m-cresol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Overall, m-cresol is not mutagenic in bacterial systems and mammalian cell systems. With respect to clastogenicity, m-cresol induced chromosomal aberrations in vitro and the respective in vivo studies yielded negative results. Taking into account the in vitro and in vivo data m-cresol is not mutagenic in vitro and not clastogenic in vivo and, consequently, should be considered as not genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: Salmonella typhimurium TA98, TA100, TA102, TA104, TA1535, TA1537 and Escherichia coli WP2uvrA and WP2uvrA/pKM101

Metabolic activation:

with and without

Metabolic activation system:

liver S9-fraction from male Sprague-Dawley rats pretreated with pheno- barbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

(1) + (2) S. typh. TA98, TA100, TA1353, TA1537 and E.coli WP2uvrA:
(1) 0.0763, 0.305, 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 µg/plate
(2) 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 µg/plate
(3) + (4) S.typh. TA102, TA104, E.coli WP2uvrA/pKM101:
(3) 0.0763, 0.305, 1.22, 4.88, 19.55, 78.1, 313, 1250, 5000 µg/plate
(4) 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, sodium acide, 9-aminoacridine, 2-nitrofluorene, 4-nitroquinoline-N-oxide, bleomycin, pyruvic aldehyde, 2-amino anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation;

Evaluation criteria:

The chemical were considered to be mutagenic when a dose-related increase of revertant colony
count was observed and the number of revertant colonies per plate with the test substance was more han twice that of the negative control and
when a reproducibility of test result was observed.

Statistics:

yes: Two-hold criteria was used for data evaluation.

Species / strain:

other: Salmonella typhimurium TA98, TA100, TA102, TA104, TA1535, TA1537 and Escherichia coli WP2uvrA and WP2uvrA/pKM101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1250 µg/plate onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

the positive controls were functional.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: no differentiation between small and large colonies, statistical evaluation not mentioned

Principles of method if other than guideline:

Similar to OECD Guideline 476, No differentiation between large and small colonies, see also freetext ME.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

no data

Species / strain / cell type:

other: mouse L 5178 Y (TK +/-) cells

Metabolic activation:

with and without

Metabolic activation system:

1254 Aroclor-iduced adult male rat liver (S9)

Test concentrations with justification for top dose:

with and without S9-mix: 52.0, 78.0, 104, 156, 260, 312, 416, 520 ug/ml in DMSO.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: without S9-Mix: Ethylmethane sulfonate (EMS) with S9-Mix: 3-Methyl-cholanthrene (MCA)

Details on test system and experimental conditions:

Mouse lymphoma assay

Evaluation criteria:

The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment is a mutant frequency that is >=2 times the concurrent background frequency. The background frequency is defined as the average mutant frequency of the solvent control.

Statistics:

no data

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: with and without S9-mix: 520 ug/ml;

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

m-cresol was evaluated as nonmutagenic in the mouse lymphoma cell system.
The positive controls were functional.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: only abstract available

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese hamster lung (CHL/IU) cells

Details on mammalian cell type (if applicable):

primary culture

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix: rat liver induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

without S9-mix: 0, 300, 500, 700, 900, 1100 µg/mL
with S9-mix: 0, 12.5, 25, 50, 100, 200, 400 µg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: without S9-Mix: 1-methyl-3-nitro-1-nitrosoguanidine; with S9-mix: 3,4-benzo[a]pyrene

Details on test system and experimental conditions:

incubation time: 6 hours
plates/test: 2

Evaluation criteria:

the test substance is considered to be positive for mutagenic activity when assay cultures with the test substnce show a significantly higher
incidence of cells with chromosomal aberrations as compared with the negative control and when this effect is reasonably reproducible or
dose-dependent.

Statistics:

Multi-sample chi² test and then Fisher's exact test

Species / strain:

other: Chinese hamster lung (CHL/IU) cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: without S9-mix: 1100µg/mL (6 hour incubation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Polyploidy was not induced in any treatment group.

With 6 hr short-term treatment, sturctural chromosomal aberrations were induced at 700 and 900 µg/mL (20 and 27.5 %) without S9-mix.

Structural chromosomal aberrations were induced at 25, 50, 100, 200, 400 µg/mL (10.5, 21.5, 17.5, 24.0, 30.5 %) with S9-mix.

