D-Xylopyranose, oligomeric, 2-octyldodecyl xylosides

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

464-320-6

EC Name:

-

Cas Number:

423772-95-6

Molecular formula:

(C5H9O4)(C5H8O4)n-1-O-C20H41, n = number of D-xylopyranose units

IUPAC Name:

D-Xylopyranose, oligomeric, 2-octyldodecyl xylosides

Details on test material:

- Identification: APX 20 P
- Supplier: SEPPIC
- Appearance: brown paste
-Batch : 270LG

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.

Test animals

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.

Administration / exposure

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.

Doses / concentrations

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

Examinations

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.