D-Xylopyranose, oligomeric, 2-octyldodecyl xylosides

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test (OECD 471, K, Rel.1): not mutagenic in bacteria with and without metabolic activation.

- In vitro gene mutation study in mammalian cells (OECD 490, K, Rel.1): not mutagenic with and without metabolic activation.

- In vitro Chromosome aberration study in mammalian cells (OECD 473, K, Rel.1): Not clastogenic with and without metabolic cativation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, OECD guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 50 ... 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50 ... 5000 µg/plate

Vehicle / solvent:

Solvent: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

solvent

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Positive controls:

yes

Positive control substance:

9-aminoacridine

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Positive controls:

yes

Positive control substance:

other: 2-amino-anthracene

Details on test system and experimental conditions:

Concentration of the test substance resulting in precipitation: 5000 µg/plate

Evaluation criteria:

The test material may be considered positive in this test system if the foliowing criteria are met:
The test material should have induced a reproducibie, dose-related and statisticaily (Dunnett’s method of linear regression(5)) significant increase in the revertant count in at ieast one strain of bacteria.

Species / strain:

other: as specified above

Metabolic activation:

with

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Species / strain:

other: as specified above

Metabolic activation:

without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Species / strain:

other: as specified above

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Species / strain:

other: as specified above

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Remarks on result:

other: other: preliminary test

Remarks:

Migrated from field 'Test system'.

Conclusions:

Under the test conditions of this study, negative the test substance is not mutagenic in bacteria with and without metabolic activation.

Executive summary:

Introduction.

The method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 471 ‘Bacterial Reverse Mutation Test’, Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.

 

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia cou strain WP2uvrK were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 ig/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

 

Results.

The vehicle (dimethyl suiphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

 

Conclusion.

The test material was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 23 July 2007 to 18 August 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

OECD 473 version 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch No. 1137JG
- Expiration date of the lot/batch: October 2007
- Storage condition of test material: Required quantity of the test substance was received from the test substance controller (TSC, IIBAT) and stored at room temperature (20± 5°C)

Species / strain / cell type:

lymphocytes: Human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:Peripheral blood lymphocytes obtained from a healthy adult male nonsmoking donor without any recent history of illness and under no medication was used for the study

Lymphocyte cultures were established from heparinised whole blood in RPMI 1640 (GIBCO) medium supplemented with 20% foetal bovine serum (GIBCO), 100 IU/ml penicillin, 100 μg/ml
streptomycin, 0.4mM L-glutamine (GIBCO) and 2% Phytohemagglutinin (PHA-M) (GIBCO).
The pH of the media was maintained between 7.2 – 7.4 using 1N HCl.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The S9 fraction prepared from Aroclor 1254-induced male Wistar rat was produced from Defense Research & Development Establishment (DRDE) , Gwalior, India
- method of preparation of S9 mix : S9 mix (10%S9° was prepared , prior to the initiation of the experiment.The components of the mix were as follows:
0.1M NADP(Sigma,USA) : 0.4 ml
1.0M D-glucose-6-phosphate (Sigma,USA) : 0.1 ml
MgCl2-KCl (Merck) : 0.2 ml
0.2M Sodium phosphate buffer (pH 7.4) (Merck) : 5.0 ml
S9 fraction : 1.0 ml
Sterile distilled water : 3.3 ml
The mixture was kept in ice until use.

Test concentrations with justification for top dose:

A range finding study was performed to assess the cytotoxicity based on mitotic index, to fix the
high dose for the main study. Three concentrations of the test substance were employed viz., 1000,
2000 and 5000 μg/ml, with (10% S9) and without metabolic activation system (S9). Cytotoxicity
was observed at all the concentrations with and without S9. Moreover, all three concentrations
exhibited hemolysis during the course of expression period post treatment. Hence, a second range
finding study was performed with further reduced concentrations viz., 18.75, 37.50, 75, 150, 300
and 600 μg/ml. The higher two concentrations 300 and 600 μg/ml exhibited cytotoxicity.
Hemolysis was seen at concentrations 150, 300 and 600 μg/ml as observed in the first range
finding study. From the results of the range finding studies, 150 μg/ml was chosen as the high dose
for the main study as it exhibited a reduction in mitotic index (MI) around 50% with and without
metabolic activation (S9) compared to the solvent control.
The concentrations chosen for the main study were 9.38, 18.75, 37.50, 75 and 150 μg/ml with
(10% S9) and without S9.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (Sigma , USA)
- Justification for choice of solvent/vehicle: Since the test substance was not freely soluble in water, 500 mg of the test substance was dissolved in 1.0 ml of DMSO (Sigma, USA).

Percentage of solvent in the final culture medium: Cultures were treated with 1 % DMSO (Sigma, USA) alone.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Cyclophosphamide (Sigma, USA) and Mitomycin C (Sigma, USA) at concentrations of 20 and 1 μg/ml with and without S9 respectively, were used as the positive controls.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration :Duplicate cultures were maintained for all the treatment conditions.

METHOD OF TREATMENT/ EXPOSURE:
Heparinized whole blood obtained from healthy male donor was used to establish lymphocyte
cultures in RPMI 1640 supplemented medium. Cultures were incubated in PHA-M (GIBCO)
containing medium for 48 h. The cultures were treated with the test substance at the above
employed concentrations with and without S9 at 48 h from initiation of the cultures and
subjected to two kinds of treatment i.e., 4 h exposure with 32 h expression period and continuous
exposure with 36 h expression period. After 4 h of exposure in the presence of the test substance,
cultures were washed of the test substance at 1600 rpm for 10 min and resuspended in freshly
prepared supplemented medium (devoid of PHA-M and S9) and incubated upto 1.5 cell cycle
times. For continuous exposure without S9, cultures were treated similarly as detailed above
except that the cultures were incubated for 1.5 cell cycle times continuously in the presence of
the test substance from the time of the test substance addition at 48 h. All the incubations were
done in a humidified atmosphere with 5% CO2 at 37 ± 0.5 °C. Duplicate cultures were
maintained for all the treatment conditions.


