Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are two Ames bacterial reverse mutation assays available, and in addition there is an in-vitro mammalian cell gene mutation assay, the (L5178 TK+/-) mouse lymphoma assay, an in-vitro micronucleus study and a chromosomal aberration assay in Chinese hamster ovary cells. All are negative.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 January 2011 to 10 February 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbiotone/¿-naphthoflavone induced S9
Vehicle / solvent:
Sterile, distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Sterile, distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
-S9 Migrated to IUCLID6: WP2uvrA; TA100; TA1535
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
-S9 Migrated to IUCLID6: TA1537
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
-S9 Migrated to IUCLID6: TA98
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: TA100; TA1537; TA1537; WPsuvrA
Remarks:
+S9
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
+S9 Migrated to IUCLID6: TA98
Details on test system and experimental conditions:
Preliminary toxicity test:
Conducted to determine the toxicity of the test article. Concentrations tested were in the range of 0 ¿ 5 mg/plate. The test was performed by mixing bacterial culture (TA100 or WP2uvrA), molten trace histidine or typtophan supplemented top agar, test material formulation and S9 or PBS and overlaying onto sterile plates of Vogel-Bonner minimal agar.  Sterility of the test item was assessed (test article formulation alone added to agar (as described above). Following 48h incubation at 37°C plates were assessed for numbers of revertant colonies sing a Domino colony counter and examined for effects on the growth of the background lawn.
 
Initial toxicity-mutation assay:
Plate incorporation method used (i.e. soft agar, bacteria, TA and S9 / buffer mixed prior to immediate incorporation on to agar plate) test followed the directions of Ames et al.,(1973, 1975). Vehicle and positive controls and seven dose levels of the test material were plated in triplicate/dose. All strains listed above were tested in the presence and absence of S9 mix. 
 
Plates were incubated for approximately 48 hours at 37°C.
 
Confirmatory mutagenicity assay:
Pre-incubation method used (i.e. bacterial strain, test article formulation and S9 or phosphate buffer were incubated for 20 minutes at 37°C). following incubation the bacterial/test article formulation etc. mix was plated on to minimal agar. Vehicle and positive controls and seven dose levels of the test material were plated in triplicate/dose, dosing being adjusted based on the results of the initial experiment. . Again plates were incubated for at 48 hours at 37°C. Means and standard deviations were calculated for the both the initial and confirmatory test.
Evaluation criteria:
The test material was considered positive: dose related increase in mutant frequency over the dose range tested, reproducible increase at 1 or more concentrations, biological relevance against in-house historical control ranges, fold increase greater than 2 times the concurrent solvent control for any tester strain.
 
Toxicity was assessed by reduction in the background lawn; dose-dependent reduction in the mutant count/plate (concurrent vehicle control).
Statistics:
Statistical analysis was performed as described by UKEMS (Mahonet al., 1989)
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
Species / strain:
E. coli WP2 uvr A
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 applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

INITIAL TOXICITY-MUTATION ASSAY:

Six doses of the test material ranging from 5 to 5000 ug/plate±S9 or 15 to 5000 ug/plate±S9 were evaluated in the plate incorporation test inSalmonellaandE.coli strains, respectively.. No precipitation was observed up to the limit dose, 5000 ug/plate. Toxicity was observed in all strains at 500 ¿g/plate and above.

 

Table 1: Bacterial mutation assay, summary of results¿ Initial (plate incorporation) test

Dose

(ug/plate)

0

5

15

50

150

500

1500

5000

Positive control

REVERTANTS/PLATE

TA1535 ¿S9

24±2.1

24±1.5

20±1.2

25±0.6

22±2.6

25±5.2S

17±3.1S

6±2.1V

921±56.0

TA1535 +S9

16±1.5

13±2.0

16±1.5

13±1.7

17±4.4

17±2.1

13±4.9S

9±2.5V

264±31.5

TA100 ¿S9

111±1.7

110±9.1

115±13.1

112±8.2

109±6.4

93±5.8S

46±16.8S

18±6.2V

379±46.8

TA100 +S9

99±6.5

99±9.5

112±6.5

106±16.1

105±20.5

82±8.0

53±3.8S

14±6.4V

1195±5.5

TA1537 ¿S9

14±3.2

14±1.2

14±1.2

17±1.2

17±3.6

13±1.2

6±3.2S

0±0.0T

798±200.3

TA1537 +S9

15±2.6

14±2.1

16±.7

15±2.3

13±1.5

15±2.6

3±2.1S

0±0.0T

208±4.6

TA98 ¿S9

29±3.5

30±1.2

32±3.8

28±2.1

27±3.1

30±1.0

7±2.6

3±1.5

214±15.7

TA98 +S9

31±5.9

33±1.0

25±1.0

31±2.6

31±6.1

2±1.7

12±6.5S

3±2.0V

208±10.5

WP2uvrA ¿S9

35±1.5

N/T

32±3.8

28±2.1

27±3.1

30±1.0

7±2.6

3±1.5

214±15.7

WP2uvrA +S9

44±5.2

N/T

37±7.9

39±3.5

39±4.0

38±1.5

33±4.4

21±9.0

332±18.0

 

CONFIRMATORY MUTAGENICITY ASSAY:

Again no precipitation was observed in any of the strains tested. Cytotoxicity was observed in allSalmonellastrains in the absence and presence of S9.

