOPD = 4-Nitro-o-phenylene diamine

2-AA = 2-Aminoanthracene

B = Reduction of background lawn

N = No background lawn

Conclusions:
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Reason / purpose:
read-across source
Target gene:
Thymidin kinase-locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
Co-factor supplemented S9 mix prepared from the livers of rats treated with phenobarbital/beta-naphthoflavone.
Test concentrations with justification for top dose:
Experiment I:
with S9 mix: 0.01, 0.05, 0.24, 0.28, 0.32, 0.36, 0.40, 0.44, 0.48 mM (incubation period: 4 h)
without S9 mix: 0.01, 0.02, 0.05, 0.10, 0.20, 0.24, 0.28, 0.32 mM (incubation period: 4 h)
Experiment II:
with S9 mix: 0.15, 0.35, 0.39, 0.43, 0.45, 0.47, 0.49, 0.53 mM (incubation period: 4 h)
without S9 mix: 0.0005, 0.001, 0.005, 0.01, 0.05, 0.10, 0.15, 0.20, 0.25 mM(incubation period: 24 h)
Vehicle / solvent:
Based on a solubility test RPMI (+5% horse serum) was used as solvent.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethanesulfonate (EMS): 200 + 300 µg/mL; Methylmethanesulfonate (MMS): 10 µg/mL; Benzo(a)pyrene (BaP): 2.5 µg/mL
Details on test system and experimental conditions:
Experiment I with and without S9 mix and Experiment II with S9 mix (incubation period: 4 h)
1 x 10E+7 cells were suspended in medium and exposed to the test item either in the presence or absence of metabolic activation in the mutation experiment for 4 h. Thereafter the test item was removed, the cells were washed and suspended in complete culture medium and grown for 2 days at 37 °C in humidified air. The cell density was determined each day and adjusted to 3 x 10E+5 cells/mL.

Experiment II without S9 mix (incubation period: 24 h)
5 x 10E+6 cells were suspended in medium and exposed to the test item in the absence of metabolic activation. After 24 h the test item was removed, cells were washed and suspended in complete culture medium and grown for 2 days at 37 °C in humidified air. The cell density was determined each day and adjusted to 3 x 10E+5 cells/mL.

After the expression period the cloning efficiency (CE) of the cells was determined by seeding a statistical number of 1.6 cells/well in two 96-well plates. The cells were incubated for at least 6 days at 37 °C in a humidified atmosphere. Additionally, cultures were seeded in selective medium. Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in selective medium with TFT. The plates were scored after an incubation period of approx. 14 days at 37 °C in humidified air.
The mutant frequency was calculated by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT. The mutant frequency is expressed as “mutants per 10E+6 viable cells”.

Suspension growth (SG) of the cell cultures reflects the number of times the cell number increases from the starting cell density.
The relative total growth (RTG) is the product of the relative suspension growth (RSG; calculated by comparing the SG of the dose groups with the SG of the control) and the relative cloning efficiency (RCE) for each culture: RTG = RSG x RCE /100.
The mutant frequencies obtained from the experiments were compared with the Global Evaluation Factor (GEF). The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation (GEF = 126 for the microwell method).
Evaluation criteria:
A mutation assay is considered acceptable if:
- At least 3/4 plates from the TFT resistance-testing portion of the experiment are scorable.
- The cloning efficiency of the negative and/or solvent controls is in the range 65% - 120%.
- The spontaneous mutant frequency in the negative and/or solvent controls is in the range 50-170 mutants per 10E+6 cells
- The cell number of the negative/solvent controls should undergo 8-32 fold increase during a 2 day growth period or 32-180 fold increase during a 3 day growth period (long-term treatment).
- The clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 10E+6 cells with at least 40% of the colonies being small colonies or with an induced small colony mutant frequency of at least 150 mutants per 10E+6 cells.
- The RTG is greater than 10%.

The test item is considered mutagenic if following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10E+6 cells.
- A dose-dependent increase in mutant frequency is detected.
Statistics:
The Poisson distribution was used to calculate the plating efficiencies for cells cloned without and with TFT selection. The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative /solvent controls.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No precipitation was observed at any concentration. The pH-value detected in medium containing the test item was within the physiological range.

None of the experimental results met the criteria for considering the test substance mutagenic, except for the highest dose level in experiment I with metabolic activation. There a significantly increased number of mutants at 0.48 mM and a dose dependency were seen. In addition the Global Evaluation Factor of 126 was exceeded by the induced mutant frequency. This effect was only seen in the highly cytotoxic range (RTG below 10%) and was not seen in the verification experiment (experiment II with metabolic activation). Therefore this effect was considered to be of no biological relevance.

