Guaiacol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gaiacol is negative in Ames tests with or without metabolic activation (published data).
Positive results observed without metabolic activation in chromosomal aberrations and SCE test with Syrian Hamster embryo (published data).

An in vivo study was done for clarification.

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The purity of the test substance was known. The authors cited the Ames et al. (1975) methods paper with a detailed protocol following the cited procedure. The results were described in details.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 pre-treated Sprague-Dawley rats.

Test concentrations with justification for top dose:

0, 0.5, 5, 50, 500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see details below

Details on test system and experimental conditions:

POSITIVE CONTROL(S) SUBSTANCE:
2-Nitrofluorene: 3 µg/plate for TA 98 and TA 1538 without metabolic activation
Sodium azide: 2 µg/plate for TA 1535 and TA 100 without metabolic activation
9-Aminoacridine: 60 µg/plate for TA 1537 without metabolic activation
2-Aminoanthracene: 5 µg/plate for all strains with metabolic activation

DETAILS ON TEST SYSTEM AND CONDITIONS:
S. Typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 were kindly supplied by Prof. B.N. Ames (University of Berkeley, Calif., USA).
The test for their reversion to histidine prototrophy was performed using the plate incorporation assay as previously described (Ames, McCann & Yamasaki, 1975). Each compound was dissolved in DMSO and tested in 5 concentrations in the presence of buffer ph 7.4 or S9 mix.

METHOD OF APPLICATION: in agar (plate incorporation)
Exposure duration: 48 hours

Evaluation criteria:

The number of histidine revertants were scored and compared to the number of spontaneously arising revertants.

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the concentration of 5000 µg/plate resulted in toxicity which was apparent as a thinning of the background lawn.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table of results:

	Concentration [µg/plate]	No. of revertants per plate	No. of revertants per plate	Cytotoxicity
				(yes/no)
		— MA	+ MA
TA 100	0	67	76	no
	0,5	66	94	no
	5	71	93	no
	50	65	81	no
	500	67	95	no
	5000	57	64	no
	Sodium azide / 2-Aminoanthracene	502	723	no
TA 1535	0	24	9	no
	0,5	21	16	no
	5	26	13	no
	50	22	19	no
	500	15	16	no
	5000	25	9	no
	Sodium azide / 2-Aminoanthracene	526	306	no
TA 1537	0	5	14	no
	0,5	7	12	no
	5	7	16	no
	50	4	13	no
	500	4	9	no
	5000	0	13	yes (-S9)
	9-Aminoacridine / 2-Aminoanthracene	810	269	no
TA 98	0	19	19	no
	0,5	20	34	no
	5	18	36	no
	50	23	14	no
	500	22	16	no
	5000	16	5	yes (+S9)
	2-Nitrofluorene/ 2-Aminoanthracene	275	1093	no
TA 1538	0	5	10	no
	0,5	13	12	no
	5	9	11	no
	50	10	11	no
	500	14	9	no
	5000	13	10	no
	2-Nitrofluorene/ 2-Aminoanthracene	284	1027	no

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

- INTERPRETATION OF RESULTS:
Regarding results and applying actual criteria evaluation, the test item presented no mutagenic activity with and without S9 mix in
Salmonella Typhimurium in all strains.
Reproductibility between the experiment and positive control results in accordance with positive criteria, validated the study.

Executive summary:

In a reverse gene mutation assay in bacteria (Pool et al., 1982), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to guaiacol at concentrations of 0 to 5000 µg/plate in the presence and absence of mammalian metabolic activation.

Guaiacol was tested up to cytotoxic concentrations and was negative.

Cytotoxicity was observed at 5000 µg/plate.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

Reference 2

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The protocol and the results were described with details. However, the purity substance was unknown and the GLP were not mentioned.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

4 bacterial strains were used instead of 5.

Principles of method if other than guideline:

Gaiacol was tested using the preincubation procedure of the Salmonella assay (Ames et al, 1975) as described by Yahagi et al. (1975).

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

see table in "details on test system"

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 3333, 11740 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: : DMSO was used because gaiacol was either insoluble or not sufficiently soluble in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see remarks

Remarks:

2-Aminoanthracene: for all strain in the presence of rat and hamster S9. 4-Nitro-o-phenylenediamine: for TA 98 without S9. Sodium azide: for TA 100 and TA 1535 without S9. 9-Aminoacridine: for TA 1537 without S9.

