2,5-xylenol

Genetic toxicity: in vivo

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

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

Test material information

Constituent 1

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 / concentrations

Doses / concentrations 1

Dose / conc.:

250 other: mg/kg bw

Remarks:

24 h

Doses / concentrations 2

Dose / conc.:

500 other: mg/kg bw

Remarks:

24 h

Doses / concentrations 3

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.