Executive summary:

m-cresol induced structural chromosome aberrations in CHL /IU cells after treatment for 6 hours with and without an exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Short description of key information:
Overall, m-cresol is not mutagenic in bacterial systems and mammalian cell systems. With respect to clastogenicity, m-cresol induced chromosomal aberrations in vitro and the respective in vivo studies yielded negative results. Taking into account the in vitro and in vivo data m-cresol is not mutagenic in vitro and not clastogenic in vivo and, consequently, should be considered as not genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: deficiencies in experimental procedure

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Principles of method if other than guideline:

In accordance with OECD Guideline 475, 5 mice/sex/dose, bone marrow cells, sacrifice 6,24,48 hrs post treatment, negative and positive controls, stat. method: Kruskal-Wallis test.

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Species:

other: mouse bone marrow cells

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

adult mice (age: 9 weeks at the time of dosing), 5 days for acclimatisation, 5 mice/cage.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

single application, application volume: 5 ml/application.

Duration of treatment / exposure:

once

Frequency of treatment:

once

Post exposure period:

no

Remarks:

Doses / Concentrations:
0, 96, 320, 960 mg/kg bw in corn oil
Basis:
actual ingested

No. of animals per sex per dose:

5 mice/sex/dose/ for the 6 hr-, for the 24 hr- and for the 48 hr- period, respectively, post dosing.

Control animals:

other: vehicle controls and positive controls (CP)

Positive control(s):

yes : CP

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

according to guideline

Evaluation criteria:

The criteria for a positive response are a statistically significant dose-related increase in the number of structural aberrations at 3 dose levels.

Statistics:

Kruskal-Wallis test

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

mortality 3/40 in the highest dose group
clinical signs not attributable to systemic availability
mitotic index in bone marrow cells of treated animals similar to those of controls

The treatment did not increase the frequency of chromosomal aberrations, indicating that m-cresol was not clastogenic under the conditions of this assay. The positive control was functional.
Mortality: 3/5 male mice in the 960 mg-group
Signs of toxicity:
960 mg-group: within 10 min after dosing: squinty eyes, scruffy coats, mild tonic convulsions and rapid breathing which ceased after 30 min., breathing difficulties.
320 mg/kg bw: slightly scruffy coats within 22 hours after dosing.
96 mg/kg bw: no signs of toxicity.

Executive summary:

m-cresol did not increase chromosomal aberrations in bone marrow cells, however mitotic index in bone marrow cells of treated animals is similar to those of controls.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: effects of m/p-cresol mixture was investigated

Qualifier:

according to guideline

Guideline:

other: MacGregor JT, Wehr CM, Henika PR, Shelby DM (1990): Fundam Appl Toxicol. 14, 513-522

Principles of method if other than guideline:

Male and female B6C3F1 mice were fed with diet containing 0, 625, 1250, 2500, 5000, and 10000 ppm m/p-cresol mixture for 13 weeks . At termination of the 13 week study peripheral blood samples were obtained by cardiac punctuire to prepare smears. slides were stained with Hoechst 33258/pyronin Y. At least10000 normochromatic erythrocytes from each animal were scored for micronuclei. In addition, the percentage of polychromatic erythrocytes (PCEs) among the total erythrocyte population was scored as measure of bone marrow toxicity.

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 5 to 6 weeks
- Housing: individually
- Diet ad libitum
- Water ad libitum
- Acclimation period: 12-13 days

ENVIRONMENTAL CONDITIONS: standard
-

Route of administration:

oral: feed

Vehicle:

test substance was given with the diet

Details on exposure:

test substance was given with the diet

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Post exposure period:

none

Remarks:

Doses / Concentrations:
0, 625, 1250, 2500, 5000, 10000 ppm
Basis:

No. of animals per sex per dose:

10 male and 10 female mice/dose

Control animals:

yes, plain diet

Positive control(s):

no data

Tissues and cell types examined:

Normochromatic erythrocytes and polychromatic eryhrocytes.

Details of tissue and slide preparation:

Peripheral blood samples were obtained by cardiac punctuire to prepare smears. Slides were stained with Hoechst 33258/pyronin Y. At least 10000 normochromatic erythrocytes from each animal were scored for micronuclei. In addition, the percentage of polychromatic erythrocytes (PCEs) among the total erythrocyte population was scored as measure of bone marrow toxicity.