TREATMENT AND HARVEST SCHEDULE:

One hour prior to harvest, 0.4 μg/ml of colchicine (Sigma, USA) was added to the cultures. The
cells were pelleted, hypotonically shocked with freshly prepared and pre incubated 0.075 M
potassium chloride (Merck) solution at 37°C, washed and fixed in 3:1 methanol (Rankem) and
acetic acid (Rankem). Slides were prepared from cells resuspended in fixative by dropping
technique and heat fixed.


FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):Heat fixed metaphase preparations were stained in Giemsa (Merck), as per IIBAT SOP. The mitotic index (MI) described as proportion of cells undergoing mitosis in 1000 cells was scored for each treatment condition.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):Two hundred well spread metaphases were scored per concentration of the test substance and solvent control, equally divided amongst the duplicates.
From the positive control a total of 50 metaphases were scored. For control of bias, all were coded by QAU and blind scoring was performed.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI);
- Any supplementary information relevant to cytotoxicity:The following factors were taken into account during evaluation:
- The overall aberration frequencies
- The percentage of cells with aberrations
- The percentage of cells with more than one aberration
- Any evidence for increased amounts of damage with increasing dose, (i.e.,) a positive
dose response.
- The estimated number of breaks involved in production of the different types of
aberrations observed (i.e.,) complex aberrations, may have more significance than simple
breaks.

Rationale for test conditions:

Before commencing the main study, the test substance was assessed for cytotoxicity based on the
mitotic index. Three test concentrations of the test substance viz., 1000, 2000 and 5000 μg/ml, were tested with and without S9 and dosed in a final volume of 0.1 ml. Cytotoxicity and hemolysis post treatment was observed in all three concentrations, hence a second range finding study was performed with concentrations 18.75, 37.50, 75, 150, 300 and 600 μg/ml. Cytotoxicity was observed at concentrations 300 and 600 μg/ml. Hemolysis was observed at concentrations 150, 300 and 600 μg/ml post treatment. The high dose for the main study was chosen based on the criteria that, it exhibited greater than 50% reduction in the mitotic index as against the solvent control. Hence, 150 μg/ml was chosen as the high dose for the main study.

Evaluation criteria:

- The overall aberration frequencies
- The percentage of cells with aberrations
- The percentage of cells with more than one aberration
- Any evidence for increased amounts of damage with increasing dose(i.e) a positive
dose response.
- The estimated number of complex aberrations
Gaps were counted, but were not used in the final assessment, since they are not considered as
significant aberrations. Open breaks were considered as indicators of genetic damage, as were
configurations resulting from the repair of breaks. Reunion figures were weighed slightly higher
than breaks since they usually resulted from more than one break and may lead to stable
configurations.
Cultures treated with positive mutagens exhibited a low mitotic index comparatively. Hence,
cytogenetic analysis of these had often to be performed with a low number of metaphases. The
positive control was taken as a reference for comparison with the solvent control and test
substance treated cultures. For determining a positive result, a concentration related increase or a
reproducible increase in the number of cells bearing chromosomal aberrations was considered.
Biological relevance of the results was considered first.
The number of cells bearing chromosomal aberrations per concentration, the type of aberration,
the statistical significance of any increase and its correlation to the test concentration in a given
time period, was considered for evaluating the mutagenic potential of the test substance. Though,
the final decision was based upon scientific judgment.

Statistics:

The percent frequencies of numerical aberrations, mean structural aberration frequencies and
mitotic indices between various treatment groups were statistically compared using Student's `t'
test using software (NCSS, 2000).

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the second range finding study six test concentrations were employed 18.75, 37.50, 75, 150, 300 and 600 μg/ml. Two of the test concentrations 300 and 600 μg/ml exhibited cytotoxicity with (10% S9) and without S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Under the test conditions employed, no apparent induction of numerical or structural aberrations was observed in any of the concentrations tested without metabolic activation in the continuous exposure time period. Cultures treated with the test substance exhibited statistically comparable values of numerical and structural aberrations to that of the solvent control group. However, the positive control group exhibited statistically increased frequencies of cytogenetic anomalies thus validating the sensitivity of the assay. Thus, the test substance was considered non genotoxic in the in vitro chromosomal aberration assay on human lymphocytes.

Summary on in vitro chromosomal aberrations in human lymphoctyes: Main experiment (4 h exposure and 32 h expression period) - Without S9

Concentration (µg/ml)	Total no.of cells scored	% Numerical aberrations	Structural aberrations (inclusive of gaps) Mean (±) SD
Solvent Control (0.1 ml DMSO)	200	1.5	5.50 (±) 2.12
9.38	200	2.0	1.00 (±) 0.00
18.75	200	0	4.50 (±) 0.71
37.50	200	0	3.50 (±) 0.71
75	200	1.0	4.50 (±) 0.71
150	200	0.5	10.50 (±) 2.12
Positive control Mitomycin C (1µg/ml)	50	4.0	49.00 (±) 1.41

* significant (p< 0.05)

Summary on in vitro chromosomal aberrations in human lymphocytes : Main experiment (4 h exposure and 32 h expression period )- With S9(10%

Concentration (µg/ml)	Total N° of the cells scored	% Numerical aberrations	Structural aberrations (inclusive of gaps)  Mean (±) SD
Solvent Control (0.1 ml DMSO)	200	1.5	4.50 (±) 0.71
9.38	200	0	5.00 (±) 1.41
18.75	200	1.5	1.50 (±) 0.71
37.50	200	1.0	2.00 (±) 1.41
75	200	1.0	5.00 (±) 0.71
150	200	0.5	6.00 (±) 1.41
*Positive control Cyclophosphamide (20 (µg/ml)	200	6.0	62.00 (±) 2.83

*significant (p< 0.05)

Summary on in vitro chromosomal abberrations in humans lymphocytes : Main experiment (continous exposure 36 h expression period)- WithoutS9

Concentration (µg/ml)	Total N°of cells scored	% Numerical aberrations	Structural aberrations (inclusive of gaps)   Mean (±) SD
Solvent Control (0.1 ml DMSO)	200	0.5	4 2.50 (±) 0.71
4.69	200	0	1.50 (±) 0.71
9.38	200	0.5	1.00 (±) 0.00
18.75	200	0.5	2.50 (±) 0.71
37.50	200	0	5.50 (±) 0.41
75	200	cytotoxic	cytotoxic
150	200	cytotoxic	cytotoxic
* Positive control Mitomycin C (µg/ml)	50	2.0	50.00 (±) 8.49

*significant (p<0.05)

Conclusions:

Under the test conditions employed, the test substance did not induce any apparent numerical and
structural aberrations both in the presence and absence of an exogenous mammalian metabolic
activation component (S9) in the 4 h exposure and continuous exposure time periods. Hence,
the test item may be considered non genotoxic in the in vitro chromosomal aberration assay on
human lymphocytes.