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours

In a preliminary study, chemical was checked for toxicity to TA 100 up to a concentration of 10 mg/plate or the limit of solubility, both with and without metabolic activation. If toxicity was no apparent, the highest dose tested is 10 mg/plate, otherwise the upper limit of solubility was used. If toxicity was observed, the doses of test chemical were chosen so that the high dose exhibited some degree of toxicity.

Evaluation criteria:

a positive response was indicated by a reproductible, dose-related increase, whether it be two-fold over background or not.

Statistics:

no data

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

10000 and 11740 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537

Table of results:

	Concentration [µg/plate]	No. of revertants per plate**	No. of revertants per plate**	No. of revertants per plate**	Cytotoxicity
					(yes/no)
		— MA	+ MA (RLI)	+ MA (HLI)
TA 100	0	92	145	150	no
	333	125	167	175	no
	1000	111	181	194	no
	3333	102	152	130	no
	10000	93	150	131	no
	11740	86	159	157	no
	positive control	432	838	966	no
TA 1535	0	13	9	16	no
	333	13	11	13	no
	1000	19	12	13	no
	3333	17	10	35	no
	10000	15	10	9	no
	11740	14	10	9	no
	positive control	756	53	162	no
TA 1537	0	8	9	9	no
	333	7	6	9	no
	1000	6	7	9	no
	3333	5	6	6	no
	10000	t	4	7	yes without MA
	11740	t	5	8	yes without MA
	positive control	623	28	51	no
TA 98	0	18	29	23	no
	333	13	28	23	no
	1000	12	26	18	no
	3333	11	24	20	no
	10000	t	18	18	yes without MA
	11740	t	21	17	yes without MA
	positive control	235	406	598	no

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

- INTERPRETATION OF RESULTS:
Regarding results and applying actual criteria evaluation, the test item presented no mutagenic activity with and without S9 mix in Salmonella
Typhimurium in all strains.
Reproductibility between the experiment and positive control results in accordance with positive criteria, validated the study.

Executive summary:

In a reverse gene mutation assay in bacteria (Haworth et al., 1983), strains TA 98, TA 100, TA 1535, TA 1537 of S. typhimurium were exposed to guaiacol at concentrations of 0 to 11740 µg/plate in the presence and absence of mammalian metabolic activation.

Guaiacol was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains.

There was no evidence of induced mutant colonies over background. Cytotoxicity was observed at 10000 and 11740 µg/plate.

This study is classified as acceptable (Klimish 2) and satisfies the requirement for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No GLP informations. Protocol and test results detailed, test only without metabolic activation system.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

no metabolic activation, no positive controls

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: Syrian Hamster embryo (SHE) cell

Details on mammalian cell type (if applicable):

- Type and identity of media: IBR Dilbecco's modified Eagle's reinforced medium (Biolab, Northbrook, IL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no

Metabolic activation:

without

Test concentrations with justification for top dose:

0; 200; 400; 800 and 1000 µM

Vehicle / solvent:

Vehicle(s)/solvent(s) used: none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

Determination of cytotoxicity:
Method: other: colony-forming efficiencies of Syrian hamster embryo cells.
Cytotoxicity of gaiacol tested was determined by the colony-forming efficiencies of SHE cells treated with this agent. SHE cells (5 x 10E5) in tertiary
culture were plated into 75-cm² flasks (Costar, Cambridge, MA, USA), incubated overnight, and treated with gaiacol at varying concentrations for 24h. After harvesting with 0.1% trypsin, the cells were replated in triplicate onto 100-mm dishes (Costar) at 2000 cells/dish and incubated for 7 days for
colony formation. The relative colony-forming efficiency was expressed as the number of colonies in the treated dishes divided by the number in the control dishes x 100. Actual colony-forming efficiency of control cells was 13.0 ± 0.7% (S.D.).

