Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 15 sep 2008 to 04 mar 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
See comments in "principles of methods" below.
Principles of method if other than guideline:
• The test item did dissolve in PEG400 but did not dissolve in an aqueous solution of 2 % CMC.
• In the first pre-experiment instead of corn oil PEG400 was used as vehicle. All other pre-experiments were performed using corn oil as vehicle.
• In the third pre-experiment the 30h observation interval for clinical signs of toxicity was not performed.
• These deviations, however, do not affect the outcome of the study.
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH D-33178 Borchen
- Age at study initiation: males and females: 8 - 12 weeks
- Weight at study initiation: the weight variation should not exceed +/- 20 % of the mean weight of each sex
- Assigned to test groups randomly: yes, the animals will be distributed into the test groups at random and identified by cage number.
- Fasting period before study: none
- Housing: single.
- Cage Type: Makrolon Type I (20 cm (l) x 10 cm (w) x 12 cm (h)), with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
- Diet: pelleted standard diet, ad libitum Harlan Winkelmann GmbH, D-33178 Borchen)
- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- temperature: 22 + 3 °C
- relative humidity: 30 - 70 %
- artificial light: 6.00 a.m. - 6.00 p.m.

IN-LIFE DATES: From 15 sep 2008 To 4 march 2009.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
The vehicle of the test item will be used as negative control.
Identity: corn oil
Supplier: Sigma-Aldrich Vertriebs GmbH
82041 Deisenhofen
Route and frequency of administration: consistent with the test item
Volume administered: consistent with the test item
Details on exposure:
Six males and six females are assigned to each test group.
At the beginning of the treatment the animals (including the controls) are weighed and the individual volume to be administered is adjusted to the
animal‘s body weight. The animals will receive the test item, the vehicle, or the positive control substance once. Twelve animals, six males and six
females, will be treated per dose group and sampling time. The animals of all dose groups, except the positive control will be examined for acute toxicsymptoms at intervals of around 1 h, 2-4 h, 6 h, 24 h, and 48h after administration of the test item. Sampling of the bone marrow is done 24 and 48
hours after treatment, respectively.
Duration of treatment / exposure:
48 hours
Frequency of treatment:
once
Post exposure period:
none
Doses / concentrations
Remarks:
Doses / Concentrations:
125, 250, 500 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
6
Control animals:
yes, concurrent no treatment
Positive control(s):
Name: CPA; Cyclophosphamide
Supplier: Sigma-Aldrich Vertriebs GmbH
82041 Deisenhofen
Catalogue no.: C 0768 (purity: > 98 %)
Dissolved in: deionised water
Dosing: 40 mg/kg b.w.
Route and frequency of administration: orally, once
Volume administered: 10 mL/kg b.w.
Solution prepared on day of administration.
The stability of CPA at room temperature is good. At 25 °C only 3.5 % of its potency is lost after 24 hours.

Examinations

Tissues and cell types examined:
Erythrocytes
Details of tissue and slide preparation:
Evaluation of the slides is performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE)
are analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes is
determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis is performed with coded slides.
Five animals per sex and test group will be evaluated as described. The remaining 6th animal of each sex will be evaluated in case of animal died in its test group spontaneously.
Evaluation criteria:
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic
erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test (8)) will be used as an aid in evaluating the results.
However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered
non-mutagenic in this system.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
The study is considered valid if the following criteria are met:
- the negative controls are in the range of our historical control data (see section 6.6).
- the positive controls are in the range of our historical control data (see section 6.6).
- at least 5 animals per group and sex are evaluable
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.

Any other information on results incl. tables

In the main experiment for the highest dose group 24 animals (12 males, 12 females) received orally a single dose of 500 mg/kg b.w. Guaiacol - CAS#90-05-1 formulated in corn oil. The volume administered was 10 mL/kg b.w.

The animals treated with 500 mg/kg b.w. expressed toxic reactions as shown in the table:

Toxic

hours post-treatment

hours post-treatment

Reactions

male / female

1 h

2-4 h

6 h

24 h

48 h*

reduction of spontaneous activity

12/12

12/12

12/12

12/12

6/6

abdominal position

3/6

0/3

0/0

0/0

0/0

eyelid closure

2/5

0/1

0/0

0/0

0/0

ruffled fur

12/12

12/12

12/12

12/12

6/6

tumbling

3/4

0/1

0/0

0/0

0/0

apathy

4/6

0/3

0/2

0/0

0/0

Bloody eyes

0/0

0/0

0/0

12/12

0/0

discoloured urine

+

+

-

-

-

death

0/0

0/0

0/1

0/0

0/0

*: data only from 6 animals per sex.

For the mid dose group 12 animals (6 males, 6 females) received orally a single dose of 250 mg/kg b.w. Guaiacol - CAS#90-05-1 formulated in corn oil. The volume administered was 10 mL/kg b.w.

The animals treated with 250 mg/kg b.w. expressed toxic reactions as shown in the table:

toxic

hours post-treatment

reactions

male / female

1 h

2-4 h

6 h

24 h

reduction of spontaneous activity

6/6

6/6

4/6

0/0

ruffled fur

6/6

6/6

6/6

2/2

For the low dose group 12 animals (6 males, 6 females) received orally a single dose of 125 mg/kg b.w. Guaiacol - CAS#90-05-1 formulated in corn oil. The volume administered was 10 mL/kg b.w..

The animals treated with 125 mg/kg b.w. expressed toxic reactions as shown in the table:

toxic

hours post-treatment

reactions

male / female

1 h

2-4 h

6 h

24 h

reduction of spontaneous activity

6/6

6/6

0/3

0/0

ruffled fur

6/6

6/6

6/6

0/0

The animals treated with the vehicle control (corn oil) did not express any toxic reactions.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Negative in micronucleus assay.
Executive summary:

The study RCC, 2009 was performed to investigate the potential of Guaiacol to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was formulated in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Twelve animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

The following dose levels of the test item were investigated:

24 h preparation interval: 125, 250, and 500 mg/kg b.w..
48 h preparation interval: 500 mg/kg b.w..

The highest dose (500 mg/kg) was estimated by a pre-experiment to be suitable. In the main study 1 female (animal no. 46) died after treatment with this dose.

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that Guaiacol - CAS#90-05-1 did not exert any cytotoxic effects in the bone marrow. However, the discoloured urine of the test item treated animals indicated the systemic distribution of the test item, thus confirming the test items bioavailability.

The analysis of the rectal temperature of the animals showed test item induced hypothermia. This effect was most prominently observed in the high dose group at the 1 h post treatment interval.

In comparison to the corresponding vehicle controls there was no biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.

40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Therefore, Guaiacol is considered to be non-mutagenic in this micronucleus assay.