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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- study according to OECD guideline 471, GLP, RL1, not mutagenic in the bacterial reverse mutation assay

- study according to OECD guideline 471, GL, RL1, not mutagenic in the bacterial reverse mutation assay, supporting data

- study according to OECD guideline 473, GL, RL1, not clastogenic in the chromosome abberation assay, read-across

- study according to OECD guideline 476, GL, RL1, not mutagenic in the gene mutation in mammalin cells assay, read-across

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-05-05 to 2017-06-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
his-
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
DNA polymerase A deficient
Metabolic activation:
with and without
Metabolic activation system:
S9 liver Mix
Test concentrations with justification for top dose:
31.6, 100, 316, 1000, 3160 and 5000 µg due to absence of cytotoxicity in a preliminary test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: purified water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Six concentrations ranging from 31.6 to 5000 µg alpha methyl glucoside/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.
- Cell density at seeding (if applicable): E008-E009 cells/mL

DURATION
- Preincubation period: 20 min
- Exposure duration: 48h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: Cytotoxicity is evidenced by a reduction in the number of spontaneous revertants by at least 50%, a clearing or diminution of the background lawn or by the degree of survival of the treated cultures.


Rationale for test conditions:
as recommended in the guideline
Evaluation criteria:
The results of the negative and positive control cultures should be within the range of the historical data generated by the testing laboratory. The range of spontaneous reversion frequencies per plate is based on Kirkland (1990):
TA98: 20 - 60
TA100: 100 - 200
TA102: 240 - 320
TA1535: 10 - 35
TA1537: 3 - 20

Where concurrent negative or positive control data fall outside the range, they may be acceptable and considered for the inclusion into the historical control distribution as long as these data are not extreme outliers.

A test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
Or
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient may be applied.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Biological relevance of the results should be considered first.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Historical control data:

positive controls

Strain S9-Mix

TA98

TA100

TA102

TA1535

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

 

2-nitro-fluorene

Benzo[a]

pyrene

sodium azide

2-amino-anthracene

Mito-mycin C

Benzo[a]

pyrene

sodium azide

2-amino-anthracene

9-amino-acidine

Benzo[a]

pyrene

Mean

151.2

150.4

952.9

948.8

1029.7

1024.3

135.6

135.4

76.7

77.6

SD

27.9

28.8

99.7

103.7

97.1

97.1

28.8

28.4

26.5

26.4

Min

91

96

677

703

781

781

51

49

28

31

Max

293

291

1213

1195

1637

1366

266

270

185

184

negative controls:

Strain S9-Mix

TA98

TA100

TA102

TA1535

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean

30.3

32.1

145.1

145.2

277.3

279.0

19.7

19.9

6.7

6.7

SD

5.6

5.9

18.4

19.2

16.5

17.2

4.4

4.6

1.7

1.8

Min

20

20

107

101

245

203

10

10

2

3

Max

49

49

195

198

323

324

34

36

10

10

Conclusions:
In the present test conducted according to OECD guideline 471, the Salmonella typhimurium strains TA 98, 100, 102, 1535, 1537 were incubated with the following concentrations of alpha methyl glucoside: 31.6, 100, 316, 1000, 3160 and 5000 µg /plate. No increases in revertant colonies were observed no cytotoxicity occurred. The test item is therefore considered non mutagenic under the present conditions.
Executive summary:

In a reverse gene mutation assay in bacteria (OECD guideline 471), strains TA 98, TA 100, TA 102, TA 1535 and TA 1537of S. typhimurium were exposed to alpha methyl glucoside (100% a.i.) in highly purified water at concentrations of (31.6, 100, 316, 1000, 3160 and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method and the preincubation method

Alpha methyl glucoside was tested up to limit concentration (5000 µg/plate)There was no increase in the number of revertants regardless of strain.  The positive controls induced the appropriate responses in the corresponding strains.  There was no evidence of induced mutant colonies over background.

 

This study is classified asacceptable.  This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Remarks:
used only for justification of read-across for other endpoints
Adequacy of study:
supporting study
Study period:
2008-07-15 to 2008-07-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
June 8, 2000
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
The Salmonella typhimurium strains used in this study were TA1535, TA1537, TA98 and TA100. The Escherichia coli strain used was WP2 uvrA. The strains TA1537 and TA98 are capable of detecting frameshift mutagens, strains TA1535, TA100 and E. coli wP2 uvrA are capable of detecting base-pair substitution mutagens.
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: tryptophane auxotroph
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine auxotroph
Metabolic activation:
with and without
Metabolic activation system:
S9-mix induced by combination of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw), rat liver micosomal enzymes prepared in house from adult male Wistar rats, which were obtained from Charles River, Sulzfeld Germany
Test concentrations with justification for top dose:
Dose-range finding test:
3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate
Main test: 33, 100, 333, 1000, 3330 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
- Stability of test substance in vehicle: unknown
- Solubility in vehicle: not indicated
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
0.1 mL dimethyl sulfoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other:
Details on test system and experimental conditions:
METHOD OF APPLICATION:in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): Salmonella typhimurium stains: histidine; E. coli stain: tryptophane

NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: reduction of the background lawn or reduction of the spontaneous reversion rate

DOSE RANGE FINDING TEST:
- selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix.- Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate were tested in triplicate.
- The highest concentration of test substance in the subsequent mutation assay was the level at which the test substance exhibited limited solubility.
- Precipitation of test substance on the plates was observed at the start of the incubation period at concentrations of 1000 µg/plate and upwards and at 3330 µg/plate and above at the end of the incubation period.
- Toxicity: to determine the toxicity of the test substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
Evaluation criteria:
No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
The preceding crlteria were not absolute and other modifying factors might enter into the final evaluation decision.
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: was observed at the start of the incubation period at concentrations of 1000 and 3330 µg/plate and at the top dose of 3330 µg/plate at the end of the incubation period.


RANGE-FINDING/SCREENING STUDIES:
Toxicity: no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Mutagenicity: no increase in the number of revertants was observed upon treatment with test substance under all conditions tested.

COMPARISON WITH HISTORICAL CONTROL DATA:
In this study, the strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
There was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
Conclusions:
Interpretation of results
negative

Based on the results of this study it is concluded that Isostearic acid, esters with methyl α-D-glucoside is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2 uvr A) were exposed to Isostearic acid, esters with methyl α-D-glucoside, at concentrations of 33, 100, 333, 1000, and 3330 µg/plate in the presence and absence of mammalian metabolic activation (plate incorporation method). Isostearic acid, esters with methyl α-D-glucoside did not induce a significant dose-related increase in the number of revertant colonies in each of the five tester strains, both in the absence and presence of mammalian metabolic activation. These results were confirmed in an independently repeated experiment. No cytotoxic effects of the test substance were observed. Precipitation was observed at concentrations of 1000 and 3330 µg/plate. The positive controls induced the appropriate responses in the corresponding strains and metabolic activation was confirmed. There was no evidence of induced mutant colonies over background.    