Table 1: Experiment I - 4 h exposure – With and without Metabolic Activation

Test group

Concentration
[mM]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants/1E+06 surviving cells

Induced mutant frequency

 

+S9 mix

Control 1

0

82.9

100

73.7

-

Control 2

0

70.6

92.8

Test Item

0.01

73.7

90.7

89.2

5.9

0.05

79.3

107.8

66.8

-16.5

0.24

71.7

97.2

93.8

10.6

0.28

80.5

93.1

98.1

14.9

0.32

72.7

78.0

88.4

5.1

0.36

80.5

60.1

70.2

-13.0

0.40

72.7

40.4

104.3

21.0

0.44

93.5

27.3

141.3 *

58.0

0.48

82.9

9.4

217.2 *

134.0

Benzo[a]pyrene

2.5

58.8

48.3

829.8 *

746.5

 

-S9 mix

Control 1

0

92.1

100.0

76.3

/

Control 2

0

84.1

83.3

/

Test Item

0.01

98.0

107.4

85.9

6.1

 

0.02

85.4

101.2

73.3

-6.5

 

0.05

82.9

109.0

82.8

2.9

 

0.10

96.5

110.7

87.0

7.2

 

0.20

110.1

79.6

70.8

-9.0

 

0.24

102.9

42.3

93.5

13.7

 

0.28

132.6

31.0

137.4*

57.6

 

0.32

114.0

23.6

126.4*

46.5

EMS

300

77.0

63.5

713.3*

633.5

MMS

10

79.3

64.3

447.5*

367.7

*: p <0.05

EMS   Ethyl methane sulphonate,       MMS Methyl methane sulphonate

Induced mutant frequency = mutant frequency of Sample – mean value of mutant frequency in corresponding controls

 

Table 2: Experiment II - 4 h Exposure - With Metabolic Activation

Test group

Concentration
[mM]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants/1E+06 surviving cells

Induced mutant frequency

Control 1

0

68.7

100.0

115.5

/

Control 2

0

86.6

75.6

/

Test Item

0.15

93.5

121.1

89.6

-5.9

0.35

67.7

77.0

130.3

34.7

0.39

92.1

82.2

94.5

-1.0

0.43

93.5

60.7

118.5

22.9

0.45

116.0

60.8

102.5

7.0

0.47

108.2

36.1

148.6*

53.0

0.49

89.3

18.9

169.4*

73.9

0.53

89.3

11.1

220.6*

125.1

Benzo[a]pyrene

2.5

68.7

68.3

691.7*

596.1

*: p <0.05

Induced mutant frequency = mutant frequency of Sample – mean value of mutant frequency in corresponding controls

 

Table 3: Experiment II - 24 h exposure - Without Metabolic Activation

Test group

Concentration
[mM]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants/1E+06 surviving cells

Induced mutant frequency

Control 1

0

93.5

100.0

75.3

/ 

Control 2

0

95.0

108.4

/ 

Test Item

0.0005

118.1

124.7

77.4

-14.4

0.001

118.1

130.7

82.6

-9.2

0.005

89.3

98.1

74.9

-16.9

0.01

106.4

98.3

72.7

-19.1

0.05

127.4

94.1

69.3

-22.5

0.10

125.0

84.9

69.5

-22.4

0.15

89.3

43.7

92.0

0.2

0.20

92.1

26.0

113.6

21.8

0.25

80.5

7.9

188.6*

96.8

EMS

200

38.9

12.2

3002.5*

2910.7

MMS

10

34.2

10.1

2321.3*

2229.5

*: p <0.05

EMS   Ethyl methane sulphonate;      MMS  Methyl methane sulphonate

Induced mutant frequency = mutant frequency of Sample – mean value of mutant frequency in corresponding controls

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

Genetic toxicity in vivo

Description of key information
Mammalian Bone Marrow Chromosome Aberration Test (CA / OECD 475): negative
Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Qualifier:
according to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
chromosome aberration assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
According to Guideline.
Route of administration:
oral: gavage
Vehicle:
Distilled water
Duration of treatment / exposure:
n.a.
Frequency of treatment:
Once
Post exposure period:
test group: 10, 24 and 48 h
positive control: 26 h
Remarks:
Doses / Concentrations:
0, 1000, 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (50 mg/kg bw)
Tissues and cell types examined:
Femoral bone marrow cells
Details of tissue and slide preparation:
SAMPLING TIMES
test groups: 10, 24 and 48 h after treatment
positive control group: 26 h after treatment
Evaluation criteria:
According to Guideline.
Statistics:
Yes
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
negative
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Reason / purpose:
read-across source
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
According to Guideline.
Route of administration:
oral: gavage
Vehicle:
Distilled water
Duration of treatment / exposure:
n.a.
Frequency of treatment:
Once
Post exposure period:
test group: 10, 24 and 48 h
positive control: 26 h
Remarks:
Doses / Concentrations:
0, 1000, 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (50 mg/kg bw)
Tissues and cell types examined:
Femoral bone marrow cells
Details of tissue and slide preparation:
SAMPLING TIMES
test groups: 10, 24 and 48 h after treatment
positive control group: 26 h after treatment
Evaluation criteria:
According to Guideline.
Statistics:
Yes
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

No data on genetic toxicity are available for AES (C9-11, 1-2.5 EO) Na (CAS 160901-28-0). Therefore this endpoint is covered by read across from structurally related AES, i.e. AES (C8-10, 1-2.5 EO) Na and AES (C12-14; 1-2.5 EO) Na (CAS 68891-38-3). The AES reported within the AES category show similar structural, physico-chemical, environmental and toxicological properties. The approach of grouping different AES for the evaluation of their effects on human health and the environment was also made by the Danish EPA (2001) and HERA (2003), supporting the read across approach between structurally related AES. For further details on the suitability of the read-across please refer to the AES Category Approach Justification.

