Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In absence of experimental data on Tar acids, Xylenol fraction (CAS 84989-06-0) an analogue read-across approach was conducted:

Gene mutation (bacteria):

WoE - mixed xylenols (RL1; according to OECD 471): negative with and without metabolic activation;

WoE - m-cresols (RL1; according to OECD 471): negative with and without metabolic activation;

WoE - o-cresols (RL1; according to OECD 471): negative with and without metabolic activation;

WoE - p-cresols (RL1; according to OECD 471): negative with and without metabolic activation;

Mammalian chromosome aberration (in vitro):

WoE - mixed xylenols (RL1; according to OECD 473): positive with and without metabolic activation;

WoE - mixed ethylphenols (RL1; according to OECD 473): positive with and without metabolic activation;

WoE – o-cresol (RL1; according to OECD 473): positive with and without metabolic activation;

WoE – p-cresol (RL1; according to OECD 473): positive with and without metabolic activation;

WoE – m-cresol (RL2; similar to OECD 473): positive with metabolic activation and negative without metabolic activation;  

Mammalian gene mutation (in vitro):

WoE - mixed xylenols (RL1; according to OECD 476): negative with and without metabolic activation;

WoE – o-cresol (RL2; according to OECD 476): negative with and without metabolic activation;

WoE – p-cresol (RL2; according to OECD 476): negative with and without metabolic activation;

WoE – m-cresol (RL2; similar to OECD 476): negative with and without metabolic activation;

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese Hamster ovary (CHO) cells
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
treatment time: 20 hrs:
-S9-mix, 100, 150, 200, 301 µg/mL performed twice; +S9-mix: 301, 601, 902 µg/mL;
treatment time: 10 hrs:
+S9-mix: 150, 225, 300 µg/mL performed twice
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: MitomycinC (for nonactivation assay); Cyclophosphamide (in the metabolic activated assay)
Details on test system and experimental conditions:
doses were chosen following a range-finding assay
Evaluation criteria:
positive if an significant increase in chromosoally aberrrant cells were observed
Statistics:
Fisher's Exact Test with an adjustment for multiple comparisons
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: Preliminary range-finding assays were performed (3.01 - 3010 µg/mL) to determine cytotoxicity: -S9-mix: >=301 µg/mL; +S9-mix: >=100 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

non-activation assay and incubation for 20 hrs:
Increases in chromosomally aberrant cells ranging between 6.5% and 11% cells with aberrations (versus 1.0% of solvent control) or between 4% and 14 % cells with aberrations (versus 2.0% of solvent control), respectively. 
Positive control was functional in each trial.

Incubation for 20 hours with metabolic activation
Increases in the chromosomally aberrant cells ranging between 18% and 40.5% cells with aberrations (902 µg/mL was toxic; versus 1.5% of  solvent control) and between 17% and 43% cells with aberrations (902 µg/mL was toxic, versus 3.0% of solvent control), respectively. 
Posivive control was functional in each trial.

Incubation for 10 hours  in the presence of S9-mix:

no significant  difference to the solvent controls; positive controls were functional

Conclusions:
p-Cresol induced chromosome aberrations in a test according to OECD TG 473 with Chinese Hamster Ovary cells in the presence and in the absence of a metabolic activation system and tested up to cytotoxicity.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No differentiation between large and small colony mutants; statistical evaluation not mentioned; no cytotoxicity data presented
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
No differentiation between large and small colony mutants
GLP compliance:
yes
Type of assay:
other: mammalian cell gene mutation assay
Target gene:
TK-gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of rat liver homogenate and necessary co-factors
Test concentrations with justification for top dose:
with activation: 0.256 µg/mL, 0.511 µg/mL, 0.767 µg/mL, 1.02 µg/mL, 1.53 µg/mL, and 3.07 µg/mL.
without activation: 51.1 µg/mL, 102 µg/mL, 153 µg/mL, 204 ug/ml, 307 µg/mL, and 409 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: ethylmethane sulfonate, 3-methylcholantrene
Details on test system and experimental conditions:
no further details
Evaluation criteria:
The experimental mutant frequency will be considered acceptable for evalualtion only if the relative colony efficiency is 10 percent or greater and the total number of viable clones exceeds about 60. A test result is evaluated positive if a dose-related increase in mutant frequency should be observed or if a >= two-fold increase over the concurrent background frequency is observed.