Based on the results of this study it is concluded that Isostearic acid, esters with methyl α-D-glucoside is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.  

This study is classified as acceptable. It satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-07-16 to 2008-09-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
published June 8, 2000
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
Whole blood (0.4 mL) treated with heparin was added to culture medium. Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added.
Blood was collected from healthy adult, non-smoking, male volunteers. The Average Generation Time (AGT) 01 the cells and the age of the donor at the time the AGT was determined (December 2007):
Dose range finding study: age 42, AGT =14.5 h
First cytogenetic assay: age 29, AGT =16.3 h
Second cytogenetic assay: age 31, AGT =14.5 h
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fractions obtained from livers of adult male Wistar rats induced with Phenobarbital (80 mg/kg bw) and beta-Naphtoflavone (100 mg/kg bw) in corn oil.
Test concentrations with justification for top dose:
Dose range finding test:
- at 3 h exposure time: 3, 10, 33, 100, 333 µg/mL culture medium with and without S9-mix;
- at 24 h and 48 h exposure time: 3, 10, 33, 100, 333, 1000 µg/mL without S9-mix.
First cytogenetic assay:
- without and with S9-mix: 33, 100 and 333 µg/mL culture medium (3 h exposure time, 24 h fixation time).
Second cytogenetic assay:
- without S9-mix: 100, 300, 400, 500, 600, 700 and 800 µg/mL culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time)
- with S9-mix: 33, 100 and 300 µg/mL culture medium (3 h exposure time, 48 h fixation time).
Scoring of chromosome aberrations:
- without S9-mix: 100, 500 and 600 µg/mL culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time)
- with S9-mix: 33, 100 and 300 µg/mL culture medium (3 h exposure time, 48 h fixation time).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
- Stability of test substance in vehicle: unknown
- Solubilily in vehicle: not indicated
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
indirect acting mutagen, requiring metabolic activation, at a final concentration of 10 µg/mL for a 3 h exposure period (24 h fixation time)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
direct acting mutagen at a final concentration of 0.5 µg/mLfor a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

Cell culture:
- Blood samples
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection Iymphocyte cultures were started.
- Culture medium
Culture medium consisted of RPMI 1640 medium (Invitrogen Corporation), supplemented with 20% (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum (Invitrogen Corporation), L-glutamine (2 mM) (Invitrogen Corporation), penicillin/streptomycin (50 U/mLand 50 µg/mL respectively) (Invitrogen Corporation) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).
- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mLor 4.8 ml culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL(9 mg/ml) phytohaemagglutinin (Remel, Europe Ud., United Kingdom) was added.

DURATION
- Exposure duration: 3, 24, 48 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 , 48 h


SPINDLE INHIBITOR (cytogenetic assays):
- During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindel inhibitor colchicine (0.5 µg/ml medium) (Acros Organics, Belgium)

NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS SCORED: approx. 200


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells.
- At least three analysable concentrations were used for scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analysed from those cultures with an inhibition of the mitotic index of about 50% or above whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control.

OTHER EXAMINATIONS:
- Determination of polyploidy: the number of polyploid cells were scored in presence and absence of S9-mix.
- Determination of endoreplication: the number of endoreduplicated chromosomes were observed in presence and absence of S9-mix


Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p< 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.
Species / strain:
other: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 600 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:

DOSE-FINDING STUDY:
- At a concentration of 333 µg/mL the test substance precipitated in the culture medium.

FIRST CYTOGENETIC ASSAY
- Both in the absence and presence of S9-mix, the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberratlons
- Both in the absence and presence of S9-mix, the test substance did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

SECOND CYTOGENETIC ASSAY
- Both in the absence and presence ot S9-mix, the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations:
- Both in the absence and presence of S9-mix, the test substance did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.
COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range:
- The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range .

POSITIVE CONTROL CHEMICALS:
The positive control ehemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells.
Remarks on result:
other: all strains/cell types tested

Chromosome aberrations:

- Both in the absence and presence of S9-mix the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

- No effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Conclusions:
Interpretation of results
negative

Finally, it is concluded that this test is valid and that Isostearic acid, esters with methyl α-D-glucoside is not clastogenic in human Iymphocytes under the experimental conditions described in this report.
Executive summary:

In a mammalian cell cytogenetics assay, peripheral human lymphocyte cultures were exposed to Isostearic acid, esters with methyl α-D-glucoside (80% Methyl Glucoside Isostearate Esters (mainly Di-), 16% Isostearic Acid, 4% Methyl glucoside), in dimethyl sulfoxide at concentrations of 0, 33, 100 and 333 µg/mL culture medium (3 h exposure time , 24 h fixation time with and without metabolic activation in the first cytogenetic assay. In the second cytotgenetic assay concentrations of 0, 100, 300, 400, 500, 600, 700 and 800 µg/mL (24 h and 48 h exposure time and fixation time) without S9-mix and 0, 33, 100 and 300 µg/ml (3 h exposure time, 48 h fixation time) with S9-mix were tested respectively.

Isostearic acid, esters with methyl α-D-glucoside was tested up to cytotoxic and precipitating concentration. In the first cytogenetic assay, the test substance was tested up to 333 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Isostearic acid, esters with methyl α-D-glucoside precipitated in the culture medium at this dose level. In the second cytogenetic assay, Isostearic acid, esters with methyl α-D-glucoside was tested up to 800 µg/mL for a 24 h and 48 h continuous exposure time with a 24 hand 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at 600 µg/ml. In the presence of S9-mix Isostearic acid, esters with methyl α-D-glucoside was tested up to 300 µg/mL for a 3 h exposure time with a 48 h fixation time. The test substancce precipitated in the culture medium at this dose level.

Isostearic acid, esters with methyl α-D-glucoside did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

No biologically relevant effects of Isostearic acid, esters with methyl α-D-glucoside on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that Isostearic acid, esters with methyl α-D-glucoside is not clastogenic in human Iymphocytes under the experimental conditions described in this report.

Positive controls induced the appropriate response. There was no evidence of Chromosome aberration induced over background. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range.