Executive summary:

According to the OECD Guidelines for Testing of Chemicals (Section 4, No. 473, Adopted 21st July 1997):

The ability of the test substance in inducing numerical and structural cytogenetic anomalies was evaluated by enumerating the incidence of chromosomal aberrations in cultured human peripheral blood lymphocytes both in the presence and absence of a mammalian S9 activation system.The peripheral blood was obtained from a healthy adult male non-smoking donor without any recent history of illness.

A range finding study was performed to assess the cytotoxicity based on mitotic index, to fix the

high dose for the main study. Three concentrations of the test substance were employed viz., 1000,

2000 and 5000 μg/ml, with (10% S9) and without metabolic activation system (S9). Cytotoxicity

was observed at all the concentrations with and without S9.Cytotoxicity was observed at all the concentrations with and without S9. Moreover, all three concentrations exhibited hemolysis during the course of expression period post treatment. Hence, a second range finding study was performed with further reduced concentrations viz., 18.75, 37.50, 75, 150, 300 and 600 μg/ml. The higher two concentrations 300 and 600 μg/ml exhibited cytotoxicity. Hemolysis was seen at concentrations 150, 300 and 600 μg/ml as observed in the first range finding study. From the results of the range finding studies, 150 μg/ml was chosen as the high dose for the main study as it exhibited a reduction in mitotic index (MI) around 50% with and without metabolic activation (S9) compared to the solvent control.

The concentrations chosen for the main study were 9.38, 18.75, 37.50, 75 and 150 μg/ml with

(10% S9) and without S9. Concurrent solvent and positive controls were maintained. The solvent

control received 1 % of DMSO (Sigma, USA). Positive controls employed for with and without S9

were cyclophosphamide (Sigma, USA) and mitomycin C (Sigma, USA) respectively, at

concentrations 20 and 1 μg/ml respectively. The cultures were maintained in duplicates for all the

treatment conditions.

The PHA-M (GIBCO) stimulated cultures were maintained in a humidified atmosphere of 5% CO2

at 37 ± 0.5 °C. After the test substance addition at the 48th hour with and without S9, cultures were

allowed for an exposure period of 4 h and an expression period of 1.5 cell cycle times.

One hour prior to harvest, the cultures were treated with a metaphase arresting chemical -

colchicine (Sigma, USA) at a concentration of 0.4 μg/ml. Metaphase preparations were made from

cells in fixative, after hypotonic shock with pre incubated 0.075M KCl (Merck) at 37 °C and a

series of washes with fixative (methanol: acetic acid at 3:1) at 1600 rpm for 10 min. Heat fixed

preparations were stained with Giemsa (Merck). Thousand consecutive cells were scored and cells

in metaphase were enumerated for deriving the percentage mitotic index. Two hundred well spread

intact metaphases with 46 ± 2 centromeres were analyzed for aberrations. The observations were

recorded and summarized as percentage numerical aberrations and mean structural aberrations.

Hemolysis was observed at the high concentration 150 μg/ml post treatment.

Under the test conditions employed, no apparent induction of numerical or structural aberrations

was observed in any of the concentrations tested with and without metabolic activation in the 4 h

exposure time period. Cultures treated with the test substance exhibited statistically comparable

values of numerical and structural aberrations to that of the solvent control group with and without

S9. However, the positive control groups exhibited statistically increased frequencies of

cytogenetic anomalies thus validating the sensitivity of the assay.

Based on the negative results observed in the 4 h exposure time period, an additional experiment

was performed with continuous exposure in the presence of the test substance for 1.5 cell cycle

time without S9. The concentrations employed were 4.69, 9.38, 18.75, 37.50, 75 and 150 μg/ml

and the experiment was performed in a similar pattern as detailed in the 4 h exposure time period.

The two high concentrations 75 and 150 μg/ml exhibited evident cytotoxicity and hemolysis was

observed post treatment. The concentration 37.50 μg/ml exhibited a reduction in MI around 50%

without metabolic activation (S9) compared to the solvent control.

Under the test conditions employed, the test substance did not induce any apparent numerical and

structural aberrations both in the presence and absence of an exogenous mammalian metabolic

activation component (S9) in the 4 h exposure and continuous exposure time periods. Hence,

the test item may be considered non genotoxic in the in vitro chromosomal aberration assay on

human lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2019-11-21 to 2020-03-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 476 and in compliance with GLP without any deviations.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

Version 2016

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

OECD GLP (inspected from 01 to 02, December 2016 / Signed on 27, April 2017)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Storage conditions: at room temperature (+20°±5°C).

Target gene:

Thymidine Kinase locus.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y TK+/- mouse lymphoma cells / Cellswere provided by ATCC (American Type Culture Collection - Rockville, MD 20852 - USA).
For cell lines:
- Absence of Mycoplasma contamination: Yes. Contamination by mycoplasma is checked using Mycoalert mycoplasma detection kit for each batch of
the cells. Only the batches, which contain no mycoplasma, are used in the mutagenicity test.
- Others: the cells have a stable caryotype (40, X0) and are deficient for p53. They have a 11-12 hour doubling time.
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
1- RPMI 0 medium: the basic culture medium used was the RPMI 0 medium containing sodium bicarbonate, non-essential aminoacids, penicillin, streptomycin and HCl.
2- RPMI 10 medium: to formulate RPMI 10, the RPMI 0 medium was supplemented with 10% inactivated horse serum, pluronic acid, L-Glutamine, sodium pyruvate and Amphotericin B.
3- RPMI 20 medium: to formulate RPMI 20, the RPMI 0 medium was supplemented with 20% inactivated horse serum, L-Glutamine, sodium pyruvate and Amphotericin B.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 / Batch number : the S9 fraction is prepared at Institut Pasteur de Lille (IPL) / IPL 18-L.
- method of preparation of S9 mix : preparation is carried out using the method described by Ames et al. (1975) in male rat Sprague Dawley OFA induced by Aroclor 1254 (origin - Monsanto, Saint Louis, U.S.A) according to the standard operating procedures of the Institut Pasteur de Lillle.
- concentration or volume of S9 mix and S9 in the final culture medium:
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):
- Sterility test: 0 colonies of contaminating microflora observed per 50 µL of S9 after a 44-h incubation on Vogel-Bonner medium at ca. 37°C.