 

Table 2A: Bacterial mutation assay, summary of results¿ confirmatory (pre-incubation) test ¿ TA1535 & TA100 ¿S9

Dose

(ug/plate)

0

1.5

5

15

50

150

500

1500

Positive control

REVERTANTS/PLATE

TA1535 -S9

25±0.6

20±1.0

25±7.1

26±2.9

23±8.4

26±0.6S

12±2.1V

0±0.0V

708±82.1

TA100 -S9

93±10.5

91±4.2

95±7.2

97±7.2

103±8.5

101±3.6S

83±22.6V

44±5.9V

1799±198.6

 

Table 2B: Bacterial mutation assay, summary of results¿ confirmatory (pre-incubation) test ¿ TA1537, TA98 & WP2uvrA¿S9

Dose

(ug/plate)

0

5

15

50

150

500

1500

5000

Positive control

REVERTANTS/PLATE

TA1537 -S9

9±2.9

8±0.6

10±1.0

7±4.0

6±2.3

6±1.7S

0±0.0T

0±0.0V

2079±280.0

TA98 -S9

26±4.4

31±3.1

22±1.7

15±1.5

16±1.7

9±1.5S

5±3.2V

0±0.0V

187±15.5

WP2uvrA -S9

26±4.5

N/T

31±4.6

25±3.5

28±1.0

26±3.2

27±3.5

15±3.8

976±62.4

 

Table 2C: Bacterial mutation assay, summary of results¿ confirmatory (pre-incubation) test All strains +S9

Dose

(ug/plate)

0

5

15

50

150

500

1500

5000

Positive control

REVERTANTS/PLATE

TA1535 +S9

16±7.09

12±1.7

15±3.2

14±0.6

15±1.2

8±0.6S

7±1.2V

0±0.0V

258±16.8

TA100 +S9

99±8.4

75±11.0

81±9.1

87±2.5

87±5.3

51±4.9S

40±0.6V

0±0.0V

938±63.0

TA1537 +S9

11±2.1

12±2.5

11±1.5

13±1.5

6±1.2

7±1.0S

2±1.0

0±0.0V

221±51.2

TA98 +S9

30±6.7

34±4.0

31±0.6

27±2.0

31±8.4

12±4.0

9±1.7V

0±0.0V

231±17.3

WP2uvrA +S9

31±7.8

N/T

28±5.5

31±22.6

29±9.6

38±0.6

36±8.3

16±0.6

353±17.1

 

N/T      Not treated

S         Sparse background lawn

T         Toxic, no background lawn

V         Very weak background lawn

 

The test material was tested up to either the maximum recommended dose level (5000 ¿g/plate) or the toxic limit, depending on the bacterial strain type. No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Based on the results from this study,sodium lauroyl methyl isethionatewas not mutagenic in the bacterial strains tested, either in the presence or absence of metabolic activation when tested up either 5000 ug/plate (the maximum dose in accordance with regulatory guidelines) or cytotoxic concentrations.
Executive summary:

In a reverse gene mutation assay in bacteria,Salmonella typhimuriumstrains TA98, TA100, TA1535, and TA1537 andEscherichiacoli WP2uvrAwere exposed to sodium lauroyl methyl isethionate formulated in sterile, distilled water. The assay was performed in two phases, using the plate incorporation and the pre-incubation method.

 

In an initial test, the plate incorporation method was used with dose levels of 5 to 5000 ug/plate (depending on strain) in the presence and absence of S9 activation. In the confirmatory test, the pre-incubation method was used. The same dose range was used.  No precipitation was observed in any strain tested. The test material was tested up to either the maximum recommended dose level (5000 ¿g/plate) or the toxic limit, depending on the bacterial strain type. No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.