This study is classified as acceptable. This study satisfies the requirement for OECD Test Guideline 473.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source substance is constituted of the target substance (alpha methyl glucoside) esterified to Isostearic acid and therefore exhibit partially similar toxicological behaviour due to similarity in their structure. During synthesis of Isostearic acid esters with methyl-α-D-glucose the four OH-groups of the methyl glucose are partially esterified with isostearate groups. The molar ratio of methyl glucose and isostearic acid is chosen close to 1:2 which means that the products may be a mixture of non reacted methyl glucose and its 4 monostearates, 6 distearates, 4 tristearates and 1 tetrastearate isomers. These numbers will increase accordingly, if the cyclic form of glucose is in equilibrium with its linear form and the other anomer (hemiacetalic carbon). A possible hydrolysis results in alpha methyl glucoside and excess Isostearic acid.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance alpha methyl glucoside is a monoconstituent substance composed of a methyl-group which is o-glycosidic bound to the α-D-glucose core structure.

The source substance Isostearic acid esters with Methyl-α-D-glucose is an UVCB with a composition of 80% alpha methyl glucoside isostearate esters (mainly di-), 16% isostearic acid and 4% alpha methyl glucoside .

3. ANALOGUE APPROACH JUSTIFICATION
For detailed information please refer to the attached justification for read-across.

4. DATA MATRIX
For detailed information please refer to the attached justification for read-across.
Reason / purpose:
read-across source
Key result
Species / strain:
other: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>=600µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
DOSE-FINDING STUDY:
- At a concentration of 333 µg/ml the test substance precipitated in the culture medium.

FIRST CYTOGENETIC ASSAY
- Both in the absence and presence of S9-mix, the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberratlons
- Both in the absence and presence of S9-mix, the test substance did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

SECOND CYTOGENETIC ASSAY
- Both in the absence and presence ot s9-mix, the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations:
- Both in the absence and presence of ss-mb, the test substance did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.
COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range:
- The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range .

POSITIVE CONTROL CHEMICALS:
The positive control ehemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells.
Remarks on result:
other: all strains/cell types tested

Chromosome aberrations:

- Both in the absence and presence of S9-mix the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

- No effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Conclusions:
It is concluded that this test is valid and that Isostearic acid, esters with methyl α-D-glucoside is not clastogenic in human Iymphocytes under the experimental conditions described in this report. Based on the structural similarities between Isostearic acid, esters with methyl α-D-glucoside and alpha methyl glucoside the target substance is considered to be non-clastogenic.
Executive summary:

In a mammalian cell cytogenetics assay, peripheral human lymphocyte cultures were exposed to Isostearic acid, esterswith methyl α-D-glucoside (80% Methyl Glucoside Isostearate Esters (mainly Di-), 16% Isostearic Acid, 4% Methyl glucoside), in dimethyl sulfoxide at concentrations of 0, 33, 100 and 333 µg/mL culture medium (3 h exposure time, 24 h fixation time with and without metabolic activation in the first cytogenetic assay. In the second cytotgenetic assay concentrations of 0, 100, 300, 400, 500, 600, 700 and 800 µg/mL (24 h and 48 h exposure time and fixation time) without S9-mix and 0, 33, 100 and 300 µg/mL (3 h exposure time, 48 h fixation time) with S9-mix were tested respectively.

Isostearic acid, esters with methyl α-D-glucosidewas testedup to cytotoxic and precipitating concentration.In the first cytogenetic assay, the test substance was tested up to 333 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Isostearic acid, esters with methyl α-D-glucoside precipitated in the culture medium at this dose level. In the second cytogenetic assay,Isostearic acid, esters with methyl α-D-glucoside was tested up to 800 µg/mL for a 24 h and 48 h continuous exposure time with a 24 hand 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at 600 µg/mL. In the presence of S9-mixIsostearic acid, esters with methyl α-D-glucoside was tested up to 300 µg/mL for a 3 h exposure time with a 48 h fixation time. The test substancce precipitated in the culture medium at this dose level.

Isostearic acid, esters with methyl α-D-glucoside did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

No biologically relevant effects of Isostearic acid, esters with methyl α-D-glucoside on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that Isostearic acid, esters with methyl α-D-glucoside is not clastogenic in human Iymphocytes under the experimental conditions described in this report.

Positive controls induced the appropriate response.There was no evidence of Chromosome aberration induced over background.The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range.

This study is classified as acceptable. This study satisfies the requirement for OECD Test Guideline 473.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-09-17 to 2008-11-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
31 May 2008
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
L5178Y mouse lymphoma cells are used because they are sensitive indicators of mutagenic activity of a broad range of chemical classes. The TK mutational system is able to detect base pair alterations, frame shift mutations and small deletions, and clastogenic effects.
Cells deficient in thymidine kinase (TK), due to the forward mutation (TK+/- to TK-/-) are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT). TK deficient cells can not incorporate the analogue into its phosphorylated derivative (nucleotide); the nucleotides needed
for cellular metabolism are obtained solely from de novo synthesis. In the presence of TK, TFT is converted into nucleotides, which are lethal to the cells. Thus, cells, which will survive in culture medium containing TFT, are mutated, either spontaneously or by the action of the test substance, giving rise to a TK deficient phenotype. Furthermore, by applying the TFT-selection procedure it is possible to discriminate between the two different classes of TFT-resistant mutants (small and large colonies). The large colonies are believed to be the result of mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. The small colonies are believed to be the result of chromosomal damage to the TK and adjacent genes.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 Hepes buffered medium (Dutch modification), supplemented with 10 % (v/v) heat inactivated horse serum (=R10 medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:
with and without
Metabolic activation system:
S9 fractions obtained from livers of male Wistar rats which were induced with Phenobarbital (80 mg/kg bw) and ß-Naphtoflavone (100 mg/kg bw) in corn oil. Conc. S9-fraction in exposure medium: 8% (v/v), in dose range finding test + 1. exp.: 12 % (v/v)
Test concentrations with justification for top dose:
The selection of the concentrations was based on data from a dose range finding test.
Dose range finding test: 0, 3, 10, 33, 100, 333, 500 µg/mL
Experiment 1:
- without metabolic activation: 0, 1, 3, 10, 33, 100, 150, 180, 200, 220, 240, 280, 300 µg/mL
- with 8 % (v/v) metabolic activation: 0, 1, 3, 10, 33, 100, 200, 250, 300, 350, 400, 425, 500 µg/mL
Experiment 2:
- without metabolic activation: 0, 10, 25, 33, 50, 100, 115, 150, 175, 180, 200, 210, 220, 240, 260 µg/mL
- with 12 % (v/v) metabolic activation: 0, 1, 3, 10, 33, 100, 200, 250, 300, 350, 375 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- Stability of test substance in vehicle: unknown
- Solubilily in vehicle: not indicated
- The final concentration of the solvent in the exposure medium was 0.8% (v/v).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24 h
- Expression time (cells in growth medium): 2 days
- Fixation time (start of exposure up to fixation or harvest of cells): 11 or 12 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 1


