Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test: negative in S. typhimurium bacterial strains with and without metabolic activation
- Chromosome aberrations: negative in cultured human lymphocytes with and without metabolic activation for structural chromosome aberrations
- Mouse lymphoma assay: negative with and without metabolic activation

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:
August 1996 - January 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Bacterial strains tested not in accordance with the current recommendations (TA1538 instead of TA102), less than five different analysable concentrations of the test substance used
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
His gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor 1254-induced rat liver
Test concentrations with justification for top dose:
0.002, 0.002, 0.2, 2 and 20 µL test solution/plate (1st experiment)
0.02, 0.06, 0.2, 0.6 and 2 µL test solution/plate (2nd experiment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: 50% aqueous solution of test substance used
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene, 2-aminofluorene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not applicable
- Exposure duration: incubation at 37°C for 2 days

SELECTION AGENT (mutation assays): 0.05 mM L-histidine

NUMBER OF REPLICATIONS: 3
Evaluation criteria:
A test material is considered mutagenic if there is a reproducibly increasing dose-response curve of induced revertant colonies for at least 3 test concentrations. The minimal criteria for a positive response are a 2- to 3-fold increase in the number of revertants (at least 15 colonies) over the spontaneous number for the TA1535, TA1537, TA1538 and TA98 strains, and a 50% increase for the TA100 strain. In addition, a positive response must not be observed only at concentrations near toxic dose levels.
Statistics:
Mean and standard deviation calculation
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:
cytotoxicity
Remarks:
from and above 0.6 µL/plate in the absence of S9, from and above 2 µL/plate (0.6 µL/plate for TA100) in the presence of S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from and above 0.6 µL/plate in the absence of S9, from and above 2 µL/plate in the presence of S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Concentration

(µL test solution)

TA98

TA100

TA1535

TA1537

TA1538

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean

SD

Ratio vs. controls

Mean

SD

Ratio vs. controls

Mean

SD

Ratio vs. controls

Mean

SD

Ratio vs. controls

Mean

SD

Ratio vs. controls

Mean

SD

Ratio vs. controls

Mean

SD

Ratio vs. controls

Mean

SD

Ratio vs. controls

Mean

SD

Ratio vs. controls

Mean

SD

Ratio vs. controls

0 (water)

18

1

1.0

27

7

1.0

169

10

1.0

175

13

1.0

11

4

1.0

13

1

1.0

13

2

1.0

12

5

1.0

9

2

1.0

14

2

1.0

0.002

19

3

1.0

28

9

1.1

152

14

0.9

165

29

0.9

12

2

1.2

11

2

0.9

14

5

1.1

12

4

0.9

12

3

1.3

19

3

1.4

0.02

22

2

1.2

23

6

0.9

173

8

1.0

162

5

0.9

14

1

1.3

10

3

0.8

16

6

1.2

16

2

1.3

8

3

0.9

17

3

1.2

0.2

19

6

1.1

27

2

1.0

148

33

0.9

196

24

1.1

14

3

1.3

16

1

1.3

12

4

0.9

18

6

1.4

15

2

1.6

22

7

1.6

2

0*

0

0.0

0*

0

0.0

0*

0

0.0

0*

0

0.0

0*

0

0.0

0*

0

0.0

0*

0

0.0

0*

0

0.0

0*

0

0.0

0*

0

0.0

20

0*

0

0.0

0*

0

0.0

0*

0

0.0

0*

0

0.0

0*

0

0.0

0*

0

0.0

0*

0

0.0

0*

0

0.0

0*

0

0.0

0*

0

0.0

Positive controls

2-Nitrofluorene

565

21

30.8

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

1600

50

177.8

-

-

-

2-Aminofluorene

-

-

-

318

18

11.9

-

-

-

1175

65

6.7

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Sodium azide

-

-

-

-

-

-

526

27

3.1

-

-

-

439

12

41.2

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

2-Aminoanthracene

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

177

7

13.6

-

-

-

145

10

11.7

-

-

-

1375

56

98.2

9-Aminoacridine

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

263

14

19.7

-

-

-

-

-

-

-

-

-

* Cytotoxicity

First experiment: Summary of numbers of revertants per plate

Conclusions:
Cocamidopropyl hydroxysultaine, as a 50% aqueous solution, was not mutagenic in a bacterial reverse mutation assay up to cytotoxic concentrations, with and without metabolic activation.
Executive summary:

Cocamidopropyl hydroxysultaine, as a 50% aqueous solution, was tested in a bacterial reverse mutation (Ames) test using the Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100 strains. The bacterial strains were exposed on minimal agar plates (using a plate incorporation method) to a range of concentrations up to 20 µL of test solution per plate, both in the presence or absence of an exogenous metabolic activation system, consisting of S9 mix from Aroclor 1254 -induced rat liver. Two independent experiments were performed in triplicate.

In this Ames test, no significant increase in the mean number of revertants over the respective vehicle controls was observed in any of the bacterial strains tested, either in the presence or in the absence of metabolic activation, up to 0.2 or 0.6 µL test solution/plate depending on the strain. Cyotoxic effects were observed at higher dose levels.

Under the conditions of this assay, Cocamidopropyl hydroxysultaine, as a 50% aqueous solution, was not mutagenic up to cytotoxic concentrations.

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:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
C8-C18 AAPHS and C12-18 AAPHS have the same functional groups, and general composition. The main variable resides in the alkyl chain distribution.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical = C8-18 cocamidopropyl hydroxysultaine (EC 939-455-3)
Target chemical = C12-18 cocamidopropyl hydroxysultaine (EC 939-457-4)

3. ANALOGUE APPROACH JUSTIFICATION
The alkyl C-chain distribution of the source chemical significantly overlaps with the one of the target chemical, the C12-alkyl derivative being the major constituent in both chemicals. The structural differences in side C-chains is not expected to lead to significant differences on phys-chem properties.

4. DATA MATRIX: see "Documentation and scientific justification of the read-across approach" in section 13.2
Reason / purpose:
read-across source
Target gene:
His gene
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:
cytotoxicity
Remarks:
from and above 0.6 µL/plate in the absence of S9, from and above 2 µL/plate (0.6 µL/plate for TA100) in the presence of S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from and above 0.6 µL/plate in the absence of S9, from and above 2 µL/plate in the presence of S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
C8-18 Cocamidopropyl hydroxysultaine, as a 50% aqueous solution, was not mutagenic in a bacterial reverse mutation assay up to cytotoxic concentrations, with and without metabolic activation. By analogy, C12-18 Cocamidopropyl hydroxysultaine is not considered mutagenic in bacteria.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 24 February to 21 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
N° 2011/40
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: Cultures of human lymphocytes were prepared from whole blood samples obtained from healthy, non-smoking donors and collected into heparinized sterile tubes.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route.
Test concentrations with justification for top dose:
1st experiment without S9 mix (3h treatment): 0; 39.06; 78.13; 156.3; 312.5; 625; 1250; 2500; 5000 µg/mL
2nd experiment without S9 mix (20h treatment): 0; 9.38; 18.18; 37.5; 75; 150; 300; 600 µg/mL
2nd experiment without S9 mix (44h treatment): 0; 9.38; 18.18; 37.5; 75; 150; 300; 600 µg/mL
1st experiment with S9 mix (3h treatment): 0; 39.06; 78.13; 156.3; 312.5; 625; 1250; 2500; 5000 µg/mL
2nd experiment with S9 mix (3h treatment): 0; 9.4; 18.8; 37.5; 75; 150; 300 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water for injections, batch No. 1F1731 (CDM Lavoisier, France)
- Justification for choice of solvent/vehicle: based on solubility data for test substance
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: without Sç mix: 3; 20 or 44h / with S9 mix: 3h
- Expression time (cells in growth medium): 20 or 44h
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 20 or 44h

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): colcemid (10µg/mL) added 3h before the harvest time
STAIN (for cytogenetic assays): After harvest, the cells were collected by centrifugation and submitted to a hypotonic treatment (KCl 0.075 M). The cells were then fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa.