Test concentrations with justification for top dose:

CYTOTOXICITY ASSAY:
* Without S9-mix: 250 – 125 –62.5– 31.25 – 15.63 – 7.81 – 3.91 µg/mL (3-hour treatment) and 250 – 125 –62.5– 31.25 – 15.63 – 7.81 – 3.91 µg/mL (24-hour treatment).
* With S9-mix : 250 – 125 –62.5– 31.25 – 15.63 – 7.81 – 3.91 µg/mL (3-hour treatment)
MUTAGENICITY ASSAYS:
* Without S9 mix : 30 – 26.09 – 22.7 – 19.72 – 13.15 – 6.57 µg/mL (3 and 24-hour treatment)
* With S9 mix : 60 – 52.17 – 45.36 – 39.45 – 26.30 – 13.15 µg/mL (3-hour treatment)
COMPLEMENTARY MUTAGENICITY ASSAY:
* Without S9 mix: 60 – 52.17 – 45.36 – 39.45 – 34.3 – 29.83 – 25.94 – 22.56 – 19.61 – 17.05 µg/mL (24-hour treatment).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (Merck, batch K50927578 920)
- Justification for choice of solvent/vehicle: The Final Study Plan indicates that the test item Xylopyranoside, 2-octyldodecyl was soluble in sterile water (from 2.3 mg/L) and dimethylsulfoxide (DMSO).
- Justification for percentage of solvent in the final culture medium: trials for solubility in DMSO were performed. They demonstrated that D-Xylopyranoside, 2-octyldodecyl was soluble at 200 mg/mL in DMSO, but it induced a too heavy precipitate incompatible with the test system at 2000 and 1000 μg/mL (i.e. when used at 1% (v/v) in culture medium). The immediately lower final concentration of 250 μg/mL in culture medium induced a homogenous cloudy precipitate, compatible with the test system; it was thus retained for pH and osmolality assessment.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

10 μg/mL (3-h treatment), 2 μg/mL (24-h treatment) (Aldrich, batch MKBL6789V and MKBX5165V for the main and the complementary assay, respectively)

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

2 μg/mL (Sigma, batch MKCF1756

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : duplicate
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:

- Exposure duration/duration of treatment: Without metabolic activation: 3 hours (short-term treatment) and 24 hours (continuous treatment). With metabolic activation: 3 hours.
- Harvest time after the end of treatment (sampling/recovery times): 10 - 14 days.

SELECTION AGENT (mutation assays): trifluorothymidine (TFT) 3 μg/mL) at 2 x103 cells per well (4*96-well microtiter plates per dose)

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Selection time (if incubation with a selective agent): NA
- Fixation time (start of exposure up to fixation or harvest of cells): 10-14 days.
- Method used: microwell plates for the mouse lymphoma assay.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
- Criteria for small (slow growing) and large (fast growing) colonies:
Each well of the mutation plates (in selective medium containing TFT) was scored as containing either a small colony, a large colony or no colony according to the following criteria:
SMALL COLONIES: colonies having a diameter less than 25% of the diameter of the well. A small colony should also have a dense colonal morphology and a clear contour.
LARGE COLONIES: colonies having a diameter greater than 25% of the diameter of the well. A large colony should show less densely packed cells, and blurred contour.
Any well containing one or more small colonies was scored as positive for small colony.
Any well containing one or more large colonies was scored as positive for large colony.
Any well containing a combination of large and small colonies was scored at the same time as large colony and small colony.
An empty well was one which contains no cell growth.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: relative total growth (RTG)
Assessment of cloning efficiency
Plating efficiency on Day 2 at the end of the expression period was used to calculate the relative
survival (RS2) on Day 2.
From the Poisson distribution, the probable number of clones/well (P(2)) on microtiter plates in which there were empty wells (without clones) out of a total ofwells was given by:
P(2) = Number of empty wells/Total plated wells
The plating efficiency (PE) in any given culture was therefore: PE = - Ln P(2)
Number of cells per well and as an average of 1.6 cells per well were plated on all survival and viability plates, PE = - Ln P(2) /1.6
The percentage of relative survival (%RS2) of each test culture was therefore determined by comparing
plating efficiencies of the test culture and then control culture: %RS2 =PE(treated)X 100/PE(control)
The %RS2 was calculated using the PE at the end of the expression time (PE2).
Assessment of growth
The total 2-day suspension growth of each culture was calculated and each treated culture was compared to the control. This value was referred to as the relative suspension growth (RSG).
The relative suspension growth (RSG) was calculated as follows: %RSG = Cell Loss during treatment x Daily growth at day 1 x Daily growth at day 2 (treated) x 100
Cell Loss during treatment x Daily growth at day 1 x Daily growth at day 2 (control) where
Cell Loss during treatment = number of cells at the end of the treatment period/number of cells at the end of the beginning period
Daily growth at day 1 = Cell concentration at day 1/Adjusted cell concentration at day 0
Daily growth at day 2 =Cell concentration at day 2/Adjusted cell concentration at day 1
The relative total growth (RTG) was obtained by multiplying the relative plating efficiency at day 2 for each culture (relative to the negative control) and the RSG.
The relative total growth (RTG) was calculated as: RTG = RSG x %RS2