NUMBER OF CELLS EVALUATED: per culture 8E+06 cells (E+06 c/mL for 3 hours treatment) or 5E+06 cells (1.25E+05 c/mL for 24 hours treatment) were used


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


Evaluation criteria:
- A test substance is considered positive (mutagenic) in the mutation assay if:
a) It induces a MF of more than MF(controls) + 126 in a dose-dependent manner; or
b) In case a positive result will be repeated, the positive response should be reproducible in at least one repeated experiment.
An observed increase should be biologically relevant and will be compared with the historical control data range.
- A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reach a mutation frequency of MF (controls) + 126.
b) The results are confirmed in an independently repeated test.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 130 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
3 hour treatment: In the absence of S9-mix, no toxicity in the relative suspension growth was observed up to concentrations of 100 µg/mL compared to the relative suspension growth of the solvent control. In the presence of S9-mix, the relative suspension growth was 5% at the test substance concentration of 500 µg/mL compared to the relative suspension growth of the solvent control.

24 hour treatment without metabolic activation: No toxicity in the relative suspension growth was observed up to concentrations of 100 µg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test substance concentrations of 333 µg/mL and above. The dose level of 10 µg/ml showed an unexplainable low relative suspension growth of 33%. However, since no dose relationship was observed, the low RSG is not considered to be caused by toxicity of the test substance.

Experiment 1:
- In the absence of S9-mix, the relative total growth of the highest test substance concentration (240 µg/mL) was reduced by 88% compared to the total growth of the solvent controls.
- In the presence of S9-mix, the relative total growth of the highest test substance concentration (350 µg/mL) was reduced by 86% compared to the total growth of the solvent controls.
- Evaluation of the mutagenicity: no significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the cultures treated with the test substance were comparable to the numbers of small and large colonies of the solvent controls.

Experiment 2:
- A concentration of 130 µg/mL the test substance already precipitated in the exposure medium. Isostearic acid, esters with methyl α-D-glucoside was tested beyond the limit of the solubility to obtain adequate mutagenicity data.
- Evaluation of toxicity: in the absence of S9-mix, no dose level with a cell survival of 10-20% was reached; this part of the experiment was repeated with the following dose range: 1, 25, 50, 75,100, 150, 175, 200, 210, 220, 230 and 240 µg/mL. In this first repeat experiment (2A) the dose levels of 150 to 240 µg/ml were too toxic for further testing. Since too many dose levels showed severe toxicity, this part of the experiment was again repeated with the following dose range: 1, 10, 25, 50, 75, 100, 115, 130, 150, 165, 180, 200, 210, 220, 230 and 240 µg/mL. In the second repeat experiment (2B), the dose levels of 1 to 165 µg/mL showed no cytotoxicity. Therefore, the dose levels of 1, 75, 130 and 150 µg/mL were not regarded relevant for mutation frequency measurement. The dose levels of 220 to 240 µg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
In the presence of S9-mix, the dose levels of 200 to 350 µg/mL showed similar cell growth delay, therefore the dose levels of 250 and 350 µg/mL were not regarded relevant for mutation frequency measurement.
- The dose levels selected to measure mutation frequencies at the TK-Iocus were:
Without S9-mix: 10, 25, 50, 100, 115, 180,200 and 210 µg/mL exposure medium.
With S9-mix: 1, 3, 10, 33, 100, 200, 300 and 375 µg/mL exposure medium.
- In the absence of S9-mix, the relative total growth of the highest test substance was reduced by 81% compared to the total growth of the solvent controls.
- In the presence of S9-mix, the relative total growth at the highest test substance concentration was reduced by 59% compared to the total growth of the solvent controls.
- Evaluation of mutagenicity: no significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the Isostearic acid, esters with methyl α-D-glucoside treated cultures were comparable to the numbers of small and large colonies of the solvent controls.


COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range. Although the mutation frequency of one of the solvent control cultures in the first experiment observed in the absence of S9-mix was just above the historical control data range, the observed mutation frequency of this solvent control culture was within the acceptability criteria of this assay.

Solubility:

The test substance precipitated in the exposure medium at concentrations of 333 µg/mL and above. Isostearic acid, esters with methyl α-D-glucoside was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 500 µg/mL.

Conclusions:
Interpretation of results
negative

In conclusion, Isostearic acid, esters with methyl α-D-glucoside is not mutagenic in the TK mutation test system under the experimental conditions described in this report.
Executive summary:

In a mammalian cell gene mutation assay, mouse lymphoma L5178Y cells cultured in vitro were exposed to Isostearic acid, esters with methyl α-D-glucoside (100% UVCB-Substance (80% Methyl Glucoside Isostearate Esters (mainly Di-), 16% Isostearic Acid, 4% Methyl Glucoside (not soluble in Olive Oil) in DMSO. The test was performed in two independent experiments in the absence and presence of S9-mix.

Experiment 1: 0, 1, 3, 10, 33, 100, 150, 180, 200, 220, 240, 280, 300 µg/mL in absence of mammalian metabolic activation and 0, 1, 3, 10, 33, 100, 200, 250, 300, 350, 400, 425, 500 µg/mL with 8 % (v/v) metabolic activation (experiment 1).

Experiment 2: 0, 10, 25, 33, 50, 100, 115, 150, 175, 180, 200, 210, 220, 240, 260 µg/mL without metabolic activation and 0, 1, 3, 10, 33, 100, 200, 250, 300, 350, 375 µg/mL with 12 % (v/v) metabolic activation.

Isostearic acid, esters with methyl α-D-glucoside was tested beyond the limit of the solubility to obtain adequate mutagenicity data.

In the first segment of experiment 2, Isostearic acid, esters with methyl α-D-glucoside was tested up to concentrations of 240 µg/mL in the absence of 8% (v/v) S9-mix and up to concentrations of 350 µg/ml in presence of 8% (v/v) S9-mix with an incubation time of 3 hours. Isostearic acid, esters with methyl α-D-glucoside was tested up to cytotoxic levels of 12 and 14% in the absence and presence of S9-mix, respectively.