NUMBER OF REPLICATIONS: 2 independent experiments, 2 cultures in parallel (same donor) per concentration

NUMBER OF CELLS EVALUATED: 200 metaphases/dose-level (on metaphases which contained 44 to 46 chromosomes). Whenever possible, 100 metaphases were scored for each culture. Only 50 metaphases/culture were analysed when at least 10% cells with structural chromosome aberration were observed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (The number of cells in mitosis is scored on a total of 1000 cells per culture)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

OTHER: according to the purity of the test substance (36.2%), a correction factor of 2.76 was applied to prepare the substance to be tested in order to assess thegenotoxicity of the substance at 100% (in accordance with the REACh requirements).
For each experiment, both cultures were prepared from the blood of one distinct donor.
Evaluation criteria:
This study was considered valid since the following criteria were met:
- the frequency of cells with structural chromosome aberration in the vehicle controls was consistent with (but not necessary within) the historical data. In any case this frequency was ≤ 5%,
- the frequency of cells with structural chromosome aberration in the positive controls was significantly higher than that of the vehicle controls (p ≤ 0.05) and consistent with (but not necessary within) the historical data.

A test item is considered positive for inducing chromosomal aberrations if a reproducible and statistically significant increase in the frequency of cells with structural chromosome aberration is observed at one or more dose levels and at one or two harvest times.
A test item is considered negative for inducing chromosomal aberrations if no significant increase is observed in the number of cells with chromosomal aberrations for any of the dose levels and at any harvest times.
Statistics:
For each experiment and for each harvest time, the frequency of cells with structural chromosome aberration (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. This comparison was performed using the Che 2 test unless treated culture data were lower than or equal to the vehicle control data. p = 0.05 was used as the lowest level of significance.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
as defined in the evaluation criteria. However numerical aberrations were observed with and without metabolic activation.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see below
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: At 5000 µg/mL, the pH of the culture medium was approximately 7.1 (7.4 for the vehicle control)
- Effects of osmolality: At 5000 µg/mL, the osmolality was equal to 319 mOsm/kg H2O (289 for the vehicle control).
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no precipitate was observed at the end of the treatment periods at any dose levels.

RANGE-FINDING/SCREENING STUDIES: no range-finding study was performed

COMPARISON WITH HISTORICAL CONTROL DATA:The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered to be valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Without S9 mix:
Following the 3-hour treatment, a slight to severe toxicity was observed at all dose-levels, as shown by a 35-100% decrease in the mitotic index.
Following the 20-hour treatment, a slight to severe toxicity was noted at dose-levels ≥ 150 µg/mL, as shown by a 39-100% decrease in the mitotic index.
Following the 44-hour treatment, a moderate to severe toxicity was noted at dose-levels ≥ 150 µg/mL, as shown by a 43-100% decrease in the mitotic index.
- With S9 mix:
At the 20-hour harvest time in the first experiment, a moderate to severe toxicity was observed at dose-levels ≥ 78.13 µg/mL, as shown by a 42-100% decrease in the mitotic index.
At the 20-hour harvest time in the second experiment, a slight to severe toxicity was observed at dose-levels ≥ 9.4 µg/mL, as shown by a 30-99% decrease in the mitotic index.
At the 44-hour harvest time, a slight to severe toxicity was observed at dose-levels ≥ 18.8 µg/mL, as shown by a 32-92% decrease in the mitotic index.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 7.6.1/1: Results of chromosome analysis for the experiment 1 without S9 mix (3h treatment)

 Dose Level (µg/mL)

Control

Low dose (78.3)

Mid dose (156.3)

High dose (312.5)

Positive control (3µg/mL)

Cytotoxicity

no

yes

yes

yes

yes

Culture

C1

C2

C1

C2

C1

C2

C1

C2

C1

C2

Chromatid aberrations

breaks

0

0

0

0

0

0

 1

4

20

18

interchanges

0

0

0

0

0

0

 0

0

6

6

Isochromatid aberrations

breaks

0

0

0

0

0

0

 0

0

4

3

interchanges

0

0

0

0

0

0

0

0

0

Multiple aberrations

0

0

0

0

0

0

0

0

4

3

Pulverization

0

0

0

0

0

0

0

0

0

0

Total number of structural aberration (- gap)

0

0

0

5

64

Frequency of Cells with structural chromosome aberrations (- gap)

0%

0%

0%

2%

39%

Mitotic index

100%

62%

56%

44%

36%

Numerical aberrations

 1

0

 0

0

 1

0

 1

0

0

0

Table 7.6.1/2: Results of chromosome analysis for the experiment 1 with S9 mix (3h treatment)

 Dose Level (µg/mL)

Control

Low dose (39.06)

Mid dose (78.3)

High dose (156.3)

Positive control (12.5µg/mL)

Cytotoxicity

no

yes

yes

yes

yes

Culture

C1

C2

C1

C2

C1

C2

C1

C2

C1

C2

Chromatid aberrations

breaks

0

0

0

0

0

0

0

1

12

21

interchanges

0

0

0

0

0

0

 0

0

2

3

Isochromatid aberrations

breaks

0

0

0

0

0

0

 0

0

8

3

interchanges

0

0

0

0

0

0

0

0

0

Multiple aberrations

0

0

0

0

0

0

0

0

0

0

Pulverization

0

0

0

0

0

0

0

0

0

0

Total number of structural aberration (- gap)

0

0

0

1

49

Frequency of Cells with structural chromosome aberrations (- gap)

0%

0%

0%

0.5%

32%

Mitotic index

100%

81%

58%

44%

62%

Numerical aberrations

 0

0

 0

0

 1

0

 2

0

0

0

Table 7.6.1/3: Results of chromosome analysis for the experiment 2 without S9 mix (20h treatment)

 Dose Level (µg/mL)

Control

Low dose (78.3)

Mid dose (150)

High dose (300)

Positive control (0.3µg/mL)

Cytotoxicity

no

yes

yes

yes

no

Culture

C1

C2

C1

C2

C1

C2

C1

C2

C1

C2

Chromatid aberrations

breaks

0

0

1

0

1

2

3

0

5

8

interchanges

0

0

0

0

0

0

 0

1

2

0

Isochromatid aberrations

breaks

0

0

0

0

0

0

 0

1

1

0

interchanges

0

0

0

0

0

0

0

0

0

Multiple aberrations

0

0

0

0

0

0

0

0

0

0

Pulverization

0

0

0

0

0

0

0

0

0

0

Total number of structural aberration (- gap)

