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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
20 October 1997 to 28 October 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
2,6-xylenol
EC Number:
209-400-1
EC Name:
2,6-xylenol
Cas Number:
576-26-1
Molecular formula:
C8H10O
IUPAC Name:
2,6-dimethylphenol

Test animals

Species:
mouse
Strain:
NMRI
Details on species / strain selection:
no data
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH, WIGA, Sulzfeld, Germany
- Age at study initiation: not stated
- Weight at study initiation: not stated; however mean wt of animals for the study was 26.9 g
- Assigned to test groups randomly: yes, under following basis: according to a randomisation plan prepared by computer programme
- Fasting period before study: not stated
- Housing: individually during study period
- Diet: standarised pellet feed, ad libitum
- Water: tap water, ad libitum
- Acclimation period: not stated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24˚C
- Humidity (%): 30 - 70%
- Air changes (per hr): not stated
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
Details on exposure:
no data
Duration of treatment / exposure:
Single oral administration
Frequency of treatment:
Single oral administration
Post exposure period:
24 and 48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
250 other: mg/kg bw
Remarks:
24 h
Dose / conc.:
500 other: mg/kg bw
Remarks:
24 h
Dose / conc.:
1 000 other: mg/kg bw
Remarks:
24 h and 48 h
No. of animals per sex per dose:
5 animals/dose/sex
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide; vincristine
- Route of administration: oral
- Doses / concentrations: cyclophosphamide: 20 mg/kg bw and vincristine: 0.15 mg/kg

Examinations

Tissues and cell types examined:
Clinical observations: post dosing (no further details presented); Polychromatic erthyrocyte (PCE) examined for the presence of micronuclei. PCE and normochromatic erthyrocytes (NCE) counted as a measure of toxicity.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The maximum dose assessed was deeemed the maximum tolerated dose (MTD)

METHOD OF ANALYSIS: The presence of micronculei were examined in 2000 PCE. In this count, the number of NCE were scored as a measure of toxicity.

Evaluation criteria:
Acceptance criteria:
- at least 2000 PCE we available for assessing MN
- The proportion of MN PCE for the negative control were within the historical conrol (stated as 1.1 to 3 MN PCE/1000 PCE scored, equivalent to 2.2 to 6.0 MN PCE/2000 PCE scored).
- The positive control induced a significant increase in the number of MN PCE over that of the concurrent control

Evaluation citeria
For a positive response:
- dose related increase in the number of MN PCE at any interval
- proportion of MN PCE exceeded the both the concurrent vehicle control and the historical control data

For a negative response:
- No signficiant increase in the number of MN PCE at any dose level
- The frequency of MN PCE were within the historical control
Statistics:
Comparison of the dose group with the vehicle control using the Wilcoxon test (one-sided).

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Clinical signs of toxicity - body weight loss.
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY: A range-finder experiment was undertaken to determine an MTD. Doses were selected from a pilot toxicity study. Doses of 750 and 1000 mg/kg bw were administered. Mortality (1 male) was observed at each dose, and clincial signs of toxicity were evident. The MTD was therefore deemed to be 1000 mg/kg by the SD.

Any other information on results incl. tables

Table 1: Male and female combined data - 24hr time point

Dose (mg/kg)

PCE

NCE

Total

%PCE

MN PCE

0

20000

7942

27942

39.7

1.5

250

20000

7509

27509

37.5

0.8

500

20000

6318

26318

31.2

1.0

1000

20000

8927

28927

44.6

0.6

CPA 20

10000

4610

104610

46.1

11.6**

VIN 0.15

10000

4721

14721

47.2

60.9**

CPA - cyclophosphamide

VIN - Vincristine

** p<0.01

Table 2: Male and female combined data - 48hr time point

Dose (mg/kg)

PCE

NCE

Total

%PCE

MN PCE

0

20000

7963

27963

39.8

0.6

1000

20000

9178

29178

63

0.7

Deaths (1 male and 1 female) at 1000 mg/kg, 48 hr sample point were observed. Clinical signs of toxicity included piloerection, squatting posture at the maximum dose. There were no marked decreases in mean PCE/total erythrocyte ratio observed for any of the 2,6 xylenol treated groups compared to the vehicle control group for either time point. Analysis of the mean MN PCE group data indicated that there was no statistically significant increases MN PCE frequency compared to concurrent control values for either sex. Individual animal and group mean MN PCE frequencies were consistent with both the concurrent vehicle control values and the historical control. Positive control treatment induced the appropriate response. 

Formulation analysis confirmed the suitability of the doses prepared.

Applicant's summary and conclusion

Conclusions:
It is concluded that 2,6-xylenol did not induce micronuclei in the polychromatic erthrocytes of the bone marrow following sampling at 24 and 48 hours post dosing of both male and female mice when tested at a dose of 1000 mg/kg. Whilst this dose was deemed a maximum tolerated dose by the SD under the conditions of the assay described, due to the mortality observed this would confirm that the MTD had been exceeded.
Executive summary:

In a bone marrow micronucleus assay using NMRI mice, a single oral gavage of 2,6-xylenol was administered to groups of male and female animals, employing a dose volume of 10 mL/kg. Doses were selected from a pilot toxicity study and doses of 750 and 1000 mg/kg bw were administered. Mortality (1 male) was observed at each dose, and clincial signs of toxicity were evident. The MTD was therefore deemed to be 1000 mg/kg by the SD.

 

Negative control groups were treated with vehicle only (olive oil) and positive control groups were treated with the clastogen, cyclophosphamide (CPA, 20 mg/kg bw) or the aneugen, vincristine (0.15 mg/kg). Mouse bone marrow was sampled at 24 and 48 hours after dosing for the vehicle and 2,6 -xylenol dosed groups. A single sampling time of 24 hours after dosing was used for both positive control groups. Slides of bone marrow cells were prepared from five animals/sex/time point for each group and scored for the occurrence of micronucleated polychromatic erythrocytes (MN PCE) and PCE/total erythrocyte ratios.

 

Deaths (1 male and 1 female) at 1000 mg/kg, 48 hr sample point were observed. Clinical signs of toxicity included piloerection, squatting posture at the maximum dose. There were no marked decreases in mean PCE/total erythrocyte ratio observed for any of the 2,6 xylenol treated groups compared to the vehicle control group for either time point.

 

Analysis of the mean MN PCE group data indicated that there was no statistically significant increases MN PCE frequency compared to concurrent control values for either sex. Individual animal and group mean MN PCE frequencies were consistent with both the concurrent vehicle control values and the historical control. Positive control treatment induced the appropriate response. 

Formulation analysis confirmed the suitability of the doses prepared.

It is concluded that 2,6-xylenol did not induce micronuclei in the polychromatic erythrocytes of the bone marrow following sampling at 24 and 48 hours post dosing of both male and female mice when tested at a dose of 1000 mg/kg. Whilst this dose was deemed a maximum tolerated dose by the SD under the conditions of the assay described, due to the mortality observed this would confirm that the MTD had been exceeded.