In the second segment of experiment 2, Isostearic acid, esters with methyl α-D-glucoside was tested up to concentrations of 210 µg/mL in the absence of 12% (v/v) S9-mix and up to concentrations of 375 µg/mL in the presence of 12 (v/v) S9-mix. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. Isostearic acid, esters with methyl α-D-glucoside was tested up to cytotoxic levels of 19 and 41% in the absence and presence of S9-mix, respectively.

In the absence of S9-mix, Isostearic acid, esters with methyl α-D-glucoside did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, Isostearic acid, esters with methyl α-D-glucoside did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9-mix for metabolic activation.

Isostearic acid, esters with methyl α-D-glucoside is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

The positive controls did induce the appropriate response. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity data.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source substance is constituted of the target substance (alpha methyl glucoside) esterified to Isostearic acid and therefore exhibit partially similar toxicological behaviour due to similarity in their structure. During synthesis of Isostearic acid esters with methyl-α-D-glucose the four OH-groups of the methyl glucose are partially esterified with isostearate groups. The molar ratio of methyl glucose and isostearic acid is chosen close to 1:2 which means that the products may be a mixture of non reacted methyl glucose and its 4 monostearates, 6 distearates, 4 tristearates and 1 tetrastearate isomers. These numbers will increase accordingly, if the cyclic form of glucose is in equilibrium with its linear form and the other anomer (hemiacetalic carbon). A possible hydrolysis results in alpha methyl glucoside and excess Isostearic acid.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance alpha methyl glucoside is a monoconstituent substance composed of a methyl-group which is o-glycosidic bound to the α-D-glucose core structure.

The source substance Isostearic acid esters with Methyl-α-D-glucose is an UVCB with a composition of 80% alpha methyl glucoside isostearate esters (mainly di-), 16% isostearic acid and 4% alpha methyl glucoside.


3. ANALOGUE APPROACH JUSTIFICATION
For detailed information please refer to the attached justification for read-across.

4. DATA MATRIX
For detailed information please refer to the attached justification for read-across.
Reason / purpose:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 130 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
3 hour treatment: In the absence of S9-mix, no toxicity in the relative suspension growth was observed up to concentrations of 100 µg/mL compared to the relative suspension growth of the solvent control. In the presence of S9-mix, the relative suspension growth was 5% at the test substance concentration of 500 µg/mL compared to the relative suspension growth of the solvent control.

24 hour treatment without metabolic activation: No toxicity in the relative suspension growth was observed up to concentrations of 100 µg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test substance concentrations of 333 µg/mL and above. The dose level of 10 µg/mL showed an unexplainable low relative suspension growth of 33%. However, since no dose relationship was observed, the low RSG is not considered to be caused by toxicity of the test substance.

Experiment 1:
- In the absence of S9-mix, the relative total growth of the highest test substance concentration (240 µg/mL) was reduced by 88% compared to the total growth of the solvent controls.
- In the presence of S9-mix, the relative total growth of the highest test substance concentration (350 µg/mL) was reduced by 86% compared to the total growth of the solvent controls.
- Evaluation of the mutagenicity: no significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the cultures treated with the test substance were comparable to the numbers of small and large colonies of the solvent controls.

Experiment 2:
- A concentration of 130 µg/mL the test substance already precipitated in the exposure medium. Isostearic acid, esters with methyl α-D-glucoside was tested beyond the limit of the solubility to obtain adequate mutagenicity data.
- Evaluation of toxicity: in the absence of S9-mix, no dose level with a cell survival of 10-20% was reached; this part of the experiment was repeated with the following dose range: 1, 25, 50, 75,100, 150, 175, 200, 210, 220, 230 and 240 µg/mL. In this first repeat experiment (2A) the dose levels of 150 to 240 µg/mL were too toxic for further testing. Since too many dose levels showed severe toxicity, this part of the experiment was again repeated with the following dose range: 1, 10, 25, 50, 75, 100, 115, 130, 150, 165, 180, 200, 210, 220, 230 and 240 µg/mL. In the second repeat experiment (2B), the dose levels of 1 to 165 µg/mL showed no cytotoxicity. Therefore, the dose levels of 1, 75, 130 and 150 µg/mL were not regarded relevant for mutation frequency measurement. The dose levels of 220 to 240 µg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
In the presence of S9-mix, the dose levels of 200 to 350 µg/mL showed similar cell growth delay, therefore the dose levels of 250 and 350 µg/mL were not regarded relevant for mutation frequency measurement.
- The dose levels selected to measure mutation frequencies at the TK-Iocus were:
Without S9-mix: 10, 25, 50, 100, 115, 180,200 and 210 µg/mL exposure medium.
With S9-mix: 1, 3, 10, 33, 100, 200, 300 and 375 µg/mL exposure medium.
- In the absence of S9-mix, the relative total growth of the highest test substance was reduced by 81% compared to the total growth of the solvent controls.
- In the presence of S9-mix, the relative total growth at the highest test substance concentration was reduced by 59% compared to the total growth of the solvent controls.
- Evaluation of mutagenicity: no significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the Isostearic acid, esters with methyl α-D-glucoside treated cultures were comparable to the numbers of small and large colonies of the solvent controls.


COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range. Although the mutation frequency of one of the solvent control cultures in the first experiment observed in the absence of S9-mix was just above the historical control data range, the observed mutation frequency of this solvent control culture was within the acceptability criteria of this assay.
Remarks on result:
other:
Remarks:
all strains/cell types tested

Solubility:

The test substance precipitated in the exposure medium at concentrations of 333 µg/mL and above.Isostearic acid, esterswith methyl α-D-glucosidewas tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 500 µg/mL.

Conclusions:
In conclusion, Isostearic acid, esters with methyl α-D-glucoside is not mutagenic in the TK mutation test system under the experimental conditions described in this report. Based on the structural similarities between Isostearic acid, esters with methyl α-D-glucoside and alpha methyl glucoside the target substance is considered to be non-mutagenic in the TK mutation test system under described experimental conditions.
Executive summary:

In a mammalian cell gene mutation assay, mouse lymphoma L5178Y cells cultured in vitro were exposed to Isostearic acid, esters with methyl α-D-glucoside(100% UVCB-Substance (80% Methyl Glucoside Isostearate Esters (mainly Di-), 16% Isostearic Acid, 4% Methyl Glucoside (not soluble in Olive Oil) in DMSO. The test was performed in two independent experiments in the absence and presence of S9-mix.

Experiment 1:0, 1, 3, 10, 33, 100, 150, 180, 200, 220, 240, 280, 300 µg/mL in absence of mammalian metabolic activation and 0, 1, 3, 10, 33, 100, 200, 250, 300, 350, 400, 425, 500 µg/ml with 8 % (v/v) metabolic activation (experiment 1).