0

1

3

6

16

Frequency of Cells with structural chromosome aberrations (- gap)

0%

0.5%

1.5%

2.5%

14%

Mitotic index

100%

89%

61%

39%

110%

Numerical aberrations

 1

0

 3

7

 1

3

 2

3

1

0

Table 7.6.1/4: Results of chromosome analysis for the experiment 2 without S9 mix (44h treatment)

 Dose Level (µg/mL)

Control

 300

Cytotoxicity

no

yes

Culture

C1

C2

C1

C2

Chromatid aberrations

breaks

2

0

2

0

interchanges

0

0

0

0

Isochromatid aberrations

breaks

1

1

1

2

interchanges

0

0

0

0

Multiple aberrations

0

0

0

0

Pulverization

0

0

0

0

Total number of structural aberration (- gap)

4

5

Frequency of Cells with structural chromosome aberrations (- gap)

2%

2%

Mitotic index

100%

43%

Numerical aberrations

0

0

7

10

Table 7.6.1/5: Results of chromosome analysis for the experiment 2 with S9 mix (3h treatment)

 Dose Level (µg/mL)

Control

Low dose (9.4)

Mid dose (18.8)

High dose (37.5)

Positive control (12.5µg/mL)

Cytotoxicity

no

yes

yes

yes

yes

Culture

C1

C2

C1

C2

C1

C2

C1

C2

C1

C2

Chromatid aberrations

breaks

1

1

0

1

4

0

1

0

15

14

interchanges

0

0

0

0

0

0

0

0

6

5

Isochromatid aberrations

breaks

1

0

0

1

0

0

0

0

1

4

interchanges

0

0

0

0

0

0

0

0

0

0

Multiple aberrations

0

0

0

0

0

0

0

0

0

0

Pulverization

0

0

0

0

0

0

0

0

0

0

Total number of structural aberration (- gap)

3

2

4

1

45

Frequency of Cells with structural chromosome aberrations (- gap)

1.5%

1%

1.5%

0.5%

32%

Mitotic index

100%

65%

70%

50%

38%

Numerical aberrations

0

0

 2

2

 1

1

7

11

0

0

Experiments without S9 mix

No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3-, 20- as well as 44-hour treatments. These results met the criteria for a negative response.

In the second experiment only, increases in the numerical aberrations were noted when compared to the vehicle control cultures, without any clear evidence of a dose-response relationship or consistency between cell cultures. These numerical aberrations exclusively consisted of polyploidy.

Experiments with S9 mix

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in either experiment, at either harvest time. These results met the criteria for a negative response.

As in the absence of S9 mix,increases in the numerical aberrations were noted when compared to the vehicle control cultures in the second experiment only, without any clear evidence of a dose‑response relationship. These numerical aberrations were exclusively polyploidy.

 

Conclusions:
Under the experimental conditions of this study, the test item, Cocamidopropyl hydroxysultaine, did not induce structural chromosome aberrations in cultured human lymphocytes, exposed for 3 hours to up to 156.3 µg/mL in the presence of a rat metabolizing system, or exposed for 3 hours to up to 312.5 µg/mL and for up to 44 hours to up to 300 µg/mL in the absence of metabolizing system. However numerical chromosome aberrations were observed with and without metabolic activation.
Executive summary:

In an in vitro chromosome aberration study, performed according to the OECD No.473 guideline and in compliance with the GLP, Cocamidopropyl hydroxysultaine (as an aqueous solution of purity 36.2%) diluted in water was tested in cultured human lymphocytes in the presence and the absence of exogenous mammalian metabolic activation (S9 mix).

The dose-levels used for treatments were 39.06, 78.13, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL for the first experiment, both with and without S9 mix; 9.38, 18.8, 37.5, 75, 150, 300 and 600 µg/mL for the second experiment without S9 mix, and 9.4, 18.8, 37.5, 75, 150 and 300 µg/mL for the second experiment with S9 mix. The amount used for the test was calculated as concentration of Cocamidopropylhydroxysultaine and therefore a correction factor of 2.76 was applied. Without metabolic activation, cells were exposed to the test substance for 3 (exp 1), 20 or 44h (exp 2) whereas with metabolic activation the treatment period was of 3 hours in both experiments. In Experiment 1 without S9 mix and in both experiments with S9 mix, cells were rinsed after the 3hrs of treatment with the test substance and placed in fresh medium culture until the harvest time. Cells were harvested 20 or 44h after the beginning of the experiment, corresponding to approximately 1.5 normal cell cycles and 24 hours later. Three hours before harvest, each culture was treated with a Colcemid® solution to block cells at the metaphase-stage of mitosis. Analysis for clastogenicity and aneuploidy was undertaken on 200 (100 per culture; 2 parallel cultures) metaphases/dose-level (on metaphases which contained 44 to 46 chromosomes). Cytotoxicity of the test substance was assessed by the mitotic index: the number of cells in mitosis was scored on a total of 1000 cells per culture.

Positive controls such as mitomycin C and Cyclophosphamid were used to check the sensitivity of the test system. They gave appropriate response, so that the test was considered as valid.

The test substance induced cytotoxicity. Indeed, a decrease of mitotic index was observed in both experiments with and without metabolic activation. The highest tested dose level for metaphase analysis induced around 50% cytotoxicity. Cocamidopropyl hydroxysultaine did not induce structural chromosome aberrations in cultured human lymphocytes with and without metabolic activation at any treatment time. In the second experiment only, increases in the numerical aberrations were noted when compared to the vehicle control cultures. These numerical aberrations exclusively consisted of polyploidy. However, the relevance of such findings was limited as they were observed without any clear evidence of a dose-response relationship or consistency between cell cultures.

Under the experimental conditions of this study, the test item, Cocamidopropyl hydroxysultaine, did not induce structural chromosome aberrations in cultured human lymphocytes, exposed for 3 hours to up to 156.3 µg/mL in the presence of a rat metabolizing system, or exposed for 3 hours to up to 312.5 µg/mL and for up to 44 hours to up to 300 µg/mL in the absence of metabolizing system.

Endpoint:
in vitro cytogenicity / chromosome aberration 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:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
C8-C18 AAPHS and C12-18 AAPHS have the same functional groups, and general composition. The main variable resides in the alkyl chain distribution.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical = C8-18 cocamidopropyl hydroxysultaine (EC 939-455-3)
Target chemical = C12-18 cocamidopropyl hydroxysultaine (EC 939-457-4)

3. ANALOGUE APPROACH JUSTIFICATION
The alkyl C-chain distribution of the source chemical significantly overlaps with the one of the target chemical, the C12-alkyl derivative being the major constituent in both chemicals. The structural differences in side C-chains is not expected to lead to significant differences on phys-chem properties.