METHODS FOR MEASUREMENTS OF GENOTOXICIY :
Calculation of mutation frequency
Scoring of empty wells in plates incubated in presence of TFT-selective medium was used to calculate
mutation frequency.
Methods for calculating plating efficiency after incubation in presence of TFT-selective medium,
mutation frequency per 106 surviving cells for the microtiterplate assay are based on P(2), i.e. the
proportion of wells in which a colony has not grown (Kraemer et al.,1980 et Cole et al., 1983)
P(2) = Number of empty wells/Total wells plated
Plating efficiency (PE) = - Ln P(2)/Number of cells per well
Mutation frequency x 10-6 cells = PE in selective medium x 106 (Mutation)/PE in non-selective medium (Viability)

Evaluation criteria:

The test item is considered as mutagenic in this system if:
1. The induced mutation frequency for at least one tested concentration is higher than the mutation frequency in the vehicle control cultures by at least the global evaluation factor of 126 x10-6 (Moore et al., 2006) and,
2. A statistical trend test demonstrates a positive concentration related increase in the mutation frequency (Moore et al., 2006).
It is noteworthy that biological significance is to be considered at first.
Consideration of whether the observed values are within or outside of the historical control range can provide guidance when evaluating the biological significance of the response.
If none of the two criteria mentioned above is fulfilled, the tested item is considered as not mutagenic in this study system.
The criteria are not absolute but do constitute an aid to decision, which makes it possible to reach a conclusion in most cases.

Statistics:

Test for linear trend of mutant frequency with dose was performed. The slope b and its variance var (b) were calculated to form the test statistic b2/var (b) that should be compared with tabulated critical values of chi2 with 1 degree of freedom.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 30 to 60 μg/mL (Factor limiting the maximum concentration tested for mutagenicity tests).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: the pH was superior to 6, and did not vary for more than one unit from the one of the solvent control at the 3 highest concentrations tested ranging from 250 to 62.5 µg/mL.
- Effects of osmolality: no effect, determined in the solvent control (culture medium + 1% (v/v DMSO)) and at 62.5, 125 and 250 µg/mL. These concentrations induced no variation in osmolarity higher than 50 mOsmol/kg when compared to the solvent control.
- Evaporation from medium: the test material is not a VOC
- Water solubility: soluble in sterile water (from 2.3 mg/L)
- Precipitation: The test item formed a clear phase separation in culture medium at 250 μg/mL
- Other confounding effects: none

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
* Without metabolic activation: after a 3-hour treatment, D-Xylopyranoside, 2-octyldodecyl revealed a very strong cytotoxicity in L5178Y mouse lymphoma cells at the 3 highest concentrations tested ranging from 250 to 62.5 μg/mL (with a total absence of cells). The lower concentrations of 31.25 and 15.63 μg/mL induced a strong and a moderate level of cytotoxicity, respectively with RTGs of 1.3 and 66%. The concentration of 30 μg/mL was retained for the mutagenicity assay and a narrowed range of concentrations was chosen in order to reach a RTG comprised between 10 and 20%.
* With metabolic activation: after a 3-hour treatment, D-Xylopyranoside, 2-octyldodecyl revealed a very strong cytotoxicity in L5178Y mouse lymphoma cells at the 2 highest concentrations tested of 250 and 125 μg/mL (with a total absence of cells). The lower concentrations of 62.5 and 31.25 μg/mL induced a strong and a moderate level of
cytotoxicity, respectively with RTGs of 1.2 and 51.8%. The concentration of 60 μg/mL was retained for the mutagenicity assay and a narrowed range of concentrations was chosen in order to reach a RTG comprised between 10 and 20%.
* Without metabolic activation: after a 24-hour treatment, D-Xylopyranoside, 2-octyldodecyl revealed a very strong cytotoxicity in L5178Y mouse lymphoma cells at the 3 highest concentrations tested ranging from 250 to 62.5 μg/mL (with a total absence of cells). The lower concentrations of 31.25 and 15.63 μg/mL induced a strong and no cytotoxicity, respectively with RTGs of 1.5 and 114%. The concentration of 30 μg/mL was retained for the mutagenicity assay and a narrowed range of concentrations was chosen in order to reach a RTG comprised between 10 and 20%.

Other: MUTAGENICITY ASSAYS:
In the assays with and without metabolic activation (following a short-term or continuous treatment), no biologically significant increases in the induced mutation frequency were noted at any of the concentrations tested. Otherwise, in the assay with metabolic activation, a statistically significant linear trend of mutant frequency was observed on the range of concentrations tested. Nevertheless, the highest induced mutation frequency (IMF) reached only +89.7 x10-6 mutants, that is to say clearly under the global evaluation factor (GEF) of +126 x10-6 for a biologically significant effect (Moore et al., 2006). In that case, this result was not considered as biologically relevant. Therefore, the test item was not mutagenic in both short-term assays with and without metabolic activation in the experimental condition employed. However, despite a narrow range of concentrations was chosen in the continuous treatment without metabolic activation, no concentrations allowing to reach a RTG within or even below the recommended range of 10-20% was obtained, and no definitive conclusion could be drawn regarding the mutagenic potential of the test item in this condition. Therefore, a complementary assay was performed with a higher and narrow range of concentrations.
In the complementary assay using a 24-hour continuous treatment without metabolic activation, as no concentrations inducing a RTG comprised between 10 and 20% were reached, the concentration of 45.37 μg/mL that induced a high level of cytotoxicity with 8.2% of RTG was retained for mutagenicity assessment. Regarding mutagenicity, no biologically significant increases in the induced mutation frequency were noted at any of the concentrations tested.

Conclusions:

Under these experimental conditions, the test item D-Xylopyranoside, 2-octyldodecyl, induced no mutagenic activity at the TK locus in L5178Y mouse lymphoma cell culture either with or without metabolic activation using short-term and long-term treatments, in duplicate conditions.

Executive summary:

In a mammalian cell gene mutation assay performed according to the OECD test guideline No. 490 and in compliance with GLP, L5178Y mouse lymphoma cells of the Chinese hamster were exposed to D-Xylopyranoside, 2-octyldodecyl diluted in Dimethylsulfoxide (DMSO), in duplicate in the presence and absence of metabolic activation (S9-mix).

