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REACH

1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-C12-14 acyl derivs., hydroxides, inner salts

EC number: 293-878-1 | CAS number: 91648-19-0

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A battery of in vitro assays have been conducted with the submission substance or with a read-across substance, which address all relevant genetic toxicity endpoints.

Link to relevant study records

Reference 1

Endpoint:: in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:: Type of genotoxicity: chromosome aberration
Type of information:: experimental study
Adequacy of study:: weight of evidence
Study period:: From 24 February to 21 November 2012
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:: no
Qualifier:: according to guideline
Guideline:: EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:: no
GLP compliance:: yes (incl. QA statement)
Remarks:: N° 2011/40
Type of assay:: other: in vitro mammalian 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.
: Key result
Species / strain:: lymphocytes: human
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: not applicable
Positive controls validity:: valid
Additional information on results:: 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.
Conclusions:: The study was negative for structural chromosome aberrations. However numerical chromosome aberrations were observed with and without metabolic activation. 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.
Executive summary:: In an in vitro chromosome aberration study, performed according to OECD 473 and in compliance with 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 concentrations 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/concentration (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 the metabolizing system.

Reference 2

Endpoint:: in vitro gene mutation study in mammalian cells
Remarks:: Type of genotoxicity: gene mutation
Type of information:: experimental study
Adequacy of study:: weight of evidence
Study period:: From 15 May to 14 December 2012
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:: no
Qualifier:: according to guideline
Guideline:: EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:: no
GLP compliance:: yes (incl. QA statement)
Remarks:: N° 2011/40
Type of assay:: other: 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.
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'.
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 OECD 476 and 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.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

21 July 2017 to 04 January 2018 (TBC)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test article name: Betadet SHR S
Chemical name: 1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-C12-14 acyl derivs., hydroxides, inner salts
CAS number: 91648-19-0
Batch number: 2657890104
Appearance: white powder
Storage conditions: 15-25°C, protected from light
Purity: 97.6%
Expiry date: 30 May 2018

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 fraction

Test concentrations with justification for top dose:

5000 µg/plate; highest cocnentraiton cited in OECD test guideline.

Vehicle / solvent:

Purified water (water for irrigation).

Untreated negative controls:

Negative solvent / vehicle controls:

yes

Remarks:

Purified water

True negative controls:

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

Betadet SHR S was tested for mutation (and toxicity) in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in two separate experiments, at a range of concentrations up to 5000 µg/plate, using triplicate plates without and with S-9 for test article, vehicle and positive controls. These platings were achieved by the following sequence of additions to molten agar at 45±1°C:
0.1 mL bacterial culture
0.1 mL of test article solution/vehicle control or 0.05 mL of positive control
0.5 mL 10% S-9 mix or buffer solution
followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1°C protected from light for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.

As the results of Mutation Experiment 1 were negative, treatments in the presence of S-9 in Mutation Experiment 2 included a pre-incubation step. Quantities of test article, vehicle control solution or positive control, bacteria and S-9 mix detailed above, were mixed together and incubated for 20 minutes at 37±1°C, with shaking, before the addition of 2 mL molten agar at 45±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.

The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response. Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as bubbles or splits in agar or a thinning of the background bacterial lawn affected the accuracy of the automated counter. Individual plate counts were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined. Control counts were compared with the laboratory’s historical control ranges. Data were considered acceptable if the vehicle control counts fell within the calculated historical control ranges and the positive control plate counts were comparable with the historical control ranges.

The assay was considered valid if all the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges
2. The positive control chemicals induced increases in revertant numbers of ≥1.5 fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control confirming discrimination between different strains, and an active S-9 preparation.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
2. Any observed response was reproducible under the same treatment conditions.

The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.

Statistics:

The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 µg/plate). However, adequate interpretation of biological relevance was of critical importance.