Experiment 2: 0, 10, 25, 33, 50, 100, 115, 150, 175, 180, 200, 210, 220, 240, 260 µg/ml without metabolic activation and 0, 1, 3, 10, 33, 100, 200, 250, 300, 350, 375 µg/mL with 12 % (v/v) metabolic activation.

Isostearic acid, esters with methyl α-D-glucoside was tested beyond the limit of the solubility to obtain adequate mutagenicity data.

In the first segment of experiment 2,Isostearic acid, esters with methyl α-D-glucoside was tested up to concentrations of 240 µg/mlin the absence of 8% (v/v) S9-mix and up to concentrations of 350 µg/mL in presence of 8% (v/v) S9-mix with an incubation time of 3 hours.Isostearic acid, esterswith methyl α-D-glucosidewas tested up to cytotoxic levels of 12 and 14% in the absence and presence of S9-mix, respectively.

In the second segment of experiment 2,Isostearic acid, esterswith methyl α-D-glucosidewas tested up to concentrations of 210 µg/mL in the absence of 12% (v/v) S9-mix and up to concentrations of 375 µg/mL in the presence of 12 (v/v) S9-mix. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. Isostearic acid, esters with methyl α-D-glucoside was tested up to cytotoxic levels of 19 and 41% in the absence and presence of S9-mix, respectively.

In the absence of S9-mix,Isostearic acid, esters with methyl α-D-glucoside did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, Isostearic acid, esters with methyl α-D-glucoside did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9-mix for metabolic activation.

Isostearic acid, esters with methyl α-D-glucoside is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

The positive controls did induce the appropriate response. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

This study is classified asacceptable.This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity data.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Data are available for alpha methyl glucoside from a study conducted according to OECD guideline 471, GLP, RL1. No data are available for alpha methyl glucoside regarding clastogenicity and gene mutation in mammalian cells. However, reliable data from a chromosome aberration assay and a gene mutation in mammalian cells study conducted with the closely related source substance Isostearic acid, esters with methylα-D-glucoside are available. A justification for read-across is given below.

 

Bacterial reverse mutation assay:

In the present test conducted according to OECD guideline 471, the S. typhimurium strains TA 98, 100, 102, 1535, 1537 were incubated with the following concentrations of alpha methyl glucoside: 31.6, 100, 316, 1000, 3160 and 5000 µg /plate in the presence and absence of mammalian metabolic activation (plate incorporation and preincubation method). No increases in revertant colonies were observed, no cytotoxicity up to the limit concentration (5000 µg/plate) occurred. The positive controls induced the appropriate responses in the corresponding strains and metabolic activation was confirmed. There was no evidence of induced mutant colonies over background.

The test item is therefore considered non mutagenic under the present conditions in the Salmonella typhimurium reverse mutation assay reverse mutation assay.

 

In a reverse gene mutation assay in bacteria according to OECD guideline 471, strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2 uvr A) were exposed to Isostearic acid, esters with methylα-D-glucoside, at concentrations of 33, 100, 333, 1000, and 3330 µg/plate in the presence and absence of mammalian metabolic activation (plate incorporation method). Isostearic acid, esters with methylα-D-glucoside did not induce a significant dose-related increase in the number of revertant colonies in each of the five tester strains, both in the absence and presence of mammalian metabolic activation. These results were confirmed in an independently repeated experiment. No cytotoxic effects of the test substance were observed. Precipitation was observed at concentrations of 1000 and 3330 µg/plate. The positive controls induced the appropriate responses in the corresponding strains and metabolic activation was confirmed. There was no evidence of induced mutant colonies over background.

Based on the results of this study it is concluded that Isostearic acid, esters with methylα-D-glucoside is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.  

 

Chromosome aberration assay:

In a mammalian cell cytogenetics assay, peripheral human lymphocyte cultures were exposed to Isostearic acid, esters with methylα-D-glucoside (80% Methyl Glucoside Isostearate Esters (mainly Di-), 16% Isostearic Acid, 4% Methyl glucoside), in dimethyl sulfoxide at concentrations of 0, 33, 100 and 333 µg/mL culture medium (3 h exposure time , 24 h fixation time with and without metabolic activation in the first cytogenetic assay. In the second cytotgenetic assay concentrations of 0, 100, 300, 400, 500, 600, 700 and 800 µg/mL (24 h and 48 h exposure time and fixation time) without S9-mix and 0, 33, 100 and 300 µg/mL (3 h exposure time, 48 h fixation time) with S9-mix were tested respectively.

Isostearic acid, esters with methylα-D-glucoside was tested up to cytotoxic and precipitating concentration. In the first cytogenetic assay, the test substance was tested up to 333 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Isostearic acid, esters with methylα-D-glucoside precipitated in the culture medium at this dose level. In the second cytogenetic assay, Isostearic acid, esters with methylα-D-glucoside was tested up to 800 µg/mL for a 24 h and 48 h continuous exposure time with a 24 hand 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at 600 µg/mL. In the presence of S9-mix Isostearic acid, esters with methylα-D-glucoside was tested up to 300 µg/mL for a 3 h exposure time with a 48 h fixation time. The test substancce precipitated in the culture medium at this dose level.

Isostearic acid, esters with methylα-D-glucoside did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

No biologically relevant effects of Isostearic acid, esters with methylα-D-glucoside on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore, it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that Isostearic acid, esters with methylα-D-glucoside is not clastogenic in human Iymphocytes under the experimental conditions described in this report.

Positive controls induced the appropriate response. There was no evidence of Chromosome aberration induced over background. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range.

 

Gene mutation in mammalian cells:

In a mammalian cell gene mutation assay, mouse lymphoma L5178Y cells cultured in vitro were exposed to Isostearic acid, esters with methylα-D-glucoside (100% UVCB-Substance (80% Methyl Glucoside Isostearate Esters (mainly Di-), 16% Isostearic Acid, 4% Methyl Glucoside (not soluble in Olive Oil) in DMSO. The test was performed in two independent experiments in the absence and presence of S9-mix.

Experiment 1: 0, 1, 3, 10, 33, 100, 150, 180, 200, 220, 240, 280, 300 µg/mL in absence of mammalian metabolic activation and 0, 1, 3, 10, 33, 100, 200, 250, 300, 350, 400, 425, 500 µg/mL with 8 % (v/v) metabolic activation (experiment 1).

Experiment 2: 0, 10, 25, 33, 50, 100, 115, 150, 175, 180, 200, 210, 220, 240, 260 µg/mL without metabolic activation and 0, 1, 3, 10, 33, 100, 200, 250, 300, 350, 375 µg/mL with 12 % (v/v) metabolic activation.