4. DATA MATRIX: see "Documentation and scientific justification of the read-across approach" in section 13.2
Reason / purpose:
read-across source
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
as defined in the evaluation criteria. However numerical aberrations were observed with and without metabolic activation.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Under the experimental conditions of this study, the test item, C8-18 Cocamidopropyl hydroxysultaine, did not induce structural chromosome aberrations in cultured human lymphocytes, exposed for 3 hours to up to 156.3 µg/mL in the presence of a rat metabolizing system, or exposed for 3 hours to up to 312.5 µg/mL and for up to 44 hours to up to 300 µg/mL in the absence of metabolizing system. However numerical chromosome aberrations were observed with and without metabolic activation. By analogy, similar results are expected with C12-18 Cocamidopropyl hydroxysultaine.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 15 May to 14 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
N° 2011/40
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase locus (Chromosome 11)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium supplemented by heat inactivated horse serum at 10%, v/v
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route.
Test concentrations with justification for top dose:
Preliminary study: (cytotoxicity assessment)
10, 100, 500, 1000, 2500 and 5000 μg/mL.

Main study (mutagenicity experiments)
Without S9 mix:
first experiment (3-hour treatment): 6.25, 12.5, 25, 50, 75, 100 and 200 μg/mL
second experiment (24-hour treatment): 3.13, 6.25, 12.5, 25, 50 and 100 μg/mL
With S9 mix (3h treatment):
first experiment: 12.5, 25, 50, 100, 200, 300 and 400 μg/mL
second experiment: 6.25, 12.5, 25, 50, 100 and 200 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water for injections: batch Nos. 2F0284 and 2F0703 (CDM Lavoisier, Paris, France).
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Remarks:
-S9 mix: MMS at 25 μg/mL (3-hour treatment) or 5 μg/mL (24-hour treatment) / +S9 mix: CPA at 3 μg/mL. Positive controls were diluted in water for injections.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 3h (first experiment without S9 mix, both experiments with S9 mix) or 24h (second experiment without S9 mix)
- Expression time (cells in growth medium): for 2 days after the end of the treatment period
- Selection time (if incubation with a selection agent): 2 days after the end of the treatment period (at the end of the expression time). Cell were incubated with TFT for 11-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): TFT (trifluorothymidine): 4 μg TFT/mL culture medium
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 2 independent experiments, 2 parallel cultures/dose-level

NUMBER OF CELLS EVALUATED: not applicable.

DETERMINATION OF CYTOTOXICITY
- Method: Adjusted relative total growth (Adj. RTG); to define the number of viable cells (CE2: Cloning Efficiency at the end of the expression period), an average of 1.6 cells/well were seeded in two 96-well plates/culture (four plates/dose-level). After at least 7 days of incubation, in a 37°C, 5% CO2 humidified incubator, the clones were counted.

Adj. RTG (Adjusted Relative Total Growth) = (Adjusted RSG x RCE2) / 100

With RSG= Relative suspension growth calculated as follows: RSG = SG treated / SG vehicle control x 100
Suspension Growth (SG) for the 3-hour treatment = Daily growth on day 1 x daily growth on day 2
Suspension Growth (SG) for the 24-hour treatment = Cell growth during the 24-hour treatment x daily growth on day 1 x daily growth on day 2
Adjusted RSG = Cell count factor x (SG treated/SG vehicle control) x 100
with Cell count factor = treated post-treatment cell count / vehicle control post-treatment cell count

RCE2 = (CE2 treated/ CE2 vehicle control) x 100
CE2 = - Ln [empty wells / total wells] / number of cells per wells (ca. 1.6)

OTHER EXAMINATIONS:
For scoring of colonies in mutant plates, the following parameters were considered:
. well containing mutant colony (small or large),
. well not containing mutant colony,
. when both small and large colonies are present in the same well both mutant colonies were counted (one small and one large).
size of small colonies: < 25% of the diameter of the well (sign of inter-gene mutations: chromosomal rearrangement, translocation)
size of large colonies: > 25% of the diameter of the well (sign of intra-gene mutations: point mutations, base deletions)
Evaluation criteria:
Acceptance criteria:
Criteria for the vehicle controls:
. the Cloning Efficiency (CE2) of the vehicle controls at the end of the expression time should be between 0.65 and 1.2,
. the mutation frequency of the vehicle controls should fall within the normal range of 50 x 10-6 - 170 x 10-6,
. the suspension growth of the vehicle controls should be between 8 and 32 for the 3-hour treatment period, and between 32 and 180 for the 24-hour treatment period.
Criteria for the positive controls:
. the increase above the vehicle control mutation frequency (IMF) should be at least 300 x 10-6, the increase in the small colony mutation frequency accounting for at least 40%,
. or the increase in the small colony mutation frequency should be at least 150 x 10-6 above that seen in the concurrent vehicle control.
In addition, the upper limit of cytotoxicity observed in the positive control culture should have an Adj. RTG greater than 10%.

Evalution criteria:
a positive result, which should fulfill the following criteria:
. at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the Global Evaluation Factor (GEF) of 126 x 10-6,
. a dose-response relationship is demonstrated by a statistical analysis.

Noteworthy increases in the mutation frequency observed only at high-levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose-levels with Adj. RTG between 10 and 20%, are not considered as positive results.

A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if (Moore et al., 2002):
. there is at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG,
. there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj. RTG.
Statistics:
A linear regression was performed to assess the linear trend between the mutation frequency and the dose. This statistical analysis was performed using SAS Enterprise Guide software version 4.2 (SAS version 9.2, SAS Institute Inc; Appendix 4). Only individual mutation frequencies obtained from cultures showing an Adj. RTG ≥ 10% were used in this analysis.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
see Tables 7.6.1/1 to 7.6.1/4
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see Tables 7.6.1/1 to 7.6.1/4
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: at 5000 µg/mL, the pH was approximately 7.4 (7.1 for the vehicle control)
- Effects of osmolality: at 5000 µg/mL, the osmolality was equal to 331 mOsm/kg H2O (298 mOsm/kg H2O for the vehicle control).
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no data but a priori the test substance is freely soluble in water

RANGE-FINDING/SCREENING STUDIES:To assess the cytotoxicity of the test item, six dose-levels (one culture/dose-level) were tested both with and without metabolic activation.
Following the 3-hour treatment without S9 mix, a severe toxicity was induced at dose-levels ≥ 100 μg/mL, as shown by a 82-100% decrease in the Adjusted Relative Total Growth. Following the 24-hour treatment without S9 mix, a moderate to severe toxicity was induced at dose-levels ≥ 10 μg/mL, as shown by a 41-100% decrease in Adj. RTG. Following the 3-hour treatment with S9 mix, a moderate to severe toxicity was induced at dose-levels ≥ 100 μg/mL, as shown by a 45-100% decrease in Adj. RTG.