Species / strain:

S. typhimurium TA 98

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:

S. typhimurium 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:

S. typhimurium TA 1535

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:

S. typhimurium TA 1537

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:

S. typhimurium TA 102

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

Treatment (µg/plate)		Mean revertants/plate ± standard deviation
		-S9 mix		+S9 mix
		Exp 1	Exp 2	Exp 1	Exp 2
Strain TA98
Water	0	22.0 ± 2.0	20.0 ± 4.6	46.7 ± 1.5	39.0 ± 11.5
Betadet SHR S	5	32.7 ± 7.6	NT	39.3 ± 5.1	NT
	16	33.0 ± 7.0	NT	41.7 ± 11.0	NT
	50	29.7 ± 2.9	NT	39.0 ± 2.6	NT
	160	33.7 ± 3.8	21.0 ± 6.1	39.7 ± 11.5	37.7 ± 4.9
	300	NT	28.7 ± 11.0	NT	40.7 ± 9.0
	500	33.0 ± 7.2	NT	37.3 ± 5.1	NT
	625	NT	34.0 ± 1.7	NT	49.0 ± 1.5
	1250	NT	32.7 ± 2.1	NT	32.7 ± 3.8
	1600	37.3 ± 15.5	NT	55.0 ± 3.5	NT
	2500	NT	35.7 ± 15.9	NT	32.7 ± 2.1
	5000	17.0 ± 5.2	10.0 ± 4.4	33.7 ± 10.3	29.3 ± 6.1
Positive control		1352.7 ± 112.6	702.0 ± 44.8	386.3 ± 40.8	326.7 ± 20.7
Strain TA100
Water	0	125.7 ± 15.0	112.0 ± 21.4	125.0 ± 5.6	154.7 ± 18.6
Betadet SHR S	5	141.0 ± 19.1	NT	134.0 ± 7.0	NT
	16	148.3 ± 4.0	NT	134.7 ± 8.7	NT
	50	125.0 ± 6.2	108.3 ± 3.8	156.7 ± 7.1	154.0 ± 7.8
	160	144.7 ± 11.0	112.0 ± 2.6	138.7 ± 7.6	166.3 ± 13.1
	300	NT	121.0 ± 19.0	NT	169.0 ± 15.6
	500	129.7 ± 2.3	NT	152.7 ± 8.5	NT
	625	NT	91.3 ± 17.6	NT	149.7 ± 6.4
	1250	NT	29.3 ± 6.1	NT	117.3 ± 14.2
	1600	39.0 ± 12.1	NT	116.7 ± 22.2	NT
	2500	NT	23.3 ± 7.5	NT	42.0 ± 6.0
	5000	Toxic	4.3 ± 2.5	18.0 ± 14.7	10.7 ± 6.5
Positive control		1188.0 ± 116.9	831.3 ± 22.2	2690.7 ± 29.0	1235.7 ± 28.5
Strain TA1535
Water	0	11.3 ± 5.0	19.0 ± 1.7	19.0 ± 5.3	23.3 ± 1.2
Betadet SHR S	5	14.0 ± 2.0	NT	19.0 ± 2.0	NT
	16	16.7 ± 4.5	NT	17.0 ± 2.6	NT
	50	14.0 ± 1.7	20.7 ± 0.6	20.7 ± 5.0	NT
	160	13.7 ± 2.9	37.3 ± 9.2	23.0 ± 4.4	27.0 ± 4.6
	300	NT	30.3 ± 12.9	NT	27.7 ± 4.9
	500	20.7 ± 5.7	NT	25.7 ± 4.0	NT
	625	NT	34.0 ± 1.7	NT	26.7 ± 4.2
	1250	NT	12.7 ± 4.9	NT	26.0 ± 5.0
	1600	14.7 ± 1.2	NT	11.7 ± 4.9	NT
	2500	NT	8.3 ± 1.2	NT	15.7 ± 6.5
	5000	Toxic	8.7 ± 2.5	11.7 ± 3.1	9.7 ± 6.0
Positive control		832.3 ± 935	801.7 ± 24.9	199.7 ± 32.2	181.0 ± 33.8
Strain TA1537
Water	0	9.7 ± 0.6	12.0 ± 2.6	11.0 ± 0.0	11.3 ± 2.5
Betadet SHR S	5	10.0 ± 3.6	NT	13.3 ± 3.2	NT
	16	9.0 ± 3.6	NT	18.7 ± 4.0	NT
	50	10.7 ± 1.5	10.0 ± 3.5	14.3 ± 2.1	NT
	160	12.0 ± 4.4	10.3 ± 1.2	18.7 ± 5.8	11.0 ± 1.0
	300	NT	19.3 ± 7.6	NT	12.3 ± 5.7
	500	8.7 ± 3.2	NT	20.7 ± 0.6	NT
	625	NT	9.3 ± 3.1	NT	10.3 ± 5.5
	1250	NT	6.3 ± 4.5	NT	10.3 ± 1.5
	1600	13.7 ± 4.2	NT	16.7 ± 2.1	NT
	2500	NT	Toxic	NT	9.0 ± 3.0
	5000	Toxic	Toxic	15.7 ± 4.7	8.7 ± 2.5
Positive control		1061.3 ± 398.1	222.3 ± 45.5	220.0 ± 35.7	300.3 ± 55.9
Strain TA102
Water	0	290.0 ± 12.3	340.0 ± 4.4	234.0 ± 33.0	366.3 ± 11.0
Betadet SHR S	5	310.3 ± 7.6	NT	228.0 ± 13.0	NT
	16	290.7 ± 7.1	NT	253.0 ± 19.5	NT
	50	289.7 ±25.4	319.0 ± 18.4	258.0 ± 31.1	NT
	160	302.3±18.0	352.7 ± 9.3	253.0 ± 31.1	347.7 ± 26.3
	300	NT	325.0 ± 45.9	NT	329.7 ± 31.1
	500	260.7 ± 30.7	NT	249.7 ± 16.3	NT
	625	NT	327.7 ± 7.0	NT	286.3 ± 18.4
	1250	NT	234.3 ± 11.6	NT	268.7 ± 31.5
	1600	119.0 ± 13.0	NT	189.7 ± 8.0	NT
	2500	NT	110.3 ± 36.1	NT	186.7 ± 23.4
	5000	Toxic	Toxic	62.0 ± 14.4	58.0 ± 10.5
Positive control		1361.3 ± 60.0	688.7 ± 65.0	1228.3 ± 141.0	1238.7 ± 133.0