Evaluation criteria:

In the micronucleus test , an individual trial is considered positiveif the trend test P value is less than or equan to 0.025 or if the P valuu for any single exposed group is less than or equal to 0.025 devided by the number of exposure groups.
A final call of positive for micronucleus induction is preferably based on reproducibly positive trials.

Statistics:

One-tailed Cochran-armitage trend test, followed by a pairwise comparison between each exposed group and the control group.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

increase in relative liver weights at the highest test dose without histopathological findings

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not specified

Conclusions:

Interpretation of results: negative.

Executive summary:

Male and female B6C3F1 mice were fed with diet containing 0, 625, 1250, 2500, 5000, and 10000 ppm m/p-cresol mixture for 13 weeks . At termination of the 13 week study peripheral blood samples were obtained by cardiac punctuire to score micronuclei. No increases in the frequencies of micronucleated erythrocytes were seen in male or female mice in either study (US Department of Health and Human Services 1991, 2007; Witt 2000).

Reference 3

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Sister chromosome exchange

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: only one dose tested and no information on GLP.

Principles of method if other than guideline:

m-Cresol was administered to 2 or 3 intact or hepatectomized male mice by single intraperitoneal injection. After 30 min, DNA labelling was initiated using BrdU. After a further 21 hr the animals were killed, cells isolated and harvested and sister chromatid exchange (SCE) frequency in bone marrow cells, alveolar macrophages and regenerating liver cells analysed. Some of the mice were partially hepatectomized to induce liver cell regeneration.

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay

Species:

mouse

Strain:

DBA

Sex:

male

Route of administration:

intraperitoneal

Vehicle:

sunflower oil

Details on exposure:

<=0.35 ml test solution was injected

Duration of treatment / exposure:

single application

Frequency of treatment:

once

Post exposure period:

21 hours post injection of the test solution.

Remarks:

Doses / Concentrations:
0, 200 mg/kg bw dissolved in sunflower oil
Basis:
analytical conc.

No. of animals per sex per dose:

3 intact male mice/dose and 3 partly hepatectomized male mice/dose

Control animals:

yes

Positive control(s):

cyclophosphamide;
- Justification for choice of positive control(s): no data
- Route of administration: i.p.
- Doses / concentrations: 5 mg/kg bw

Tissues and cell types examined:

bone marrow, alveolar macrophage, regenerating liver

Details of tissue and slide preparation:

cell isolation and harvest procedure were as described by Conner et al (1978): chromosoma 68, 303-331 (no further details given)

Evaluation criteria:

20 metaphases were analysed for each cell type from each animal. the average generation time was determined and tested for homogenicity andof variance

Statistics:

one way analyses of variance according to Snedecor and Cochran, Dunnett's test for comparisons

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

lethargy, piloerection, lacrimation

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

No increase in SCE frequencies in the intact mice as well as in the partially hepatectomized mice.
The dose tested was overtly toxic to the mice, causing lethargy, piloerection and lacrimation.
The positive control was functional.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

IN-VITRO DATA: In several Ames tests according to the respective guideline, in the absence and in the presence of a metabolic activation system, tested up to cytotoxicity, m-cresol revealed no genotoxic activity (MHLW 2001, JETOC 1998, Pool and Lin 1982, Haworth 1993) This is confirmed by the negative result in the Mouse-Lymphoma Assay performed according to the respective guideline with and without a metabolic activation system (CMA 1988). In a test for chromosome aberration in mammalian cell systems in-vitro according to guideline in the presence and in the absence of a metabolic activation system, m-cresol revealed clastogenic activity (MLHW 2001). In another chromosomal aberration test in which CHO cells m-cresol was negative (CMA1988b). Hibika et al., 2005, observed clastogenicactivity with and without S9-mix, however the evaluation criteria is not defined as requested by the respective OECD guideline. There are limited data available which indicate no induction of sister chromatid exchange in mammalian cells. Cheng and Kligerman (1984) did not observe increases in SCEs in cultured human fibroblasts but tested only in the absence of a metabolic activation system and only 20 metaphases were scored (the guideline requested 25 metaphases per plate and with and without metabolic activation system). No increase in the frequencies of SCEs in human lymphocytes were reported by Jansson et al (1986) who examined m-cresol up to 1 mM only in the absence of metabolic activation systems. There are ambiguous data in limited unscheduled DNA synthesis studies reported. Freshly isolated hepatocytes, treated for up to 18 hours with m-cresol in concentrations up to cytotoxicity, did not show unscheduled DNA synthesis (UDS) in an assay in compliance with the respective guideline, but the result was not confirmed by an independent second trial (CMA 1988f). Hamaguchi and Tsutsui (2000) observed no UDS after treatment of cultured Syrian Hamster Embryo (SHE) cells following an incubation period of up to 3 hours without metabolic activation system but noted dose-related increase in UDS induced in SHE cells (treatment time: 6 hours) in the presence of exogenous metabolic activation. However, in both trials cytotoxicity was not reached and neither negative nor positive controls were reported.