Cytotoxicity assays were carried out both without and with metabolic activation using S9 from rat liver induced by Aroclor 1254:

- Without S9-mix : 250 – 125 –62.5– 31.25 – 15.63 – 7.81 – 3.91 (3-hour treatment), 250 – 125 –62.5– 31.25 – 15.63 – 7.81 – 3.91 (24-hour treatment)

- With S9-mix : 250 – 125 –62.5– 31.25 – 15.63 – 7.81 – 3.91 (3- hour treatment)

The Factor limiting the maximum concentration tested was solubility of the test item in the test system, i.e. 250 μg/mL.

Without metabolic activation, after 3 and 24 hours of treatment, the concentration of 30 μg/mL was retained for the mutagenicity assay and a narrowed range of concentrations was chosen in order to reach a RTG comprised between 10 and 20%.

With metabolic activation, after 3 -hour treatment, the concentration of 60 μg/mL was retained for the mutagenicity assay and a narrowed range of concentrations was chosen in order to reach a RTG comprised between 10 and 20%.

Mutagenicity assays were carried out both without and with metabolic activation using S9 from rat liver induced by Aroclor 1254.

- Without S9 mix : 30 – 26.09 – 22.7 – 19.72 – 13.15 – 6.57 µg/mL(3 and 24-hour treatment)

- With S9 mix : 60 – 52.17 – 45.36 – 39.45 – 26.30 – 13.15 µg/mL (3 -hour treatment)

In the assays with and without metabolic activation (following a short-term or continuous treatment), no biologically significant increases in the induced mutation frequency were noted at any of the concentrations tested. Otherwise, in the assay with metabolic activation, a statistically significant linear trend of mutant frequency was observed on the range of concentrations tested. Nevertheless, the highest induced mutation

frequency (IMF) reached only +89.7 x10-6 mutants, that is to say clearly under the global evaluation factor (GEF) of +126 x10-6 for a biologically significant effect (Moore et al., 2006). In that case, this result was not considered as biologically relevant.

Therefore, the test item was not mutagenic in both short-term assays with and without metabolic activation in the experimental condition employed. However, despite a narrow range of concentrations was chosen in the continuous treatment without metabolic activation, no concentrations allowing to reach a RTG within or even below the recommended range of 10- 20% was obtained, and no definitive conclusion could be drawn regarding the mutagenic potential of the test item in this condition. Therefore, a complementary assay was performed with a higher and narrow range of concentrations 60, 52.17, 45.36, 39.45, 34.3, 29.83, 25.94, 22.56, 19.61, 17.05 µg/mL (without metabolic activation, 24 hours of treatment).

In the complementary assay using a 24-hour continuous treatment without metabolic activation, as no concentrations inducing a RTG comprised between 10 and 20% were reached, the concentration of 45.37 μg/mL that induced a high level of cytotoxicity with 8.2% of RTG was retained for mutagenicity assessment. Regarding mutagenicity, no biologically significant increases in the induced mutation frequency were noted at any of the concentrations tested.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

Under these experimental conditions, the test item D-Xylopyranoside, 2-octyldodecyl, induced no mutagenic activity at the TK locus in L5178Y mouse lymphoma cell culture either with or without metabolic activation using short-term and long-term treatments, in duplicate conditions.

This study is considered as acceptable and satisfies the requirement for mammalian gene mutation endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- In vivo, in micronucleus test (OECD 474, K, Rel.1): not mutagenic in rats.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 April 2007 to 04 May 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

July 1997 version

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test Item Identification: APX 20P
- Form : Paste
- Colour : Brown
- Source and lot/batch No.of test material: Batch No. 1137JG
- Expiration date of the lot/batch: October 2007
- Purity : > 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored at room temperature in darkness.
- Solubility and stability of the test substance in the solvent/vehicle: The test item was soluble in olive oil.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was diluted in olive oil.and administered under a volume of 5 mL/kg body wieght.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
The test item was administered as a solution of the test item diluted in olive oil.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

50 Sprague Dawley rats (SPF Caw) originating from Elevage JANVIER (53940 Le Genest St Isle-France)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elevage JANVIER (53940 Le Genest St Isle –France),
- Age at study initiation: 7-week old (males) and 8-week old (females).
- Weight at study initiation: The male animals weighed between 208 g and 232 g and the female animales weighted between 187 g and 210 g .
- Assigned to test groups randomly: [yes, under following basis: ]The rats were assigned to the individual group by manual randomisation.
- Fasting period before study:
- Housing:The rats were kept by five under conventional conditions in Makrolon cages. Each cage was identified by a label indicating animal number, study number and start of experimentation.
- Diet (e.g. ad libitum): The rats were fed pelletized M20 Rat/mouse maintenance (SDS)
- Water (e.g. ad libitum):Tap water ad libitum.
- Acclimation period: Period of at least five days.

ENVIRONMENTAL CONDITIONS
Temperature and humidity were checked and recorded continuously:
- Temperature (°C): between 19 °C and 23 °C,
- Humidity (%): between 38% and 54%.
- Photoperiod (hrs dark / hrs light): Light-dark phases: 12 hours light, 12 hours dark, automatically controlled.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: [olive oil ]
- Amount of vehicle: volume of 5 mL/kg
- Lot/batch no. (if required): Batch : 06040254

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The animals of Groups 4 and 5, received an effective dose of 2000 mg/kg body weight of the test item APX 20 P, diluted in olive oil (Batch 06040254 /A) and administered by force-feeding under a volume of 5 mL/kg body weight using a suitable syringe graduated fitted with an oesophageal metal canula.
Animals from Groups 1 and 2 received in the same experimental conditions the control item (Distilled water –Batch 100179301) under a volume of 2 mL/kg body weight.
Animals from Group 3 received in the same experimental conditions, an effective dose of 50 mg/kg body weight of the positive control item (Cyclophosphamide – CAS N°[6055-19-2]) diluted in distilled water and administered under a volume of 2 mL/kg body weight.
With this dose, a statistically significant increase in the number of polychromatic erythrocytes with micronuclei, as compared to the negative control, was to be expected.