NT: Not tested

Conclusions:

It was concluded that Betadet SHR S did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

A GLP compliant bacterial mutation study was conducted with the submission substance (Betadet SHR S) in accordance with OECD Guideline 471 using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102. Betadet SHR S was tested at a range of concentrations, up to a maximum of 5000 µg/plate in the absence and in the presence of metabolic activation in two independent experiments. Vehicle and positive control treatments were also included. Each concentration was tested in triplicate. Plate incorporation methodology was employed in Experiment 1 and in the absence of metabolic activation in Experiment 2. Pre-incubation methodology was used for the Experiment 2 treatments in the presence of metabolic activation.

Toxicity ranging from a diminution of the background bacterial, with or without a concurrent reduction in the number of revertants, to a complete killing of the test bacteria. There were no increases in the number of revertants which were ≥1.5-fold (strain TA102), or ≥ 2 -fold (strains TA98 and TA100) or ≥ 3 -fold (strains TA1535 and TA1537) the concurrent vehicle controls. It is concluded that Betadet SHR S did not induce mutagenicity in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102 in the absence and in the presence of metabolic activation.

Reference 4

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:: weight of evidence
Study period:: From 24 February to 21 November 2012
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Justification for type of information:: 1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source substance, C8-C18 AAPHS, has the same functional groups and general composition as the target C12-14 substance. The main variable resides in the alkyl chain distribution.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source substance = C8-18 cocamidopropyl hydroxysultaine (EC 939-455-3).
Target substance = C12-14 cocamidopropyl hydroxysultaine (EC 293-878-1).

3. ANALOGUE APPROACH JUSTIFICATION
Cf. attached Read-Across Justification Document (§13 Assessment reports).

4. DATA MATRIX
Cf. attached Read-Across Justification Document (§13 Assessment reports).
Reason / purpose for cross-reference:: read-across source
Reason / purpose for cross-reference:: read-across: supporting information
Qualifier:: according to guideline
Guideline:: OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:: no
Qualifier:: according to guideline
Guideline:: EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:: no
GLP compliance:: yes (incl. QA statement)
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.
: Key result
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'.
Conclusions:: The study was negative for structural chromosome aberrations. However numerical chromosome aberrations were observed with and without metabolic activation.