IN-VIVO DATA: There is a reliable but limited in-vivo chromosomal aberration assay in mice bone marrow and in vivo micronucleus test which can be taken into account. The chromosomal aberration test, following oral dosing of 96 -960 mg/kg bw to mice, yielded a negative result. The doses were chosen from dose-range finding study with 400-2000 mg/kg bw resulting in mortality (0/6-6/6) and difficulty in breathing and lethargy (CMA 1989). However, because of experimental deficiencies (only 50 instead of 100 metaphases were scored) and the information that the mitotic index in the target tissue is comparable to those of the controls, the reliability of the test result is questionable. Thus, this assay cannot fully compensate the result from the in-vitro assay. There is another reliable in vivo micronucleus test available (US Health and Human Services 1991, 2007, Witt 2000) using m/p-cresol mixture evaluating erythrocytes for micronuclei after a treatment period of 13 weeks, yielded a negative result, too. This, in vivo study can be considered, because - as already discussed in the section Repeated Dose Toxicity - m/p-cresol mixture in repeated dose toxicity studies caused histopathological changes which are consistent with the observed effects on cresols in general. Therefore using m/p-cresol mixture for in vivo investigations on clastogenicity, such a study can provide reliable information on in vivo clastogenicity and thus help to clear the suspicion from the in vitro study that m-cresol might be clastogenic In the respective study, male and female B6C3F1 mice were fed with diet containing 0, 625, 1250, 2500, 5000, and 10000 ppm m/p-cresol mixture for 13 weeks. At termination of the 13 week study peripheral blood samples were obtained by cardiac punctuire to prepare smears. Slides were stained with Hoechst 33258/pyronin Y. At least 10000 normochromatic erythrocytes from each animal were scored for micronuclei. In addition, the percentage of polychromatic erythrocytes (PCEs) among the total erythrocyte population was scored as measure of bone marrow toxicity. No increases in the frequencies of micronucleated erythrocytes were seen in male or female mice in either study (US Department of Health and Human Services 1991, 2007; Witt 2000). There is an additional limited in vivo SCE study available supporting that m-cresol has no in vivo genotoxicity potential. Cheng and Kligerman (1984) injected intraperitoneal 200 mg/kg bw. m-cresol to healthy and partly hepatectomized mice 21.5 hours prior to sacrifice inducing symptoms including lethargy piloerection an lacrimation. There is no information on the target tissue (bone marrow, alveolar macrophages, liver) reaction. 20 metaphases were scored for evaluation. Compared to negative control (solvent sunflower oil) bone marrow did not show an increase in SCE frequencies in intact or partly hepatectomized mice and alveolar macrophages developed only a slight not significant increase in partly hepatectomized mice. However, results from regenerating liver cannot be taken into account due to the choice of application route: intraperitoneal injection, which might implicate the damage of the liver. Overall, m-cresol is not mutagenic in bacterial systems and mammalian cell systems. With respect to clastogenicity, m-cresol induced chromosomal aberrations in vitro and the respective in vivo studies yielded negative results. Taking into account the in vitro and in vivo data m-cresol is not mutagenic in vitro and not clastogenic in vivo and, consequently, should be considered as not genotoxic.

Justification for classification or non-classification

According to Regulation (EC) No. 1272/2008 a classification is not justified.