Rationale for selection of dose

In a preliminary study, two groups of 6 Sprague Dawley rats (3 female and 3 male) received
respectively 1000 mg/kg b.w and 2000 mg/kg b.w. of the test item APX 20 P diluted in olive oil, by
oral route under a volume of 5 mL/kg body weight.
No mortality occurred. No clinical signs related to the administration of the test item were observed during the 24 hours of observation. The macroscopical examination of the animals at the end of this preliminary study did not reveal any treatment related changes.

Therefore, 2000 mg/kg was selected as the highest dose and the test selected was a limit test. In effect, 2000 mg/kg is recommended as the maximum dose (MRD) by international accepted guidelines.

Duration of treatment / exposure:

Exposition for 24 hours and 48 hours.

Frequency of treatment:

Once

Post exposure period:

No post exposure period.

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

In a preliminary study, two groups of 6 Sprague Dawley rats (3 female and 3 male) received
respectively 1000 mg/kg b.w and 2000 mg/kg b.w. of the test item APX 20 P diluted in olive oil, by
oral route under a volume of 5 mL/kg body weight.
No mortality occurred. No clinical signs related to the administration of the test item were observed during the 24 hours of observation. The macroscopical examination of the animals at the end of this preliminary study did not reveal any treatment related changes.

Therefore, 2000 mg/kg was selected as the highest dose and the test selected was a limit test. In effect, 2000 mg/kg is recommended as the maximum dose (MRD) by international accepted guidelines.

No. of animals per sex per dose:

5 females and 5 males per group.

Control animals:

yes

other: Concurante vehicle positive control ( distilled water)

Positive control(s):

Cyclophosphamide - CAS N° [6055-19-2]
- Justification for choice of positive control(s):Statistically significant increase in the number of polychromatic erythrocytes with micronuclei, as compared to the negative control, was to be expected.
- Route of administration: oral route and administered under a volume of 2 mL/kg body weight.
- Doses / concentrations:50 mg/kg body weight

Tissues and cell types examined:

Bone marrow cells were obtained from the femurs immediately following sacrifice for examination.

Details of tissue and slide preparation:

The cells were prepared and stained using established methods. Smear preparations were made and then stained by conventional stains (e.g., Giemsa – VWR – Batch 498123).
The smears were stained for 2.5 minutes with May Grünwald stain (VWR – Batch 501220). The slides were rinsed with pH 7.0 buffer solution (VWR – Batches 565650 & 558347) and counterstained for 12 minutes with Giemsa R stain diluted 1:30 (v/v). The slides were then rinsed for 10 seconds and air drying before to be analysed.


The proportion of immature among total (immature + mature) erythrocytes is determined for each
animal by counting a total of at least 200 erythrocytes for bone marrow.
All slides, including those of positive and negative controls, should be independently coded before
microscopic analysis (x 1000 magnification). At least 1000 immature erythrocytes per animal were
scored for the incidence of micronucleated immature erythrocytes. Additional information may be
obtained by scoring mature erythrocytes for micronuclei.
When analysing slides, the proportion of immature erythrocytes among total erythrocytes should not
be less than 20% of the control value.

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in the number of micronucleated cells or a clear increase in the number of micronucleated cells in a single
dose group at a single sampling time. Biological relevance of the results were considered first.
Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determining factor for a positive response. Equivocal results should be clarified by further testing preferably using a modification of experimental conditions.


Positive results in the micronucleus test indicate that the item induces micronuclei which are the result of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test species.
Negative results indicate that, under the test conditions, the test item does not produce micronuclei in the immature erythrocytes of the test species.

Statistics:

Miclonuclei number on immature erythrocytes of the dose groups were compared with those of the
control, separately for each sex, using the multiple two-sided t-test according to Dunnett.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY

- Clinical signs of toxicity in test animals: No clinical signs related to the administration of the test item were observed during the 24 hours of observation.
- High dose with and without activation: 2000 mg/kg

RESULTS OF DEFINITIVE STUDY

No mortality occurred during the study.

The clinical observations are given in the observations data sheet, tables 1 to 5 and those of body
weight evolution in tables 6 to 10, hereafter.

No clinical signs related to the administration of the test item were observed.
The body weight evolution of the animals remained normal throughout the study, similar between
treated and control animals.
The results of the macroscopical examinations are given in the necropsy data sheet, tables 11 to 16, hereafter.
The macroscopical examination of the animals at the end of the study did not reveal treatment-related changes.

The results of the micronucleated cells examinations showed that at least 1000 polychromatic erythrocytes per animal, using 5 males and 5 females per group, were evaluated.
For the treated groups, the proportion of immature erythrocytes among total erythrocytes was not less than 20% of the control value.
The proportions of immature erythrocytes among total erythrocytes are given in appendix 4.

The negative control values were all in the expected range predetermined as historical controls.
For the positive control (male and female), a statistically significant increase in polychromatic
erythrocytes with micronuclei was established (p<0.001 in the male treated group and p<0.01 in the female treated group).
For the treated group (male and female), no significant increase in the number of polychromatic erythrocytes with micronuclei was recorded at the reading 24 hours and at the reading 48 hours.

Mean numbers of polychromatic erythrocytes with micronuclei per 1000 PCE

Dose : 2000 mg/kg body weight

Route: oral

Administration date : 04 April 2007

Species: Sprague Dawley Rats

Test item	Dose mg/kg	Preparation time [h]	MN-PCE[0/00] males	MN-PCE [0/00] females
Negative control	0 0	24 48	5.4 6.4	6.6 8.2
PH-06/0391 APX 20P	2000 2000	24 48	9.4 9.2	9.4 9.2
Cyclophosphamide	50	24	29**	25*

(**) : p< 0.001% (*): p<0.01%

PCE: Polychromatic erythrocytes

Conclusions:

The results obtained, under these experimental conditions, enable to conclude that the test item was not mutagenic in the micronucleus test in rats.

Executive summary:

The miconucleus test was performed according to Commission directive 2000/32/EC (Test method

B.12) and the OECD Guideline n°474 (dated 21 July 1997).

The test item was given once orally by gavage to male and female rats, at the dose of 2000 mg/kg body weight. Rats of the negative group received the control item (distilled water) and the

positive control group were treated with an oral dose of 50 mg cyclophosphamide /kg body weight.