 

Based on the results from this study,sodium lauroyl methyl isethionatewas not mutagenic in the bacterial strains tested, either in the presence or absence of metabolic activation when tested up either 5000 ug/plate (the maximum dose in accordance with regulatory guidelines) or cytotoxic concentrations.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial forward mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Post mitochondrial supernatant (S9) prepared from livers of male Sprague Dawley rats induced with single ip injection of Arochlor 1254 at 500mg/kg.
Test concentrations with justification for top dose:
1.0, 3.3, 10, 33, 100 µg (+s9 mix)
10, 33, 100, 333, 1000 µg (-S9 mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Migrated to IUCLID6: 2- aminoanthracene, sodium azide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: 20±2 minutes
- Exposure duration: 48 hours
Plates that were not counted immediately following the incubation period were stored at 4±2¿C until colony counting could be conducted.


SELECTION AGENT (mutation assays): Number of revertants



NUMBER OF REPLICATIONS: Each treatment was plated in triplicate.
Three independent experiments were conducted


NUMBER OF CELLS EVALUATED: 1.9 x 108 cells seeded per plate

Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid

In experiment B1, no positive responses were observed in any of the tester strains in the presence or absence of metabolic activation.  As toxicity was only marginal at the highest dose without metabolic activation, the top dose was adjusted to 333 µg/plate for the subsequent confirmatory assays

In experiment B2, no positive responses were observed with tester strains TA98, TA1535 and TA1538.  Tester strains TA100 and TA1537 were not reported due to unacceptable titre values and were retested in experiment B3.

In experiment B3, no positive reactions were observed in TA100 and TA1537 in the presence or absence of metabolic activation.  A 2 fold non-dose responsive increase was observed with tester strain TA1537 in the absence of metabolic activation, however, it was considered that increases of this magnitude (less than 3 fold) with this strain and non-dose-responsive increases are not evaluated as positive.

Conclusions:
Interpretation of results: Negative

The results of the assay, indicate that under the conditions of the study, the test article LB-7819-1 did not cause a positive response with any of the tester strains in the presence or absence of metabolic activation
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
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:
other: OECD 487 (draft guideline) In vitro Mammalian Cell Micronucleus Test (MNvit)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable.
Species / strain / cell type:
lymphocytes: duplicate human lymphocyte cultures prepared from pooled blood of two female donors.
Details on mammalian cell type (if applicable):
Blood from two healthy, non-smoking female volunteers.
Range-finder: Donor age = 25, 29; Donor identity = 7811, 9740
Experiment 1: Donor age = 27, 29; Donor identity = 9679, 9740
Experiment 2: Donor age = 25, 59; Donor identity = 7811, 9740
No volunteer suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. The measured cell cycle time of the donors used at Covance falls within the range 13 +/- 1.5 hours.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9)
Test concentrations with justification for top dose:
Range-finder: 0, 2.902, 4.837, 8.062, 13.44, 22.39, 37.32, 62.21, 103.7, 172.8, 288, 480 and 800 µg/mL
Experiment 1: 0, 25, 50, 100, 200, 225, 250, 275, 300, 350, 400, 450 and 500 µg/mL (-S-9); 0, 25, 50, 100, 200, 250, 300, 350, 400, 450, 500 and 550 µg/mL (+S-9)
Experiment 2: 0, 25, 50, 100, 200, 225, 250, 275, 300, 350, 400, 450 and 500 µg/mL (-S-9); 0, 12.5, 25, 50, 100, 200, 250, 300, 325, 350, 400, 450 and 475 µg/mL (+S-9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile purified water
- Justification for choice of solvent/vehicle: An initial range-finder experiment was performed in which JORDAPON SCI (90) was formulated in dimethyl sulphoxide (DMSO) and tested at final concentrations of 7.835-185.5 µg/mL. These data were rejected primarily because an acceptable maximum concentration was not achieved as neither precipitate or toxicity was observed. Further solubility assessments were performed in which purified water was found to be a more suitable vehicle (soluble up to 800 µg/mL). All further treatments were therefore performed using purified water as the vehicle.
Untreated negative controls:
no
Remarks:
Not applicable for this assay.
Negative solvent / vehicle controls:
yes
Remarks:
sterile purified water or DMSO.
True negative controls:
no
Remarks:
Not applicable for this assay.
Positive controls:
yes
Remarks:
4-nitroquinoline-1-oxide (NQO), cyclophosphamide (CPA) and vinblastine (VIN). NQO and CPA prepared in DMSO, VIN prepared in purified water.
Remarks:
NQO at 2.50 and 5.00 µg/mL final concentration; CPA at 6.25 and 12.5 µg/mL final concentration; VIN at 0.06 and 0.08 µg/mL final concentration.
Details on test system and experimental conditions:
Metabolic activation system:
Rat liver S-9 fraction from male Sprague Dawley rats induced with Aroclor 1254.
Treatment was carried out both in the absence and presence of S-9, prepared in the following way:
Glucose-6-phosphate (180 mg/mL), NADP (25 mg/mL), potassium chloride (150 mM) and rat liver S-9 were mixed in the ratio 1:1:1:2.
For all cultures treated in the presence of S-9, an aliquot of the mix was added to each cell culture to achieve the final concentration of test article in a total of 10 mL. The final concentration of the liver homogenate in the test system was 2%. Cultures treated in the absence of S-9 received an equivalent volume of KCl (150 mM).

Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinsed blood into 9 mL (range-finder) or 8.1 mL (main experiments) HEPES-buffered RPMI medium containing 20% (v/v) heat-inactivated foetal calf serum and 50 µg/mL gentamycin, so that the final volume following addition of S-9 mix/KCl and the test article in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37 +/- 1°C and rocked continuously.
Blood cultures were incubated in the presence of PHA for the following periods prior to treatment:
Range-finder: Approximately 24 or 48 hours
Experiment 1: Approximately 24 hours
Experiment 2: Approximately 48 hours
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed.
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed.
3. A concentration-related increase in the proportion of MNBN cells was observed.
The test article was considered as positive in this assay if all of the above criteria were met.
The test article was considered as bnegative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result.
Statistics:
After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained.
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.
The proportion of MNBN cells for each treatment condition were conmpared with the proportion in negative controls by ushing Fisher's exact test. Probability values of p equal or less than 0.05 were accepted as significant.
Species / strain:
lymphocytes: human lymphocyte cultures prepared from the pooled blood of two female donors
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See Conclusions.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations equal to or greater than 480 µg/mL in the absence of S-9, and 800 µg/mL in the presence of S-9.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Treatment of cells with JORDAPON SCI (90) in the absence and presence of S-9 resulted in frequencies of MNBN cells, which were similar to and not significantly ( p <= 0.05) different from those observed in concurrent vehicle controls for the majority of concentrations analysed. The single exception to this was observed following treatment in Experiment 2 in the presence of S-9 at the highest concentration analysed (450 µg/mL), where a small increase (p <= 0.05) was observed. However, the statistical increase observed was set against a low vehicle control response, with both replicate cultures exhibiting MNBN cell frequencies within historical values. As such, it was not considered of biological importance. The MNBN cell frequency of all other JORADPON SCI (90) treated cultures fell within normal ranges.
Remarks on result:
other: other: Not applicable.
Remarks:
Migrated from field 'Test system'.

Table 1: Experiment 1 (24-hour PHA) Results summary

Treatment

Concentration (µg/mL)

Cytotoxicity (%)

Mean MNBN cell frequency (%)

Historical(%)#

Statistical significance

20+28 hour –S-9

Vehiclea

-

0.35

0.2-1.7

-

 

50.00

7

0.40

 

NS

 

200.0

39

0.40

 

NS

 

225.0

54

0.30

 

NS

 

*NQO, 2.50

ND

2.45

 

p <= 0.001

 

*Vinblastine, 0.08

ND

0.65

 

NS

3+45 hour +S-9 

Vehiclea

-

0.35

0.2-1.4

-

 

100.0

3

0.40

 

NS

 

200.0

10

0.45

 

NS

 

250.0

28

0.20

 

NS

 

300.0

34b

0.45

 

NS

 

*CPA, 6.25

ND

7.75

 

p <= 0.001

aVehicle control was purified water

* Positive control

#95thpercentile calculated range

NS = Not significant

ND = Not determined

bAlthough the highest concentration selected for analysis did not induce the target percentage cytotoxicity of 60%, it was the highest concentration that could be practicably analysed. This was due to the fact that higher test article concentrations induced large numbers of mononucleate toxic / necrotic cells that did not contain cytoplasm, and as such, were excluded from RI calculations. It was therefore considered that in this instance, the RI values underestimated the amount of cytotoxicity present and that the highest concentration selected for analysis was acceptable and valid.

 

Table 2: Experiment 2: (48-hour PHA) Results summary

Treatment

Concentration (µg/mL)

Cytotoxicity (%)

Mean MNBN cell frequency (%)

Historical(%)#

Statistical significance

20+28 hour –S-9

Vehiclea

-

0.20

0.0-1.0

-

 

50.00

0

0.45

 

NS

 

200.0

39

0.15

 

NS

 

225.0

54

0.40

 

NS

 

*NQO, 5.00

ND

4.80

 

p <= 0.001

 

*Vinblastine, 0.06

ND

2.90

 

p <= 0.001

3+45 hour +S-9 

Vehiclea

-

0.20

0.0-0.9

-

 

200.0

11

0.40

 

NS

 

300.0

20

0.30

 

NS

 

400.0

34

0.20

 

NS

 

450.0

41b

0.65

 

p <= 0.05

 

*CPA, 12.5

ND

13.60

 

p <= 0.001

aVehicle control was purified water

* Positive control

#95thpercentile calculated range

NS = Not significant

ND = Not determined

bAlthough the highest concentration selected for analysis did not induce the target percentage cytotoxicity of 60%, it was the highest concentration that could be practicably analysed. This was due to the fact that higher test article concentrations induced large numbers of mononucleate toxic / necrotic cells that did not contain cytoplasm, and as such, were excluded from RI calculations. It was therefore considered that in this instance, the RI values underestimated the amount of cytotoxicity present and that the highest concentration selected for analysis was acceptable and valid.