COMPARISON WITH HISTORICAL CONTROL DATA: With one exception which is not considered to have a biological impact on the validity of the
study, the Cloning Efficiencies (CE2), the Suspension Growths (SG) and the mutation frequencies of the vehicle controls were as specified in the acceptance criteria. Moreover, the induced mutation frequencies obtained for the positive controls met the acceptance criteria specified in the study plan. The study was therefore considered as valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Without S9 mix, following the 3-hour treatment, a slight to severe toxicity was induced at dose-levels
≥ 25 μg/mL, as shown by a 35-100% decrease in Adj. RTG. Following the 24-hour treatment, a moderate to severe toxicity was induced at dose-levels ≥ 3.13 μg/mL, as shown by a 43-100% decrease in Adj. RTG.
With S9 mix, in the first experiment, a slight to severe toxicity was induced at dose-levels ≥ 50 μg/mL, as shown by a 37-100% decrease in Adj. RTG.
In the second experiment, a slight to severe toxicity was induced at dose-levels ≥ 25 μg/mL, as shown by a 33-95% decrease in Adj. RTG.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 7.6.1/1: mutagenicity results of first experiment without S9 mix, 3-hour treatment:

Doses (µg/mL)

Individual values

Mean values of C1 and C2

Cultures

Adj RTG %

MF x 10-6

Adj RTG %

MF x 10-6

Large colony (x 10-6)

Small colony

0

C1

100

95

100

110

47

62

C2

100

126

6.25

C1

67

99

84

95

42

50

C2

100

91

12.5

C1

84

103

83

109

59

47

C2

83

114

25

C1

54

81

65

85

30

53

C2

75

90

50

C1

20

106

22

131

44

83

C2

23

156

75

C1

2

189

2

190

66

119

C2

2

192

MMS (25 µg/mL)

C1

59

351

64

385

134

 

222

C2

69

418

 

Table 7.6.1/2: mutagenicity results of second experiment without S9 mix, 24-hour treatment:

Doses (µg/mL)

Individual values

Mean values of C1 and C2

Cultures

Adj RTG %

MF x 10-6

Adj RTG %

MF x 10-6

Large colony (x 10-6)

Small colony

0

C1

100

118

100

113

39

69

C2

100

108

3.13

C1

46

141

57

153

66

82

C2

68

165

6.25

C1

33

166

50

154

44

105

C2

68

142

12.5

C1

14

205

21

199

58

130

C2

28

193

25

C1

6

320

11

212

57

149

C2

16

104

50

C1

0

227

0

200

50

142

C2

0

174

MMS (5 µg/mL)

C1

35

1058

38

929

182

587

C2

42

800

 

Table 7.6.1/3: mutagenicity results of first experiment with S9 mix, 3-hour treatment:

Doses (µg/mL)

Individual values

Mean values of C1 and C2

Cultures

Adj RTG %

MF x 10-6

Adj RTG %

MF x 10-6

Large colony (x 10-6)

Small colony

0

C1

100

145

100

147

47

95

C2

100

149

12.5

C1

94

135

92

113

49

60

C2

90

92

25

C1

122

147

102

160

47

104

C2

83

172

50

C1

63

177

63

171

52

116

C2

63

165

100

C1

18

180

15

196

58

129

C2

12

213

CPA (3 µg/mL)

C1

54

990

45

1047

238

674

C2

35

1104

 

Table 7.6.1/4: mutagenicity results of second experiment with S9 mix, 3-hour treatment:

Doses (µg/mL)

Individual values

Mean values of C1 and C2

Cultures

Adj RTG %

MF x 10-6

Adj RTG %

MF x 10-6

Large colony (x 10-6)

Small colony

0

C1

100

47

100

63

30

32

C2

100

78

6.25

C1

68

101

83

96

56

36

C2

98

91

12.5

C1

65

75

80

74

39

33

C2

96

74

25

C1

56

109

64

104

41

60

C2

73

98

50

C1

57

84

67

81

23

57

C2

76

79

100

C1

49

91

51

100

43

54

C2

52

110

200

C1

2

131

5

111

54

55

C2

8

92

CPA (3 µg/mL)

C1

31

613

46

598

152

389

C2

61

584

 

Conclusions:
Under the experimental conditions of this study, the test item, Cocamidopropyl hydroxysultaine, did not show any mutagenic activity in the mouse lymphoma assay, up to 200 μg/mL in the presence of a rat metabolizing system (3-hour treatment) or up to 75 μg/mL (3-hour treatment) or 50 μg/mL (24-hour treatment) in the absence of a rat metabolizing system.
Executive summary:

In an in vitro mammalian cell mutation assay, performed according to the OECD No.476 and in compliance with the GLP,  cocamidopropyl hydroxysultaine (as an aqueous solution of purity 36.2%) diluted in water for injections was tested in the L5178Y Tk +/- mouse lymphoma cell line in the presence and the absence of mammalian metabolic activation (S9 mix).

Prior to the mutagenicity experiments, a preliminary assay was conducted in order to assess the cytotoxicity of the test substance.The substance was tested for its toxicity at dose levels between 0 and 5000 µg/mL as recommended in the OECD guideline.The amount used for the test (preliminary and main assays) was calculated as concentration of Cocamidopropylhydroxysultaine and therefore a correction factor of 2.76 was applied.

Two independent experiments were then performed. In Experiment 1, L5178Y TK +/- mouse lymphoma cells (heterozygous at the thymidine kinase locus) were exposed to the test item at eight dose levels, in duplicate, together with vehicle (water) and positive controls (Methylmethane sulphonate (MMS) or Cyclophosphamide (CPA) without and with metabolic activation respectively) using 3-hour exposure groups both in the absence and presence of metabolic activation. In Experiment 2, the cells were exposed to the test item at seven dose levels using a 3‑hour exposure group in the presence of metabolic activation and a 24‑hour exposure group in the absence of metabolic activation.

The concentration range of test item was selected following the results of the preliminary toxicity test, and in Experiment 1 was 6.25 to 200 µg/ml in the absence of metabolic activation and 12.5 to 400 µg/mL in the presence of metabolic activation. In Experiment 2 the dose range was 3.13 to 400 µg/ml in the absence of metabolic activation, and 6.25 to 200 µg/ml in the presence of metabolic activation.

The vehicle controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

Under the experimental conditions of this study, the test item, Cocamidopropyl hydroxysultaine, did not show any mutagenic activity in the mouse lymphoma assay, up to 200 μg/mL in the presence of a rat metabolizing system (3-hour treatment) or up to 75 μg/mL (3-hour treatment) or 50 μg/mL (24-hour treatment) in the absence of a rat metabolizing system. In conclusion, the test item was considered not to be mutagenic to L5178Y cells under the conditions of the test.

 

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:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
C8-C18 AAPHS and C12-18 AAPHS have the same functional groups, and general composition. The main variable resides in the alkyl chain distribution.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical = C8-18 cocamidopropyl hydroxysultaine (EC 939-455-3)
Target chemical = C12-18 cocamidopropyl hydroxysultaine (EC 939-457-4)

3. ANALOGUE APPROACH JUSTIFICATION
The alkyl C-chain distribution of the source chemical significantly overlaps with the one of the target chemical, the C12-alkyl derivative being the major constituent in both chemicals. The structural differences in side C-chains is not expected to lead to significant differences on phys-chem properties.