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.
Executive summary:: In an in vitro chromosome aberration study, performed according to OECD 473 and in compliance with 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 concentrations 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/concentration (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 the metabolizing system.

Reference 5

Endpoint:: in vitro gene mutation study in mammalian cells
Remarks:: Type of genotoxicity: gene mutation
Type of information:: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:: weight of evidence
Study period:: From 15 May to 14 December 2012
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Justification for type of information:: 1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source substance, C8-C18 AAPHS, has the same functional groups and general composition as the target C12-14 substance. The main variable resides in the alkyl chain distribution.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source substance = C8-18 cocamidopropyl hydroxysultaine (EC 939-455-3).
Target substance = C12-14 cocamidopropyl hydroxysultaine (EC 293-878-1).

3. ANALOGUE APPROACH JUSTIFICATION
Cf. attached Read-Across Justification Document (§13 Assessment reports).

4. DATA MATRIX
Cf. attached Read-Across Justification Document (§13 Assessment reports).
Reason / purpose for cross-reference:: read-across source
Reason / purpose for cross-reference:: read-across: supporting information
Qualifier:: according to guideline
Guideline:: OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:: no
Qualifier:: according to guideline
Guideline:: EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:: no
GLP compliance:: yes (incl. QA statement)
Type of assay:: in vitro mammalian cell gene mutation tests using the thymidine kinase gene
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
Cytotoxicity / choice of top concentrations:: cytotoxicity
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.
Conclusions:: The study was negative with and without metabolic activation 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 OECD 476 and 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.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Although not required, a bone marrow rat micronucleus study is also available for the read-across substance, which was conducted as part of the repeated dose study. The study showed no evidence of aneuploidy.

Link to relevant study records

Referenceopen all close all

Reference 1

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:

other: Dedicated phase of a GLP and OECD test guideline compliant repeated-dose toxicity study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

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. QA statement)

Type of assay:

other: in vivo micronucleus assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

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
Group		(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:

Interpretation of results (migrated information): negative
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 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).

Reference 2

Endpoint:: in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:: key study
Study period:: April - December 2012
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: other: Dedicated phase of a GLP and OECD test guideline compliant repeated-dose toxicity study
Justification for type of information:: 1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source substance, C8-C18 AAPHS, has the same functional groups and general composition as the target C12-14 substance. The main variable resides in the alkyl chain distribution.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source substance = C8-18 cocamidopropyl hydroxysultaine (EC 939-455-3).
Target substance = C12-14 cocamidopropyl hydroxysultaine (EC 293-878-1).

3. ANALOGUE APPROACH JUSTIFICATION
Read-Across Justification Document (§13 Assessment reports).

4. DATA MATRIX
Read-Across Justification Document (§13 Assessment reports).
Reason / purpose for cross-reference:: read-across source
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. QA statement)
Type of assay:: other: in vivo micronucleus assay
Species:: rat
Strain:: Sprague-Dawley
Sex:: male/female
Details on test animals or test system 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
Dose / conc.:: 30 mg/kg bw/day (nominal)
Dose / conc.:: 100 mg/kg bw/day (nominal)
Dose / conc.:: 300 mg/kg bw/day (nominal)
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)
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 conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

There is no indication for mutagenicity based on a negative Ames test with the submission substance, negative studies of mammalian cell mutation and mammalian cell clastogenicity with a read-across substance and a negative mouse bone marrow micronucleus assay with the read-across substance. Classification for mutagenicity according to CLP is not required.

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Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

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	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

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	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

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	1	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

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

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

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

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

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

Registration Dossier

Registration Dossier

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Diss Factsheets

1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-C12-14 acyl derivs., hydroxides, inner salts

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Link to relevant study records

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Endpoint conclusion

Genetic toxicity in vivo

Description of key information

Link to relevant study records

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Endpoint conclusion

Additional information

Justification for classification or non-classification

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