 

Conclusions:
Interpretation of results (migrated information):
negative See Conclusions.

It is concluded that the test item did not induce micronuclei in cultured human peripheral blood lymphocytes when tested under two different experimental conditions at concentrations up to either its limit of cytotoxicity or the maximum practicable concentration, in both the absence and presence of S-9.
Executive summary:

In a study to determine the genetic toxicity potential of Jordapon SCI (90) (ie. Sodium cocoyl isethionate); It is concluded that the test item did not induce micronuclei in cultured human peripheral blood lymphocytes when tested under two different experimental conditions at concentrations up to either its limit of cytotoxicity or the maximum practicable concentration, in both the absence and presence of S-9.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Due to a technical error at time of harvest, insufficient metaphase cells were located for the evaluation of 50 spreads for the cyclophosphamide treatment at 50 ¿g/mL, for one duplicate treatment. This did not compromise the study's scientific integrity.
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
In vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Post mitochondrial supernatant (S9) prepared from livers of male Sprague-Dawley rats induced with a single intraperitoneal dose of Aroclor 1254 (500 mg/kg). S9 was mixed with 1.4 mg NADP, 2.7 mg isocitric acid and 15¿L S9 per mL medium.
Test concentrations with justification for top dose:
19, 38, 75, 150 and 300 ¿g/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
triethylenemelamine
Remarks:
Migrated to IUCLID6: ¿S9 treatment at a concentration of 0.5 ¿g/mL.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: (lot No. 67F-0155) for the +S9 treatment at a concentration of 50 ¿g/mL.
Details on test system and experimental conditions:
METHOD OF APPLICATION:
An initial toxicity test on the effect of the test substance on mitotic indices and cell cycle delay was performed to determine the dose levels for the chromosome aberration test.
In the chromosome aberration test, cultures were incubated either in the presence or absence of metabolic activation. The ¿S9 cultures were exposed for 8 or 14 hours for the ¿S9 treatment and 2 hours for the +S9 treatment.
In the ¿S9 group, the treatment medium was removed 2 hours prior to cell harvest and the cells were washed with PBS and 0.1 ¿g/mL of Colcemid was added to each flask.
In the +S9 group, the treatment medium was removed after the 2 hour exposure period and the cells were washed with PBS. Complete medium was then added and the flasks incubated for an additional 6 or 15 hours. 0.1 ¿g/mL of Colcemid was then added to each flask and then incubated for 2 hours.
Two hours after the addition of Colcemid, metaphase cells were harvested by trypsinisation, prepared, fixed and mounted on slides using Giemsa staining for cytogenetic analysis. In addition to a 10-hour harvest time dose levels of 150 and 300 ¿g/mL were harvested at 16 hours for the ¿S9 assay and 19 hours for the +S9 assay, owing to delays in cell cycle kinetics at these two dose levels


DURATION
- Preincubation period: Cultures cells were incubated at 37±1°C for 16-24 hours prior to treatment.
- Exposure duration: 2 hours
- Expression time (cells in growth medium): 6-15 hours


NUMBER OF CELLS EVALUATED: 100 metaphases containing 20±2 centromeres

Evaluation criteria:
When possible, a minimum of 100 metaphase spreads (50 per duplicate flask) were examined and scored for aberrations. The mitotic index was recorded as the percentage of cells in mitosis per 500 cells counted.
Statistics:
Fisher's Exact test
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Cytoxicity: In the absence of metabolic activation no metaphase cells were found for chromosome aberration analysis at either the 10 or 16 hour harvest at 300 ¿g/mL owing to excessive toxicity. The mitotic indices were slightly reduced relative to the solvent control at 75 ¿g/mL (10 hour harvest) and moderately reduced at 150 ¿g/mL (10 and 16 hour harvest).
In the presence of metabolic activation no metaphase cells were found for chromosome aberration analysis at 300 ¿g/mL or 150 and 300 ¿g/mL at the 10 or 19 hour harvest, respectively, owing to excessive toxicity. The mitotic indices were slightly reduced relative to the solvent control at 75 ¿g/mL (10 hour harvest), markedly reduced at 150 ¿g/mL (10 hour harvest) and moderately reduced at 150 ¿g/mL (19 hour harvest).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