Statistics:
not mentioned
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: with activation: 7.98 µg/mL, without activation: 511 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

the positive controls were functional

Conclusions:
p-cresol was not mutagenic in the mouse lymphoma forward mutation assay according to OECD TG 476 with and without metabolic activation.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
no E.coli or S. typhimurium TA 102 tested
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
other: Salmonella typhimurium TA 98, TA100, TA1535, TA1537.
Metabolic activation:
with and without
Metabolic activation system:
a 9000 x g supernatant from male Syrian Hamster liver and from male Sprague-dawley rat liver both preliminarily induced with Aroclor 1254
Test concentrations with justification for top dose:
0.0, 3.3, 10.0, 33.0, 100.0, 333.0 µg/plate in water as solvent
Vehicle / solvent:
water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 4-nitro-o-phenylene diamine, sodium azide, 9-aminoacridine
Details on test system and experimental conditions:
Ames test preincubation methodology according to Ames, Mutat. Res. 31,347 (1975) and Yahagi, Cancer Lett. 1,91 (1975)
Evaluation criteria:
POSITIVE RESPONSE: was indicated by a reproducable, dose-related increase wether it be two-fold over background or not
Statistics:
analysis based on the models presented by Margolin
Key result
Species / strain:
other: TA 98, TA100, TA1535, TA1537.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: to select dose range the chemical was checked for toxicity to S. typh. TA100
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
p-cresol yielded a negative result when tested in the Ames test according to OECD TG 471 using 4 strains of Salmonella typhimurium with and without metabolic activation systems
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
no information on GLP, no E.coli or S. typhimurium TA 102 tested
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
yes
Remarks:
no E.coli or S. typhimurium TA 102 tested
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA1538 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Arclor 1254 induced S9
Test concentrations with justification for top dose:
0, 0.5, 5, 50, 500 and 5000 ug/plate
Vehicle / solvent:
- Vehicle: DMSO
- Justification for choice of solvent/vehicle: widely used
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Sodium azide ( -S9 TA1535 & TA100); 2-Nitrofluorene (-S9 TA1538; -S9 TA98); 9-Aminoacridine (-S9 TA1537); 2-Aminoanthracene (+S9 all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Plate incoporation method

Evaluation criteria:
A dose-related significantly increased number of revertants was evaluated as a positive result.
Statistics:
Dose rsponse effects were assessed using the Joncheere test
Key result
Species / strain:
other: TA 1535, TA 1537, TA1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Observed in all strains
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

p-cresol

Conc

TA1535

TA1537

TA1538

TA98

TA100

(ug/plate)

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0

12

22

5

11

16

36

21

23

67

76

0.5

22

31

10

11

16

30

20

30

65

90

5

24

31

3

NT

21

34

18

31

68

81

50

21

26

10

10

14

38

20

31

67

87

500

29

26

6

11

15

37

18

29

64

100

5000*

26

0

0

0

7

20

29

9

3

12

 

 Positive controls

 SA

 526 ± 54

-

-

-

-

-

-

-

 502 ± 28

-

 2NF

-

-

-

-

 284 ± 60

-

 275 ± 74

-

 -

-

 9AA

-

-

 810 ± 95

-

-

-

-

-

-

-

 2AA

-

 306 ± 60

-

 269 ± 47

-

 1027 ± 199

-

 1093 ± 227

-

 723 ± 159

 * toxicity - apparent as a thinning of the background lawn

SA - Sodium azide

2NF - 2 -Nitrofluorene

9AA - 9 -Aminoacridine

2AA - 2 -Aminoanthracene

 