4. DATA MATRIX: see "Documentation and scientific justification of the read-across approach" in section 13.2
Reason / purpose:
read-across source
Key result
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
Conclusions:
Under the experimental conditions of this study, the test item, C8-18 Cocamidopropyl hydroxysultaine, did not show any mutagenic activity in the mouse lymphoma assay, up to 200 μg/mL in the presence of a rat metabolizing system (3-hour treatment) or up to 75 μg/mL (3-hour treatment) or 50 μg/mL (24-hour treatment) in the absence of a rat metabolizing system. by analogy, C12-18 Cocamidopropyl hydroxysultaine is not considered mutagenic in mouse lymphoma assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo test:
Bone marrow micronucleus test in rats as part of an OECD 422 repeated dose toxicity study, negative up to 300 mg/kg bw/d (5-6 weeks of exposure)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April - December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Principles of method if other than guideline:
Test performed based on the methods described by Schmid (Schmid W. The micronucleus test. Mutation Research 31: 9-15, 1975) and modified by Salamone et al. (Salamone M, Heddle J, Stuart E and Katz M. Toward an improved micronucleus test. Studies on 3 model agents, mitomycin C, CPA and dimethylbenzanthracene. Mutation Research 74: 347-356, 1980).
Apart from detecting chromosome breakage events (clastogenesis), the micronucleus test is capable of detecting chemicals which induce whole chromosome loss (aneuploidy) in the absence of clastogenic activity. In the bone marrow of rats exposed to a chemical which induces cytogenetic damage, chromosomal fragments or entire chromosomes which are left behind at cell division were not incorporated into the nuclei of daughter cells. Most of these fragments condense and form one or more micronuclei in the cytoplasm. The visualization of micronuclei is facilitated in erythrocytes because their nucleus is extruded during erythropoiesis. Accordingly, the basis of this test is an evaluation of the increase in the number of Micronucleated Polychromatic Erythrocytes (MPE).
Substances which inhibit either proliferation or maturation of erythroblasts and those which are toxic for nucleated cells, decrease the proportion of immature erythrocytes (polychromatic, PE) when compared to mature erythrocytes (normochromatic, NE). Thus, the cytotoxicity of a substance can be evaluated by a decrease in the PE/NE ratio.
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation:
. Main study: 9 (females) - 10 (males) weeks old
. Micronucleus phase: 15 weeks old
- Weight at study initiation:
. Main study: 216 g (females) - 392 g (males)
. Micronucleus phase: 277 g (females) - 498 g (males)
- Fasting period before study: No
- Housing: Individual (except during pairing) in polycarbonate 940 cm² cages with stainless stell lids and autoclaved dust
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days (main study) / 7 days (micronucleus phase) before dosing initiation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 50 +/- 20
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 10 May 2012 To: 20 July 2012
Route of administration:
oral: gavage
Vehicle:
Drinking water treated by reverse osmosis
Details on exposure:
PREPARATION OF DOSING SOLUTIONS (main study):
- The test item was administered as a solution in the vehicle, by mixing with the required quantity of vehicle.
- The dose formulations were prepared daily.

VEHICLE
- Concentration in vehicle: The concentration of the test item in samples of each control and test item dose formulation prepared for use in weeks 1, 3, 5 and 7 was determined.
- Administration volume: 5 mL/kg/day (main study) / 10 mL/kg (cyclophosphamide-treated group in micronucleus phase)
Duration of treatment / exposure:
The dose formulations were administered daily according to the following schedule (Day 1 corresponding to the first day of the treatment period):

MAIN STUDY:
. In the males:
- 2 weeks before pairing (from study day 1 to 14),
- during the pairing period (3 weeks, from study day 15 until study day 16 to 29),
- until sacrifice (at least 5 weeks in total, from study day 17 to 30 until study day 36).
. In the females:
- 2 weeks before pairing (from study days 1 to 14),
- during the pairing period (3 weeks, from study days 15 to 29),
- during gestation (from study days 16 to 30 until study days 36 to 50),
- during lactation until day 5 post-partum inclusive (from study days 37 to 51 until study days 42 to 56),
- until sacrifice for the non-pregnant females (at least 6 weeks in total, approximately, until study day 41 to day 45).

MICRONUCLEUS PHASE:
The positive control dose formulation was administered as a single dose on the day preceding the scheduled sacrifice (i.e. on completion of the treatment period for groups 1 to 4).
Frequency of treatment:
Once daily
Post exposure period:
None
Remarks:
Doses / Concentrations:
0, 30, 100, 300 mg/kg bw/day
Basis:
other: nominal dose levels (main study)
No. of animals per sex per dose:
10 (main study) / 5 (micronucleus phase)
Control animals:
yes, concurrent vehicle
Positive control(s):
- Nature: cyclophosphamide (CPA, CAS No. 6055-19-2, Sigma, Saint-Quentin-Fallavier, France)
- Justification for choice of positive control: In the absence of any specific recommendation in the OECD guideline No. 474, the dose level was selected based on scientific literature, in order to insure a clear positive response.
- Route of administration: oral (gavage)
- Doses / concentrations: 30 mg/kg bw as a single dose 24 hours prior to euthanasia at the end of the treatment period
Tissues and cell types examined:
Bone marrow cells (increase of the frequency of micronucleated cells) were examined in the first five animals in sultaine-treated groups and all CPA-treated animals.
Details of tissue and slide preparation:
- Preparation of the smears:
At the end of the treatment period, all animals (first five principal animals in sultaine-treated groups, all CPA-treated animals) were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination. The femurs were removed and bone marrows were flushed and suspended in fetal calf serum. The separation of anucleated erythrocytic cells from other myeloic cells was carried using a cellulose column. This elution step enables the production of slides containing only polychromatic and normochromatic erythrocytes without any nucleated cells or mast cell granules. After centrifugation of the eluate containing the cells, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air dried and stained with Giemsa.
The slides were coded so that the scorer is unaware of the treatment group of the slide under evaluation ("blind" scoring). Thereafter, CPA-treated animals were discarded without any further investigations.

- Microscopic examination of slides:
In a first instance, the micronucleus analysis was performed on the slides of the males only. Based on the toxicological results obtained in this study (as differences were not observed between males and females), the slides analysis was not performed for the females.
For each sampled male, the number of Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio were established by scoring a total of 1000 erythrocytes (PE + NE). Scoring was performed "blind".
Evaluation criteria:
All individual data of MPE/2000PE and the PE/NE ratio were presented in tabular form. Mean and standard deviations of MPE/1000PE and PE/NE ratios were given for each experimental group. Because MPE/1000PE is considered as a percentage, this parameter was statistically analyzed after Arcsine transformation. The model was validated by a comparison between the vehicle group and the positive control. Effect of the test item was determined by comparisons of each group to the control one.
For a result to be considered positive, there must be: either a dose-related increase in the frequency of MPE when compared to the vehicle control group, or an increase in the frequency of MPE in a single dose group, of at least 2-fold the frequency of the vehicle control group.
Biological relevance of the results was considered first. Statistical analysis was used as an aid in evaluating the test results.
Statistics:
Normality and homogeneity of variances were tested using a Kolmogorov-Smirnov test and a Bartlett test. If normality and homogeneity of variances are demonstrated, the statistical comparisons are performed using a Student t-test (two groups) or a one-way analysis of variance (three groups) followed by a Dunnett test (if necessary). If normality or homogeneity of variances is not demonstrated, a Mann/Whitney test (two groups) or a Kruskall Wallis test (three groups) will be performed followed by a Dunn test (if necessary). All these analyses were performed using the software SAS Enterprise Guide Version 2.05.89 (SAS Release 8.02 TS Level 02M0, SAS Institute Inc), with a level of significance of 0.05 for all tests.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The positive control cyclophosphamide induced a significant increase (p < 0.001) in the frequency of MPE when compared with the vehicle group, indicating the sensitivity of the test system under our experimental conditions. The test conditions were therefore considered to be valid.
The mean values of the PE/NE ratios in animals treated with the test item at 30, 100 or 300 mg/kg/day were not statistically significantly different from that of the vehicle control animals.
The mean frequencies of MPE in the three test item treated groups were not found significantly different from that in the vehicle group. These results met the criteria of a negative response.
(See Table below)