In the absence of metabolic activation, at the 10 hour cell harvest slight toxicity was observed at 150 ¿g/mL and excessive toxicity at 300 ¿g/mL. At the 16 hour harvest, slight to moderate toxicity was observed at 150 ¿g/mL and excessive toxicity at 300 ¿g/mL. No metaphase cells were found for chromosome aberration analysis at either the 10 or 16 hour harvest at 300 ¿g/mL. The mitotic indices were slightly reduced relative to the solvent control at 75 ¿g/mL (10 hour harvest) and moderately reduced at 150 ¿g/mL (10 and 16 hour harvest). The % of cells with structural aberrations was not significantly different from the solvent control at either harvest (p>0.05, Fisher¿s exact test). 19% of cells treated with TEM were aberrant (p <0.05 Fisher's exact test)

In the absence of metabolic activation, at the 10 hour cell harvest, slight toxicity was observed at 38 ¿g/mL, moderate toxicity at 75 ¿g/mL and excessive toxicity at 150 and 300 ¿g/mL. At the 19 hour harvest, excessive toxicity was observed at 150 and 300 ¿g/mL. No metaphase cells were found for chromosome aberration analysis at either the 10 or 19 hour harvest at 300 ¿g/mL or 150 and 300 ¿g/mL, respectively. The mitotic indices were slightly reduced relative to the solvent control at 75 ¿g/mL (10 hour harvest), markedly reduced at 150 ¿g/mL (10 hour harvest) and moderately reduced at 150 ¿g/mL (19 hour harvest). The % of cells with structural aberrations was not significantly different from the solvent control at either harvest (p>0.05, Fisher¿s exact test). 10% of cells in treated with CP were aberrant(p<0.05, Fisher¿s exact test).
The positive and negative controls fulfilled the requirements for test validity.

Summary of findings for the cytotoxicity of SCI to Chinese Hamster Ovary cells in the presence and absence of metabolic activation

Treatment(¿g a.i./mL)

S9 activation

Harvest time

Mitotic index

Cells scored

Aberrations per cella(mean ± SD)

Cells with aberrations (%)

Untreated cells

-

10

3.9

100

0.020 ± 0.141

2

Water

-

10

5.1

100

0.000 ± 0.000

0

SCI, 19

-

10

5.0

100

0.050 ± 0.261

4

SCI, 38

-

10

3.7

100

0.020 ± 0.141

2

SCI, 75

-

10

3.3

100

0.020 ± 0.141

2

SCI, 150

-

10

2.2

100

0.040 ± 0.400

1

TEM, 0.5

-

10

1.5

100

0.230 ± 0.529

19**

Water

-

16

3.1

100

0.000 ± 0.000

0

SCI, 150

-

16

0.9

100

0.020 ± 0.141

2

Untreated cells

+

10

3.9

100

0.020 ± 0.141

2

Water

+

10

3.2

100

0.020 ± 0.141

2

SCI, 19

+

10

3.3

100

0.050 ± 0.219

5

SCI, 38

+

10

2.5

100

0.060 ± 0.239

6

SCI, 75

+

10

1.7

100

0.000 ± 0.000

0

SCI, 150

+

10

0.2

7b

0.000 ± 0.000

0

CP, 50

+

10

1.5

60c

0.117 ± 0.372

10*

Water

+

19

3.8

100

0.030 ± 0.223

2

SCI, 150

+

19

1.0

45b

0.022 ± 0.149

2

aSeverely damaged cells were counted as 10 aberrations.
bToxicity precluded the analysis of additional metaphase cells.
cDue to a technical error at the time of harvest, insufficient metaphase cells were located for the evaluation of 50 spreads for one of the duplicate treatments.
* Significantly different from the control at p<0.05 (Fisher¿s exact test).
** Significantly different from the control at p<0.01 (Fisher¿s exact test).