Chromosome aberrations:
SHE cells (5 x I0E5) in tertiary culture were plated into 75-cm² flasks, incubated overnight, and treated with gaiacol at varying concentrations for 24 h. SHE cells have the doubling time of 16 h and the treatment time is appropriate for detecting chromosome aberrations by diverse chemical carcinogens. The cells were harvested with 0.1% trypsin for chromosome preparation. Three hours before harvest, Colcemid (GIBCO, Grand Island, NY, USA) was administered at 0.2 µg/ml and metaphase chromosomes were prepared as described previously. For determination of chromosome aberrations, 100 metaphases were scored per experimental group. Achromatic lesions greater than the width of the chromatid were scored as gaps unless there was displacement of the broken piece of chromatid. If there was displacement, they were scored as breaks.

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: overnight
- Exposure duration: 24 hours
- Expression time (cells in growth medium): none
- Fixation time (start of exposure up to fixation or harvest of cells): 1.5 days

SPINDLE INHIBITOR: Colcemid:
STAIN (for cytogenetic assays): 3% Giemsa
NUMBER OF REPLICATIONS: no data
NUMBER OF CELLS EVALUATED: 100 metaphases per experimental group
OTHER EXAMINATIONS: type of aberrations observed: chromatid gap, isochromatid gap, chromatid breaks, isochromatid breaks, exchanges, ring chromosomes, dicentric chromosomes, fragmentations.

Evaluation criteria:

percentage of aberrant metaphases

Statistics:

X2 test, P <0.01

Species / strain:

mammalian cell line, other: Syrian Hamster embryo (SHE) cell

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

other: strain/cell type: Syrian Hamster embryo (SHE) cell

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
positive without metabolic activation

Positive results without metabolic activation.

Executive summary:

In a Chromosomal aberration test (Hikiba, 2005), Syrian Hamster Embryo cells were exposed to guaiacol at concentrations of 0 to 1000 µM in the absence of mammalian metabolic activation. Guaiacol was positive.

The positive controls were not examined.

This study is classified as acceptable and satisfies the requirement for Test Guideline for in vitro cytogenetic mutagenicity data. 

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No GLP informations. Test performed only without metabolic activation, and 2 doses were used instead of 3.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Deviations:

yes

Remarks:

no metabolic activation, no positive controls

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

mammalian cell line, other: Syrian Hamster embryo (SHE) cells

Details on mammalian cell type (if applicable):

- Type and identity of media: IBR Dilbecco's modified Eagle's reinforced medium (Biolab, Northbrook, IL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no

Metabolic activation:

without

Test concentrations with justification for top dose:

0, 100 and 300 µM

Vehicle / solvent:

Vehicle(s)/solvent(s) used: none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

Determination of cytotoxicity:
Method: other: colony-forming efficiencies of Syrian hamster embryo cells.
Cytotoxicity of gaiacol tested was determined by the colony-forming efficiencies of SHE cells treated with this agent. SHE cells (5 x 10E5) in tertiary culture were plated into 75-cm² flasks (Costar, Cambridge, MA, USA), incubated overnight, and treated with gaiacol at varying concentrations for 24 h. After harvesting with 0.1% trypsin, the cells were replated in triplicate onto 100-mm dishes (Costar) at 2000 cells/dish and incubated for 7 days for
colony formation. The relative colony-forming efficiency was expressed as the number of colonies in the treated dishes divided by the number in the control dishes x 100.

SCEs:
Cells (5 x I0E5) were plated into 75-cm² flasks, incubated overnight, and treated with gaiacol at varying concentrations for 24 h in the presence of 5-bromodeoxyuridine (10 µg/ml) under dark conditions. Three hours before the end of treatment, Colcemid was added to give a final concentration of 0.2 µg/ml, and metaphase chromosomes were spread on glass slides. The slides were stained for 15 min in a solution of Hoechst 33258 (50 µg/ml in water), washed, dipped in PBS(-), and exposed to near UV from a row of 6 F15TB/BLB fluorescent bulbs (33 J/m²/s; Sylvania, Springfield, USA) for 1
hour at 55°C. The slides were then stained with 3% Giemsa solution for 10 min. 30 second-division metaphases with the diploid number of chromosomes were analysed for SCE frequency.

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: overnight
- Exposure duration: 24 hours
- Expression time (cells in growth medium): none
- Fixation time (start of exposure up to fixation or harvest of cells): 1.5 days

SPINDLE INHIBITOR: Colcemid:
STAIN (for cytogenetic assays): 3% Giemsa, Hoechst 33258
NUMBER OF REPLICATIONS: no data
Number of cells evaluated: 30

Evaluation criteria:

Thirty second-division metaphases with the diploid number of chromosomes were analysed for sister chromatid exchange frequency.