In general a lack of mutagenic activity for the AES category is predictable based on structural and mechanistic considerations. Mutagens are chemicals that either 1) contain highly reactive electrophilic centers capable of interacting with nucleophilic sites on DNA (direct acting agents) or 2) can be metabolized to highly reactive electrophiles. The chemical structures represented by this chemical class do not contain electrophilic functional groups or functional groups capable of being metabolized to electrophiles. AES are readily absorbed in the gastrointestinal tract in human and rat and excreted principally via the urine or faeces depending on the length of the ethoxylate chain but independently of the route of administration. Once absorbed, AES are extensively metabolized by beta- or omega oxidation. The EO-chain seems to be resistant to metabolism. Thus, AES with fully saturated carbon chains are not metabolized to reactive electrophiles.

 

There are two studies for the read-across substance AES (C8-10, 1-2.5 EO) Na and one study for the read-across substance AES (C12-14; 1-2.5 EO) Na (CAS 68891-38-3) addressing genetic toxicity available.

Mutagenicity of AES (C8-10, 2 EO) Na in bacteria was assessed in a study performed according to OECD Guideline 471 with Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 102 and TA 100 (Z&S, 2012e). The tester strains were treated using the plate incorporation and the pre incubation method both with and without the addition of a rat liver S9-mix. The concentrations for both testing methods was 3.16, 10, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate. Results achieved with vehicle (distilled water) and positive controls were valid. Cytotoxicity was seen in presence and absence of metabolic activation while no genotoxicity was observed under both circumstances.

The mutagenicity of AES (C8-10, 2 EO) Na in a mammalian cell line was investigated according to OECD guideline 476 using the mouse lymphoma L5178Y cells with and without metabolic activation (Z&S, 2012f). The test concentrations were 0.01, 0.02, 0.05, 0.10, 0.20, 0.24, 0.28, 0.32 mM without metabolic activation as well as 0.01, 0.05, 0.24, 0.28, 0.32, 0.36, 0.40, 0.44, 0.48 mM with metabolic activation in the first experiment (4 h incubation). In the second experiment the cells were incubated with concentrations of 0.15, 0.35, 0.39, 0.43, 0.45, 0.47, 0.49, 0.53 mM in the presence of metabolic activation for 4 h and at concentrations of 0.0005, 0.001, 0.005, 0.01, 0.05, 0.10, 0.15, 0.20, 0.25 mM in the absence of metabolic activation for 24 h. Results achieved with the vehicle (RPMI medium) and positive controls were valid. Cytotoxicity was seen in presence and absence of metabolic activation. No genotoxicity was observed, except for the highest dose level in experiment I with metabolic activation where a significantly increased number of mutants and a dose dependency were seen at 0.48 mM. In addition the Global Evaluation Factor of 126 was exceeded by the induced mutant frequency. This effect was observed in the highly cytotoxic range only (relative total growth below 10%) and was not verified in the verification experiment (experiment II with metabolic activation). Therefore this effect was considered to be of no biological relevance.

The in vivo clastogenic potential of AES (C12-14) Na (CAS 68891-38-3, analytical purity 27-29%, no data on grade of ethoxylation) was assessed in a mammalian bone marrow chromosomal aberration test with CD-1 mouse according to OECD Guideline 475 (BASF, 1995). The test substance was administered via gavage at doses of 1000 and 2000 mg/kg bw to five animals per sex per dose. Distilled water was used as vehicle. The post exposure period were 10, 24 and 48 h for the test group including the vehicle control and 26 h for the positive control group. Results achieved with the negative (distilled water) and positive controls were valid. No signs of toxicity and no increased number of chromosome aberration were seen at 1000 and 2000 mg/kg bw. Thus the test substance did not show clastogenicity at 1000 and 2000 mg/kg bw based on the test material and 270 to 290 and 540 to 580 mg/kg bw based on the active ingredient.

 

In conclusion, AES and their metabolites lack the structural moieties which confer mutagenic properties. This is reflected by the lack of reliable positive genotoxicity studies within the AES category. This is supported by the conclusions of the HERA report for AES were it is stated that: “In all available in vitro and in vivo genotoxicity assays, there is no indication of genetic toxicity of AES.”

 

References:

Danish EPA - Environmental and Health Assessment of Substances in Household Detergents and Cosmetic Detergent Products (2001). Environmental Project No. 615, pp. 24-28

HERA (2003). Human & Environmental Risk Assessment on ingredients of European household cleaning products Alcohol Ethoxysulphates, Human Health Risk Assessment Draft, 2003. http: //www. heraproject. com.

Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.