Isostearic acid, esters with methylα-D-glucoside was tested beyond the limit of the solubility to obtain adequate mutagenicity data.

In the first segment of experiment 2, Isostearic acid, esters with methylα-D-glucoside was tested up to concentrations of 240 µg/mL in the absence of 8% (v/v) S9-mix and up to concentrations of 350 µg/mL in presence of 8% (v/v) S9-mix with an incubation time of 3 hours. Isostearic acid, esters with methylα-D-glucoside was tested up to cytotoxic levels of 12 and 14% in the absence and presence of S9-mix, respectively.

In the second segment of experiment 2, Isostearic acid, esters with methylα-D-glucoside was tested up to concentrations of 210 µg/mL in the absence of 12% (v/v) S9-mix and up to concentrations of 375 µg/mL in the presence of 12 (v/v) S9-mix. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. Isostearic acid, esters with methylα-D-glucoside was tested up to cytotoxic levels of 19 and 41% in the absence and presence of S9-mix, respectively.

In the absence of S9-mix, Isostearic acid, esters with methylα-D-glucoside did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, Isostearic acid, esters with methylα-D-glucoside did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9-mix for metabolic activation. Isostearic acid, esters with methylα-D-glucoside is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

The positive controls did induce the appropriate response. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

Based on the available information, alpha methyl glucoside is considered to be not mutagenic. It is also considered not clastogenic and non mutagenic based on the results from the chromosome aberration assay and the a mammalian cell gene mutation assay performed with Isostearic acid, esters with methylα-D-glucoside. There are no data gaps in genetic toxicity. Even though there is no information on genetic toxicity in humans, there is no reason to believe that the absence of genetic toxicity observed in experimental conditions would not be relevant for human health.

Justification for read-across

For details on substance identity, toxicokinetics and detailed toxicological profiles, please refer also to the general justification for read-across attached as pdf document to section 13 of the IUCLID file.

Analogue approach justification

The read-across hypothesis is based on similar break down products of target and source substance, i.e. the target substance (alpha methyl glucoside) is one of two break down products of the source substance (Isostearic acid, esters with Methyl-α-D-glucose). According to scenario 1 of the Read-across Assessment Framework (RAAF) this analogue approach is based on the (bio) transformation of the source substance to common compounds, i.e. the target substance alpha methyl glucoside and Isostearic acid. Based on the available experimental data, including genotoxicity studies, the read-across strategy is supported by a similar toxicological behaviour of the two substances and their break-down products, respectively. Although there is a difference in absorption, for both substances a very low acute toxicity and mutagenicity was shown. However, in accordance with ECHA guidance on information requirements chapter R 7.c absorption of Isostearic acid, esters with methyl-α-D-glucose in the GI-tract is considered unlikely due to its size. The main metabolism pathway of the unchanged source substance would then be biotransformation by xenobiotic metabolism, i.e. functionalisation, conjugation and subsequent biliary excretion. However, unspecific hydrolysis of the source substance (Isostearic acid, esters with methyl-α-D-glucose) during GI-tract passage is more likely to occur resulting in its breakdown products, namely alpha methyl glucoside and Isostearic acid.

Alpha methyl glucoside (target substance) is a non-metabolisable glucose analogue which is used in several published studies to investigate cellular glucose uptake (Segal et al., 1973; Lee et al., 2007; Prieto et al., 1996; Genel et al., 1970). Alpha methyl glucoside enters the cell via the ubiquitously occurring SGLT transporter. Recovery of nearly 100% of labelled alpha methyl glucoside revealed a full absorption in various tissues. Due to reabsorption by the kidney, its concentration is elevated in the renal brush border membrane (Lee et al., 2007). This increase of alpha methyl glucoside is only limited by the ion gradient resulting from sodium symport. Alpha methyl-glucoside is considered to be mainly renally excreted. The other remaining break down product Isotearic acid is metabolised via β-oxidation and is thereby eliminated by the intermediary metabolism and thus, indistinguishable from fatty acids from other sources including diet.

 

Structural similarity

a. Structural similarity and functional groups

The target substance, alpha methyl glucoside, consists of alpha-D-glucose which forms an o-glycosidic bond with a methyl-group at C1. Since several studies confirm that alpha methyl glucoside should be regarded to as a non-metabolizable sugar in mammalian cells, it can be concluded that the substance is renally excreted without transformation (Segal et al., 1973; Lee et al., 2007; Prieto et al., 1996; Genel et al., 1970). Although not metabolisable by mammalian cells it was shown that alpha methyl glucoside is metabolised by several bacteria except these ones occurring most frequently in the GI flora (Devriese et al., 1996, Tittsler & Sandholzer, 1935, Koser & Saunders, 1932).

The source substance, Isostearic acid esters with methyl-α-D-glucose, consists of one to four C18 aliphatic chains which are esterified to alpha methyl glucoside. Although this substance has a very lipophilic character (logKow > 6.5, water solubility < 5E-004 g/L and a high molecular weight (weighted mean) 690.31 g/mol) it cannot be excluded that it is absorbed to little extent. Subsequent to uptake unhydrolysed Isostearic acid, esters with methyl-α-D-glucose will to some extent undergo biotransformation by xenobiotic metabolism and subsequently biliary excreted, on the other hand unspecific hydrolysis is likely to occur and two fragments will emerge of which one is readily metabolised by fatty acid metabolism, i.e. β-oxidation of the aliphatic chain and the other one, alpha methyl glucoside (= target substance), is excreted unchanged. However, since Isostearic acid, esters with methyl-α-D-glucose is an UVCB consisting of an estimated amount of 4% of methyl-α-D-glucose, it must be assumed that studies conducted with the source substance are also performed with 4% of the target substance.

However, there are some differences between the target substance and the source substance regarding their physicochemical properties and consequently their toxicological effects.