Statistics:

P <0.01, Student t test.

Species / strain:

mammalian cell line, other: Syrian Hamster embryo (SHE) cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks on result:

other: strain/cell type: Syrian Hamster embryo (SHE) cells

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
positive without metabolic activation

Positive results without metabolic activation.

Executive summary:

To evaluate the genotoxic potential of Guaiacol used in dental practice, the abilities of this agent to induce sister-chromatid exchanges (SCEs) were examined using Syrian hamster embryo (SHE) cells (Miyachi, 2005). Statistically significant increases in the frequencies of SCEs were observed in SHE cells treated with guaiacol (P 0.01; Student test) without metabolic activation.

Because SCE assays are used as a sensitive indicator for evaluating genetic toxicity of chemicals, the chemical agents that had a positive response in the present study are potentially genotoxic to mammalian cells.

This study is classified as acceptable and satisfies the requirement for Test Guideline for in vitro cytogenetic mutagenicity data. 

Genetic toxicity in vivo

Description of key information

Negative result in micronucleus assay in vivo (study report, 2009).

Link to relevant study records

Reference

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

Qualifier:

according to guideline

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. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system 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.

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

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.

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.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Fourteen studies were available concerning genotoxicity in vitro: Ames tests (8 studies), Gene Mutation Assay on Saccharomyces cerevisiae (1 study), Chomosomal aberrations (1 study), Sister chromatid exchange (2 studies), DNA damage and/or repair (1 study) and human pulp fibroblast (1 study).

Ames studies:

2 studies (Pool, 1982 and Haworth, 1983) were selected as key studies because they were well described (protocol and results), and 2 others (Florin, 1980 and Aeschbacher, 1989) were selected as supporting studies, because only one dose was tested and the results were not detailed. All these studies were well described and are reliability 2 according to Klimich ranking. In these studies, several strains of Salmonella typhimurium were tested, TA 98, TA 100, TA 1535, TA 1537, TA 1538, TA 102, and different doses from 0 to 11740 µg/plate were tested.

All results in these 4 studies were negative with and without metabolic activation system.

Other available studies have reliability 3 or 4 and were not taken into account for the assessment.

Chromosomal aberration:

Only one study was available (Hikiba, 2005) and was selected as a key study, because it was well described and had the reliability 2 according to Klimich ranking. In this study, Syrian Hamster Embryo cells were exposed to guaiacol at concentrations of 0 to 1000 µM in the absence of mammalian metabolic activation system.

The result with Guaiacol was positive.

The positive controls were not examined.

This study is classified as acceptable and satisfies the requirement for Test Guideline for in vitro cytogenetic mutagenicity data. 

Sister chromatid exchange:

Two studies were available, but only one was selected as a key study (Miyashi, 2005). In order to evaluate the genotoxic potential of Guaiacol used in dental practice, the abilities of this agent to induce sister-chromatid exchanges (SCEs) were examined using Syrian hamster embryo (SHE) cells (Miyachi, 2005). Statistically significant increases in the frequencies of SCEs were observed in SHE cells treated with Guaiacol, (P 0.01; Student test). Because SCE assays are used as a sensitive indicator for evaluating genetic toxicity of chemicals, the chemical agents that had a positive response in the present study are potentially genotoxic to mammalian cells.

This study is classified as acceptable and satisfies the requirement for Test Guideline for in vitro cytogenetic mutagenicity data. 

The other available study has reliability 3 and was not taken into account.

Other in vitro studies have reliability 3 or 4 and were not taking into account for assessment.

Micronucleus in vivo:

Only one study was available and was selected as key study, which summary is the following:

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.

Ten 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 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.

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-mutagenicin this micronucleus assay.

Gaiacol is negative in Ames tests with or without metabolic activation (published data).
Negative result in micronucleus assay in vivo (study report, 2009).
Positive results observed without metabolic activation in chromosomal aberrations and SCE test with Syrian Hamster embryo (published data).

Justification for classification or non-classification

Based on the available in vitro data and on the recent in vivo micronucleus assay, Gaiacol is not expected to be genotoxic.