Table: Results of the cytogenetic test: data summary

Group

Dose(1)

MPE/1000PE

PE/NE ratio

(mg/kg/day)

mean

(sd)

mean

(sd)

Males

Vehicle

0

0.8

(0.4)

0.42

(0.19)

Test item

30

1.3

(0.8)

0.54

(0.09)

100

1.5

(0.9)

0.38

(0.11)

300

0.8

(0.3)

0.48

(0.13)

Cyclophosphamide

30

15.1***

(3.3)

0.22*

(0.06)

(1): expressed as active material

Five animals per group

MPE: Micronucleated Polychromatic Erythrocytes

PE: Polychromatic Erythrocytes

NE: Normochromatic Erythrocytes

sd: standard deviation

*** p<0.001         * p<0.05

The mean values of the polychromatic/normochromatic erythrocytes (PE/NE) ratios in animals treated with the test item at 30, 100 or 300 mg/kg/day were not statistically significantly different from that of the vehicle control animals. However, based on the effects observed in kidneys (distant from the site of administration) in the repeated-dose toxicity phase of the study, the target tissue (bone marrow) was considered to have been exposed to the test substance and/or its metabolites or degradation products.

Conclusions:
Under the experimental conditions of this study, Cocamidopropyl hydroxysultaine, did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells up to 300 mg/kg administered daily for the whole dosing period.
Executive summary:

As part of an OECD 422 compliant study, following daily oral administration (by gavage) to male and female rats from before mating, during mating and, for the females, throughout gestation until day 5 post‑partum (p.p.)inclusive, an evaluation of the potential of the test item to induce damage to the chromosomes or the mitotic apparatus in bone marrow cells (increase in the frequency of micronucleated cells) was performed.

Three groups of ten male and ten female Sprague-Dawley rats received the test item, Cocamidopropyl hydroxysultaine, as a 36.2% aqueous solution, daily, by oral administration (gavage), over the administration period, at dose‑levels of 30, 100 or 300 mg/kg/day.An additional group of 10 males and 10 females received the vehicle control, drinking water, under the same experimental conditions. The dosing volume was 5 mL/kg/day. Another group of five males and five females received Cyclophosphamide (CPA) as a single dose of 30 mg/kg on the day preceding schedule sacrifice, and acted as a positive control group for micronuclei induction. At necropsy, the femur of the first five principal animals in groups 1 to 4 and all group 5 animals were sampled for bone marrow micronucleus analysis.

 

There were no pathological findings in animals given 30 mg/kg/day of CPA at microscopic examination.

The positive control cyclophosphamide induced a significant increase (p < 0.001) in the frequency of micronucleated polychromatic erythrocytes (MPE) when compared with the vehicle group, indicating the sensitivity of the test system under our experimental conditions. The test conditions were therefore considered to be valid.

The mean values of the polychromatic/normochromatic erythrocytes (PE/NE) ratios in animals treated with the test item at 30, 100 or 300 mg/kg/day were not statistically significantly different from that of the vehicle control animals. However, based on the effects observed in kidneys (distant from the site of administration) in the repeated-dose toxicity phase of the study, the target tissue (bone marrow) was considered to have been exposed to the test substance and/or its metabolites or degradation products.

The mean frequencies of MPE in the three test item treated groups were not found significantly different from that in the vehicle group. These results met the criteria of a negative response.

In conclusion, under the experimental conditions of the study, Cocamidopropyl hydroxysultaine did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells up to 300 mg/kg administered daily for the whole dosing period (i.e., approximately 5 to 6 weeks).

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
C8-C18 AAPHS and C12-18 AAPHS have the same functional groups, and general composition. The main variable resides in the alkyl chain distribution.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical = C8-18 cocamidopropyl hydroxysultaine (EC 939-455-3)
Target chemical = C12-18 cocamidopropyl hydroxysultaine (EC 939-457-4)

3. ANALOGUE APPROACH JUSTIFICATION
The alkyl C-chain distribution of the source chemical significantly overlaps with the one of the target chemical, the C12-alkyl derivative being the major constituent in both chemicals. The structural differences in side C-chains is not expected to lead to significant differences on phys-chem properties.

4. DATA MATRIX: see "Documentation and scientific justification of the read-across approach" in section 13.2
Reason / purpose:
read-across source
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Post exposure period:
None
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Under the experimental conditions of this study, C8-18 Cocamidopropyl hydroxysultaine, did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells up to 300 mg/kg administered daily for the whole dosing period. By analogy, similar results are expected with C12-18 Cocamidopropyl hydroxysultaine.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

As the genetic toxicity of the substance EC 939-457-4 has not been investigated experimentally, a read-across approach was followed to fill in the endpoint based on studies conducted on the structural analogue substance C8 -18 cocamidopropyl hydroxysultaine (= source substance EC 939-455-3).Cocamidopropyl hydroxysultaine was assessed for its genotoxic potential in in vitro (Ames test, chromosome aberration test, mouse lymphoma assay) and in vivo (bone marrow micronucleus test) studies.

 

In vitro:

 

Cocamidopropyl hydroxysultaine was tested for its mutagenic potential in vitro in a Klimisch score 2 bacterial reverse mutation (Ames) test (1997), used as a key study. In this study, the test substance as a 50% aqueous solution, was tested in Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100 strains. The bacterial strains were exposed to a range of concentrations up to 20 µL of test solution per plate, using the direct incorporation method both in the presence or absence of an exogenous metabolic activation system. No significant increase in the mean number of revertants over the respective vehicle controls was observed in any of the bacterial strains tested, either in the presence or in the absence of metabolic activation, up to 0.2 or 0.6 µL test solution/plate depending on the strain. Cyotoxic effects were observed at higher dose levels. Therefore, under the conditions of this assay, Cocamidopropyl hydroxysultaine, as a 50% aqueous solution, was not mutagenic up to cytotoxic concentrations.