Conclusions:
Interpretation of results (migrated information):
negative

Exposure to SCI produced a negative result for the CHO chromosomal aberration assay in the presence or absence of metabolic activation.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
Pyruvic acid was not stated as a constituent of growth medium RPMIS 1640 in error. In one control culture e proportion of small colony mutants and the increase in small colony mutant frequency was outside the recommended result range.
Principles of method if other than guideline:
Deviation from guideline: Pyruvic acid was not stated as a constituent of growth medium RPMIS 1640 in error. In one control culture (experiment 2, -S9, 24 hour treatment), the proportion of small colony mutants and the increase in small colony mutant frequency was outside the recommended result range. However, all positive control cultures showed the necessary guideline requirements had been fulfilled. Neither deviation is considered to affect the integrity of the study.
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (tk) locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Post mitochondrial supernatant (S-9) prepared from the livers of Aroclor 1254 induced male Fischer rats.
Test concentrations with justification for top dose:
Experiment 1:
-S9 (3 hour): 0, 15, 25, 35, 45, 52.5, 60, 67.5, 75, 82.5 and 90 ¿g/mL
+S9 (3 hour): 0, 20, 40, 60, 80, 90, 100, 110, 120, 130 and 140 ¿g/mL
Experiment 2:
-S9 (3 hour): 0, 20, 40, 60, 80, 90, 100, 110, 120, 130 and 140 ¿g/mL
+S9 (3 hour): 0, 40, 60, 80, 90, 100, 110, 120, 130, 140 and 150 ¿g/mL
-S9 (24 hour): 0, 60, 80, 90, 100, 110, 120, 130, 140 and 150 ¿g/mL
Vehicle / solvent:
DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION:
For experiments 1 and 2 in the absence and presence of S9, at least 107 L5178Y mouse lymphoma cells were suspended in tissue culture medium for the 3 hour treatments and at least 4 x 106 cells were used for the 24 hour ¿S9 treatment in experiment 2. Appropriate test solution was then added.
For the 3 hour treatments, S9 mix of KCl was added, incubated for 3 hours, centrifuged, washed and resuspended in fresh medium.
For the 24 hour treatment, after incubation for 24 hours, cultures were centrifuged, washed, resuspended in fresh medium and the cell densities determined.
Cells were then maintained during a 2 day expression period. 5-triflurothymidince (TFT) was added to cell cultures which were then incubated for 11 to 12 days to determine the number of mutant (tk-/-) cells.


NUMBER OF CELLS EVALUATED: 192 wells averaging 1.6 cells per well, per culture for measurement of cloning efficiency.
384 wells averaging 2 x 103 cells per well, per culture for measurement of TFT resistance



DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth
Evaluation criteria:
For valid data, the test substance was considered to be mutagenic in this assay if the mutant frequency (MF) of any test concentration exceeded the sum of the mean control mutant frequency plus the Global Evaluation Factor (GEF) and the linear trend was positive.
Statistics:
Chi squared test. Statistical analysis was used to compare the mutation frequency between each dose level and the solvent control
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY: Dose related toxicity was observed up to the highest concentrations. The highest concentrations deemed suitable for evaluation of mutagenicity demonstrated the following toxicity (expressed as % Relative Total Growth (RTG)). In experiment 1 in the presence of metabolic activation (3 hour treatment), 38% RTG at 100 ¿g/mL and 6% RTG at 115 ¿g/mL were observed, compared to results in the absence of metabolic activation in which 59% RTG at 90 ¿g/mL was seen. In experiment 2 in the presence of metabolic activation (3 hour treatment), 17% RTG was observed at 150 ¿g/mL and in the absence of metabolic activation 10% RTG was observed at 140 ¿g/mL. After 24 hours in the absence of metabolic activation, 14% RTG was seen at 140 ¿g/mL.

JORDAPON SCI (90) displayed Relative Total Growth (RTG) of 59% at 90 ¿g/mL in the absence of S9 in experiment 1 after 3 hours, and 6% RTG at 115 ¿g/mL in the presence of S9 after 3 hours. In experiment 2, after 3 hours in the absence of S9, % RTG ranged from 73% at 100 ¿g/mL to 10% RTG at 140 ¿g/mL. After 3 hours in the presence of S9, % RTG ranged from 65% at 100 ¿g/mL to 17% RTG at 150 ¿g/mL. After a 24 hour treatment in the absence of S9, 14% RTG at 140 ¿g/mL was observed. There was a statistically significant linear trend for experiment 1 in the presence of S9 (p<0.1 chi squared test).

However, since all mutant frequencies were less than the criteria for a positive mutagenic response this trend was not biologically significant. There were no other statistically significant trends observed. No evidence for mutagenicity was obtained at any concentration as all mutant frequencies were below the sum of the mean control mutant frequency plus the global evaluation factor (GEF). There was no significant increase in the proportion of either small or large colony mutants.
The positive controls gave the expected responses.

Summary of results for treatment with JORDAPON SCI (90) in the presence and absence of metabolic activation

Treatment (¿g/mL)

-S9

-S9

Treatment (¿g/mL)

+S9

+S9

% RTG

MFa

% RTG

MFa

Experiment 1 (3 hour treatment)

0

100

62.58

0

100

54.21

15

94

64.56

20

89

57.14

25

95

66.64

40

105

56.31

35

93

51.91

70

73

58.65

45

94

46.55

85

62

70.08

52.5

93

80.39

100

38

79.69

60

98

56.82

115

6

85.08

67.5

71

65.06

-

-

-

75

74

60.84

-

-

-

82.5

64

60.30

-

-

-

90

59

67.92

-

-

-

NQO, 0.15

78

283.93

BP, 2

64

244.01

NQO, 0.2

62

427.36

BP, 3

42

404.67

Experiment 2 (3 hour treatment)