 

b. Common breakdown products:

The metabolism expected to occur in the unhydrolysed source substance Isostearic acid, esters with methyl-α-D-glucose due to its size is functionalisation, conjugation and excretion. On the other hand hydrolysis of the isostearic acid moeity is likely to occur. The rate of hydrolysis is assumed to be enzyme-mediated and may thus be limited. Further degradation may be the metabolism of the fatty acid chain via β-oxidation. However, since the source substance is considered to be highly lipophilic, intestinal absorption may be limited for the unhydrolysed substance. However, the source substance is considered to be unspecifically hydrolysed in the GI-tract and subsequently absorbed and further metabolised. In case of alpha methyl glucoside (one of two breakdown products), a known non-metabolisable molecule, distribution preferentially to the kidney and in tissues exhibiting the SGLT1 and SGLT2 transporter occurs. This uptake and transient increase of concentration is also driven by renal reabsorption and slow excretion but this increase is limited by sodium symport. Finally, alpha methyl glucoside is considered to be renally excreted.

c. Differences

As described above alpha methyl glucoside and Isostearic acid, esters with methyl-α-D-glucose are distinguished by the presence or absence of the C18 alkyl-chains.Associated with the presence of these alkyl chains also differences in the physicochemical properties of both substances occur, i.e. differences in water solubility, partition coefficient, molecular weight etc. These physicochemical characteristics are also decisive for absorption of the source substance either oral (via GI-tract), dermal or by inhalation. Exposure to the source substance via inhalation route can be excluded due to its waxy appearance and its low vapour pressure. Due to its high molecular weight and log Kow the absorption of the unhydrolysed source substance is also limited via the dermal and the oral route. However, as explained above the composition of the source substance substantiates an exposure to the target substance as well. In contrast, dermal absorption of alpha methyl glucoside is likely due to its small molecular weight. Respiratory absorption of alpha methyl glucoside cannot be fully excluded due to its granular form although its median particle size is 100 µm, thus, it is considered that the substance is not inhalable according to ECHA guidance on information requirements Chapter R.7.c.

Since alpha methyl glucoside is produced and handled only as an intermediate consumer exposure is assumed to be marginal, thus, exposure via the oral route is not expected.Dermal absorption of alpha methyl glucoside is also considered to be low due to its low log Kow (-2.5) it is presumably to hydrophilic to cross the stratum corneum.

 

 

Comparison of genetic toxicity data

Endpoint

Target substance

Source substance

 

Alpha methyl glucoside

 

Isostearic acid, esters with methyl-α-D-glucoside

 

Acute oral toxicity

 

OECD 471, S. typhimurium, RL 1, GLP, 31.6, 100, 316, 1000, 3160 and 5000 µg/plate, with/without m.ac.,

 

not mutagenic

 

OECD 471, S. typhimurium & E.coli, RL 1, GLP, 33, 100, 333, 1000, 3330 µg/plate, with/without m.ac.,

 

not mutagenic

 

 

No data, read-across

OECD 473, cultured peripheral human lymphocytes, RL1, GLP, First assay:

- without/with m.ac.: 33, 100 and 333 µg/mL culture medium (3 h exposure, 24 h fixation).

Second assay:

- without m.ac.: 100, 300, 400, 500, 600, 700 and 800 µg/mL culture medium, 24 h/48 h exposure, 24 h/48 h fixation,

 

not clastogenic

 

No data, read-across

OECD 476, mouse lymphoma L5178Y cells, Exp 1:

- without m. ac.: 0, 1, 3, 10, 33, 100, 150, 180, 200, 220, 240, 280, 300 µg/mL

- with 8 % (v/v) m. ac.: 0, 1, 3, 10, 33, 100, 200, 250, 300, 350, 400, 425, 500 µg/mL

Exp 2:

- without m. ac.: 0, 10, 25, 33, 50, 100, 115, 150, 175, 180, 200, 210, 220, 240, 260 µg/mL

- with 12 % (v/v) m. ac.: 0, 1, 3, 10, 33, 100, 200, 250, 300, 350, 375 µg/mL,

 

not mutagenic

 

Genetic toxicity – no classification according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

Experimental data are available for the target substance (OECD 471, GLP, reverse bacterial mutation assay). However, no data are available regarding chromosome aberration and gene mutation in mammalian cells for the target substance.

The closely related source substance Isostearic acid, esters with methyl-α-D-glucoside is non-mutagenic and non-clastogenic in gene mutation in mammalian cells and chromosome aberration studies according to OECD guideline 476 and 473, respectively.

As the target substance alpha methyl glucoside is one of the two breakdown products of the source substance, the results of the source substance Isostearic acid, esters with methyl-α-D-glucoside are considered to be relevant for the target substance alpha methyl glucoside.

 

Quality of the experimental data of the analogues:

The source substance Isostearic acid, esters with methyl-α-D-glucoside has been tested in reliable studies according to OECD TG 471, 473 and 476. The tests have been conducted according to GLP criteria. Therefore, these data have no uncertainties and can be used in an analogue approach. The available data from the source chemical is sufficiently reliable to justify the read-across approach.

 

Conclusion for read-across

The structural similarities between the breakdown products of the source substance and the target substance as presented above support the read-across hypothesis. Adequate and reliable scientific information indicates that the source substance and its degradation products and target substance have similar toxicity profiles under the experimental conditions in the considered studies for the endpoint genetic toxicity.

Thus, the results obtained with the source substance Isostearic acid, esters with methyl-α-D-glucoside are considered to be also relevant for the target substance alpha methyl glucoside.

 

 

References:

Lee, Y. J., & Han, H. J. (2007). Regulatory mechanisms of Na+/glucose cotransporters in renal proximal tubule cells.Kidney International,72, S27-S35.

Segal, S., Genel, M., Holtzapple, P., & Rea, C. (1973). Transport of alpha-methyl-D-glucoside by human kidney cortex.Metabolism,22(1), 67-76.

Prieto, R. M., Ferrer, M., & Tur, J. A. (1996). Changes in intestinal alpha-methyl-D-glucoside uptake due to pregnancy and lactation in rats.Digestion,57(1), 16-21.

Genel, M., London, D., Holtzapple, P. G., & Segal, S. (1971). Uptake of alpha-methylglucoside by normal and diabetic human jejunal mucosa.Translational Research,77(5), 743-750.

Enoch, S. J., Madden, J. C., & Cronin, M. T. D. (2008). Identification of mechanisms of toxic action for skin sensitisation using a SMARTS pattern based approach.SAR and QSAR in Environmental Research,19(5-6), 555-578.

Koser, S. A., & Saunders, F. (1932). The Fermentation of Alpha-Methyl-D-Glucoside by Members of the Coli-areogenes Group.Journal of bacteriology,24(4), 267.

Tittsler, R. P., & Sandholzer, L. A. (1935). The fermentation of alpha-methylglucoside by bacteria.Journal of bacteriology,29(4), 363.

Devriese, L. A., Pot, B., & Collins, M. D. (1993). Phenotypic identification of the genus Enterococcus and differentiation of phylogenetically distinct enterococcal species and species groups.Journal of Applied Microbiology,75(5), 399-408.

 

Justification for classification or non-classification

Based on reliable and relevant data alpha methyl glucoside does not need to be classified as mutagenic or clastogenic according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).