 

In compliance with REACH Annex VIII requirements and based on negative results in both other in vitro tests, Cocamidopropyl hydroxysultaine was further tested for its mutagenic potential in vitro in a Klimisch score 1 mammalian gene mutation assay (2012), used also as a key study. In this study, the test substance as a 36.2% aqueous solution, was assessed in the L5178Y Tk +/- mouse lymphoma cell line in the presence and the absence of mammalian metabolic activation (S9 mix). The cytotoxicity of the test substance was determined in a preliminary study up to 5000 µg/mL. Two independent experiments were then performed. Cells were exposed to the test substance without metabolic activation for 3 hours in the experiment 1 and 24 hours in the experiment 2. In the condition with metabolic activation, cells were exposed to the test substance for 3 hours in both experiments. Under the experimental conditions of this study, the test item, Cocamidopropyl hydroxysultaine, did not show any mutagenic activity in the mouse lymphoma assay, up to 200μg/mL in the presence of a rat metabolizing system (3-hour treatment) or up to 75μg/mL (3-hour treatment) or 50μg/mL (24-hour treatment) in the absence of a rat metabolizing system. In conclusion, the test item was considered not to be mutagenic to L5178Y cells.

 

In compliance with REACH Annex VII requirements, Cocamidopropyl hydroxysultaine was also tested for its clastogenic potential in vitro in a Klimisch score 1 chromosome aberration test (2012), used as a key study. In this study, the test substance as a 36.2% aqueous solution, was assessed in cultured human lymphocytes. The dose-levels used for treatments were up to 5000 µg/mL for the first experiment, both with and without S9 mix. As cytotoxicity was observed, dose ranges were lowered in the second experiment from 9.38, to 600 µg/mL without S9 mix, and from 9.4 to 300 µg/mL with S9 mix. Without metabolic activation, cells were exposed to the test substance for 3 (exp 1), 20 or 44h (exp 2) whereas with metabolic activation the treatment period was of 3 hours in both experiments. Cells were harvested 20 or 44 h after the beginning of the experiment, corresponding to approximately 1.5 normal cell cycles and 24 hours later. Cytotoxicity of the test substance was assessed by the mitotic index. The highest tested dose level for metaphase analysis induced around 50% cytotoxicity. Under the test conditions, Cocamidopropyl hydroxysultaine did not induce structural chromosome aberrations in cultured human lymphocytes with and without metabolic activation at any treatment time.

In the second experiment only, increases in the numerical aberrations were noted when compared to the vehicle control cultures, without any clear evidence of a dose-response relationship or consistency between cell cultures. These numerical aberrations exclusively consisted of polyploidy. To explore further these numerical aberrations, a bone marrow micronucleus assay was performed in order to assess the aneugenic potential of Cocamidopropyl hydroxysultaine in vivo. Indeed, micronucleus induction may be the result of clastogenic or aneugenic activity.

 

In vivo:

 

As part of the OECD 422 compliant study on the test substance, following daily oral administration (by gavage) to male and female rats from before mating, during mating and, for the females, throughout gestation until day 5 post‑partum (p.p)inclusive,an evaluation of the potential of the test item to induce damage to the chromosomes or the mitotic apparatus in bone marrow cells (increase in the frequency of micronucleated cells) was performed. Three groups of ten male and ten female Sprague-Dawley rats received the test item, Cocamidopropyl hydroxysultaine, as a 36.2% aqueous solution, daily, by oral administration (gavage), over the administration period, at dose levels of 30, 100 or 300 mg/kg/day. An additional group of 10 males and 10 females received the vehicle control, drinking water, under the same experimental conditions. Another group of five males and five females received Cyclophosphamide (CPA) as a single dose of 30 mg/kg on the day preceding scheduled sacrifice, and acted as a positive control group for micronuclei induction. At necropsy, femur was sampled for bone marrow micronucleus analysis. The mean values of the polychromatic/normochromatic erythrocytes (PE/NE) ratios in animals treated with the test item at 30, 100 or 300 mg/kg/day were not statistically significantly different from that of the vehicle control animals. The mean frequencies of micronucleated PE in the three test item treated groups were not found significantly different from that in the vehicle group. Therefore, under the experimental conditions of the study, Cocamidopropyl hydroxysultaine did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells up to 300 mg/kg administered daily for the whole dosing period (i.e., approximately 5 to 6 weeks).

 

Particular case of the numerical abnormalities seen in the Chromosome Aberration assay:

Experiment 1 1 2 2 2 2 1 1 2 2 2 2
Culture 1 2 1 2 1 2 1 2 1 2 1 2
Treatment time (h) 3 3 20 20 44 44 3 3 3 3 3 3
Harvest time (h) 20 20 20 20 44 44 20 20 20 20 44 44
S9 mix - - - - - - + + + + + +
Concentrations (µg/mL)                
0 1 0 0 1 0 0 0 0 0 0 0 0
9.4 - - - - - - - - 2 2 - -
18.8 - - - - - - - - 1 1 - -
37.5 - - - - - - - - 7 11 - -
39.06 - - - - - - 0 0 - - - -
75 - - 3 7 - - - - - - 3 6
78.13 0 0 - - - - 0 1 - - - -
150 - - 1 3 - -    
156.3 1 0 - - - - 0 2 - - - -
300 - - 2 3 7 10 - - - - - -
312.5 1 0 - - - - - - - - - -

As shown in the summary table above, increases in the numerical aberrations when compared to the vehicle controls were noted in the chromosome aberration assay, but:

  • with no consistency between cell cultures (only in the second experiment, not in the first, for the same treatment and harvest times with S9),
  • with no clear evidence of a dose-response relationship (between 75 and 300 µg/mL without S9 for 20 hours of treatment, harvesting at 20 hours; between 37.5 and 156.3 µg/mL with S9 for 3 hours of treatment, harvesting at 20 hours; lower number of numerical abberations at 75 µg/mL than at 37.5 µg/mL without S9 for 3 hours of treatment).

Significant numerical aberrations could be the indication of an aneugenic activity. A weight-of-evidence approach was applied also taking into consideration:

  • the negative results of the Mouse Lymphoma Assay (which is technically able to detect some aneugens),
  • the negative results of the in vivo micronucleus assay, in the bone marrow of rats continuously exposed for 5 to 6 weeks to the test substance (with signs of toxicity indicative of a systemic distribution of the substance and/or its metabolites or degradation products),
  • the absence in the OECD 422 study of histopathological changes, such as atrophy and/or necrosis, in fast-renewal tissues, such as bone marrow or digestive tract, indicative of an inhibitory effect on mitosis,

Based on all these elements, increased numerical aberrations in the chromosome aberration assay were considered toxicologically irrelevant.

 

Conclusion:

 

Based on the results of in vitro (Ames test, chromosome aberration test, mouse lymphoma assay) and in vivo (bone marrow micronucleus test) studies, Cocamidopropyl hydroxysultaine is considered to be devoid of mutagenic, clastogenic or aneugenic properties.

Justification for classification or non-classification

Based on the available experimental data, Cocamidopropyl hydroxysultaine did not induce gene mutations as clear negative results were obtained in the Ames and Mouse Lymphoma assays. The test substance did not induce structural chromosome aberration in vitro in human cultured lymphocytes. No relevant aneugenic activity was observed.

 

Therefore, Cocamidopropyl hydroxysultaine as a pure substance is considered to be devoid of mutagenic, clastogenic or aneugenic properties and does not need to be classified for genetic toxicity.