0

100

65.59

0

100

67.63

20

109

55.82

40

77

55.11

40

91

71.17

60

75

63.20

60

94

63.30

80

91

52.57

80

78

76.08

90

83

63.68

90

82

66.06

100

65

55.18

100

73

55.22

110

54

52.78

110

61

59.13

120

51

61.70

120

45

49.18

130

40

46.35

130

23

58.67

140

33

45.46

140

10

66.68

150

17

63.63

NQO, 0.15

72

385.06

BP, 2

82

350.51

NQO, 0.2

60

481.31

BP, 3

43

703.75

Experiment 2 (24 hour treatment)

0

100

56.37

-

-

-

60

104

45.95

-

-

-

80

92

51.06

-

-

-

90

70

60.42

-

-

-

100

64

48.80

-

-

-

110

56

68.65

-

-

-

120

45

55.37

-

-

-

130

37

65.65

-

-

-

140

14

65.65

-

-

-

NQO, 0.01

35

506.55

-

-

-

a5-TFT resistant mutants/106viable cells 2 days after treatment.
%RTG: Relative Total Growth (adjusted by Day 0 factor for 24 hour treatment).

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of the test, JORDAPON SCI (90) did not induce mutation at the tk locus of L5178Y mouse lymphoma cells up to cytotoxic concentrations in either the absence or the presence of metabolic activation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

There are two Ames bacterial reverse mutation assays available. One for SLMI and one for SCI. Twokey studies are Klimisch 1 study carried out to GLP and OECD guideline 471. The studies included five strains of S. typhimurium (TA1538, TA1535, TA1537, TA98 and TA100). The study was negative with and without metabolic activation In the Klimisch 1, mammalian cell gene mutation assay, a Mouse lymphoma L5178Y (TK+/-) assay carried out to OECD guideline 476 under GLP, it was found that Coco fatty acids 2-sulfoethyl ester, sodium salt did not induce mutation at the tk locus of the L5178Y mouse lymphoma cells. The conditions for the study included treatments up to toxic concentrations in two independent experiments in the absence and presence of the rat liver metabolic activation system (S9 mix).

 

There are two studies available investigating the in-vitro potential to cause adverse effects on the chromosomes (clastogenicity). The first study is an in-vitro cytogentics study which is Klimisch 1 to OECD guideline 487 (a draft at the time of testing in 2007) Unilever in vitro micronucleus assay KIM070199. This study in human lymphocytes concluded that the sodium cocoyl isethionate (Coco fatty acids 2-sulfoethyl ester, sodium salt) did not induce micronuclei in the culture human peripheral blood lymphocytes when tested under two different experimental conditions at concentrations up to either its limit of cytotoxicity or the maximum practicable concentration, in both the absence and present of S-9.

 

There is also a chromosomal aberration study in Chinese hamster ovary cells, Unilever Chromosomal Aberration LB-7818. This study was Klimisch 1 as it was carried out according to OECD guideline 473 in full GLP compliance. The Coco fatty acids 2-sulfoethyl ester, sodium salt was tested up to concentration that produced 57% and 71% reductions in the mitotic index at the 10 and 16 hour harvests at the highest concentration of 150µg/ml. Reduction was 94% and 74% at the 10 and 19 hour harvest for the S-9 activated study. This confirmed that sufficiently high concentrations were tested. No statistically significant increase in chromosome aberrations was observed in either the non-activated or S-9 activated test systems, the test substance Sodium Cocoyl Isethionates Coco fatty acids 2-sulfoethyl ester, sodium salt was concluded to be negative.

 


Justification for selection of genetic toxicity endpoint


For the genetic toxicology end point it is difficult to select a single key study as there are three types of study, the Ames bacterial reverse mutation assay, which investigates the potential for mutations to DNA in bacteria, three of these are available. There is also an in-vitro mammalian cell gene mutation assay, the (L5178 TK+/-) mouse lymphoma assay. In addition there are two clastogenicity assays looking for damage to the chromosomes, an in-vitro micronucleus study and a chromosomal aberration assay in Chinese hamster ovary cells. The key Ames test study has been selected for the end point, but the others are all relevant.

Justification for classification or non-classification

There are two Ames bacterial reverse mutation assays, a Mouse lymphoma L5178Y (TK+/-) mammalian cell mutation assay and two assays for clastogenicity, the in-vitro micronucleus assay and the Chromosomal Aberration assay in Chinese hamster ovary. All studies are of high quality Klimisch 1 they are all consistently negative, so there is a significant weight of evidence that SCI and subsequently SLMI are not mutagenic or clastogenic. 

There is no data to indicate any requirement to classify SLMI for genetic toxicity.