Bone marrow smears were prepared from femurs of each animal and stained with Giemsa’s solution.

Result:

The treated group (male and female) showed no significant increase in the number of polychromaticerythrocytes with micronuclei, at the reading time 24 hours and 48 hours.

The positive treated group (male and female) showed the expected significant increase in the number of polychromatic erythrocytes with micronuclei (p<0.001 in the male treated group and p<0.01 in the female treated group).

Conclusion:

The results obtained, under these experimental conditions, enable to conclude that the test item

was not mutagenic in the micronucleus test in rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

 

Table 7.6/1: Summary of genotoxicity tests (In vitro):

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   Safepharm, 2003	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537 TA 98 TA 100 WP2 uvrA	-S9 +S9	Up to 5000 µg/plate	-S9 : non mutagenic +S9 : non mutagenic
2   Institut Pasteur, 2020	ML/TK (OECD 490) K, rel. 1)	Gene mutation	mouse lymphoma L5178Y cells	-S9 +S9	Up to 30 µg/mL	-S9 : non mutagenic +S9 : non mutagenic
3  IIBAT , 2007	CA/HL test (OECD 473) K, rel. 1	Chromosomal aberration	Human Lymphocytes	-S9 +S9	Up to 300 µg/mL	-S9 : non mutagenic +S9 : non mutagenic

 

Gene mutation Assays (Tests n° 1 ):

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD 471 test guideline with the test material (See Table 1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.Therefore, the test material does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies.The test material is therefore considered as non-mutagenic according to the Ames test.

 

Inability to produce gene mutation was confirmed in mammalian cells using an in vitro forward mutation assay in mouse lymphoma TK L5178Y cells (ML/TK test) (Test n°2). None of the dose levels up to the cytotoxicity limit with the test material, either with or without metabolic activation, induced significant mutant frequency increases in the initial or repeat tests. The test material does not induce forward mutations at the TK locus in L5178Y mouse lymphoma cells under activation and non activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases.The test material is therefore considered as negative for inducing forward mutations at the TK locus in L5178Y mouse lymphoma cells under activation and non-activation conditions used in this assay. This result confirms the results of Ames test and extends the non-mutagenic effect of the test material from bacteria to mammalian cells.

 

Gene mutation test (Test n°2)

In a mammalian cell gene mutation assay performed according to the OECD test guideline No. 490 and in compliance with GLP, L5178Y mouse lymphoma cells of the Chinese hamster were exposed to D-Xylopyranoside, 2-octyldodecyl diluted in Dimethylsulfoxide (DMSO), in duplicate in the presence and absence of metabolic activation (S9-mix).

Mutagenicity assays were carried out both without and with metabolic activation using S9 from rat liver induced by Aroclor 1254.

- Without S9 mix : 30 – 26.09 – 22.7 – 19.72 – 13.15 – 6.57 µg/mL(3 and 24-hour treatment)

- With S9 mix : 60 – 52.17 – 45.36 – 39.45 – 26.30 – 13.15 µg/mL (3 -hour treatment)

In the assays with and without metabolic activation (following a short-term or continuous treatment), no biologically significant increases in the induced mutation frequency were noted at any of the concentrations tested. Otherwise, in the assay with metabolic activation, a statistically significant linear trend of mutant frequency was observed on the range of concentrations tested. Nevertheless, the highest induced mutation frequency (IMF) reached only +89.7 x10-6 mutants, that is to say clearly under the global evaluation factor (GEF) of +126 x10-6 for a biologically significant effect (Moore et al., 2006). In that case, this result was not considered as biologically relevant.

Therefore, the test item was not mutagenic in both short-term assays with and without metabolic activation in the experimental condition employed. However, despite a narrow range of concentrations was chosen in the continuous treatment without metabolic activation, no concentrations allowing to reach a RTG within or even below the recommended range of 10- 20% was obtained, and no definitive conclusion could be drawn regarding the mutagenic potential of the test item in this condition. Therefore, a complementary assay was performed with a higher and narrow range of concentrations 60, 52.17, 45.36, 39.45, 34.3, 29.83, 25.94, 22.56, 19.61, 17.05 µg/mL (without metabolic activation, 24 hours of treatment).

In the complementary assay using a 24-hour continuous treatment without metabolic activation, as no concentrations inducing a RTG comprised between 10 and 20% were reached, the concentration of 45.37 μg/mL that induced a high level of cytotoxicity with 8.2% of RTG was retained for mutagenicity assessment. Regarding mutagenicity, no biologically significant increases in the induced mutation frequency were noted at any of the concentrations tested.

Under these experimental conditions, the test item D-Xylopyranoside, 2-octyldodecyl, induced no mutagenic activity at the TK locus in L5178Y mouse lymphoma cell culture either with or without metabolic activation using short-term and long-term treatments, in duplicate conditions.

 

Chromosomal aberration (Test n°3)

The clastogenic potential of the test material was determined using an in vitrochromosome aberration test in human lymphocytes, which measures the potential of a substance to increase the incidence the of structural chromosome aberrations in cultured human lymphocytes.

None of the dose levels up to the cytotoxicity limit with the test material, either with or without metabolic activation, induced significant increases in the frequency of cells with aberrations in either of two experiments. The test material does not induce structural aberrations in the chromosomes of human lymphocytes under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells.The test material is therefore considered as negative for inducing chromosomal mutations in human lymphocyte cells under activation and non-activation conditions used in this assay.

 

In vivo Micronucleus test (OECD 474):

 

One study was available and considered as key study (OECD 474, Rel.1). In this study, the test item was given once orally by gavage to male and female rats, at the dose of 2000 mg/kg body weight. The treated group (male and female) showed no significant increase in the number of polychromaticerythrocytes with micronuclei, at the reading time 24 hours and 48 hours.

The positive treated group (male and female) showed the expected significant increase in the number of polychromatic erythrocytes with micronuclei (p<0.001 in the male treated group and p<0.01 in the female treated group). The test substancewas not mutagenic in the micronucleus test in rats.

 

Conclusion: based on in vitro and in vivo studies, the test substance is considered as not genotoxic.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

 

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the Annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.