Conclusions:
p-cresol was found to be negative in the Ames study when tested up to a concentration of 5000 ug/plate (the maximum recommended concentration in accordance with currenty regulatory guidelines) in S. typhimurium strains (TA1535, TA1537, TA1538, TA98, TA100). Signs of toxicity was evident at the maximum dose tested, manifest by a slight thinning of the background lawn.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
+S9-mix: 2 hour treatment time too short
Principles of method if other than guideline:
Method: preliminary range finding studies; in accordance with OECD Guideline 473
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
without S9-mix (17.25 hrs / duplicate): (1)+(2): 198, 297, 398, 495 µg/mL DMSO; with S9-mix (2 hrs): (1)+(2): 250, 500, 749, 999 µg/mL, (3) 699, 749, 799, 898, 998, 1100 µg/mL 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: with S9-mix: Cyclophosphamide (CP); without S9-Mix: Mitomycin C
Details on test system and experimental conditions:
no further data
Statistics:
Fisher's Exact test with an adjustment for multiple comparisons
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: Preliminary range finding assays were performed with and without metabolic activation to determine cytotoxicity: >=898 µg/mL: toxic
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Preliminary range finding assays were performed with and without metabolic activation to determine cytotoxicity: >=898 µg/mL: toxic
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
see section "remarks on results "

m-Cresol was considered negative for inducing chromosomal aberrations when tested in CHO cells in-vitro without a metabolic activation system.

When the test was performed in the presence of a metabolic activation system m-Cresol induced a significant increase in aberrations at the highest doses tested despite the short treatment time.

The positive controls were functional.

Conclusions:
m-Cresol was considered negative for inducing chromosomal aberrations when tested in CHO cells in-vitro without a metabolic activation system. However, when the test was performed in the presence of a metabolic activation system m-cresol induced a significant increase in aberrations at the highest doses tested despite the short treatment time.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
no differentiation between small and large colonies, statistical evaluation not mentioned
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
no differentiation between large and small colonies
GLP compliance:
yes
Type of assay:
other: mammalian cell gene mutation assay
Target gene:
TK gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
1254 Aroclor-induced 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 µg/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:
no further data
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
Key result
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 µg/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.
Conclusions:
m-cresol was evaluated as nonmutagenic in the mouse lymphoma cell system. The positive controls were functional.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
no data on GLP
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his and trp operon
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Activation system prepared from rat liver induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
0, 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: without S9-mix: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, Sodium azide, 9-Aminoacridine;
Details on test system and experimental conditions:
Pre-incubation method,
Plates/test 3,
Replicates 2
Evaluation criteria:
The test substance is considered to be positive for mutagenic activity when assay plates with the test substance a show significant increase in revertant colony count as compared with that on negative control plates and when this effect is reasonably reproducible or dose dependent.
Statistics:
yes, but method not mentioned
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9-mix: >2500 µg/plate; with S9-mix: 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with and without S9-mix: >2500 µg/plate;
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
m-cresol yielded a negative result when tested in the Ames test according to OECD TG 471 using 4 strains of Salmonella typhimurium (Salmonella typhimurium TA 98, TA100, TA1535, TA1537) and E.coli WP2 uvr Awith and without a metabolic activation system. The positive controls were functional.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
no data on GLP
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
yes
Remarks:
no E.coli or S. typhimurium TA 102 tested
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA1538 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Arclor 1254 induced S9
Test concentrations with justification for top dose:
0, 0.5, 5, 50, 500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle: DMSO
- Justification for choice of solvent/vehicle: widely used
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Sodium azide ( -S9 TA1535 & TA100); 2-Nitrofluorene (-S9 TA1538; -S9 TA98); 9-Aminoacridine (-S9 TA1537); 2-Aminoanthracene (+S9 all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Plate incoporation method
Evaluation criteria:
A dose-related significantly increased number of revertants was evaluated as a positive result.
Statistics:
Dose rsponse effects were assessed using the Joncheere test
Key result
Species / strain:
other: TA 1535, TA 1537, TA1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Observed in all strains
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

m-cresol

Conc

TA1535

TA1537

TA1538

TA98

TA100

(ug/plate)

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0

12

9

5

11

8

18

16

14

91

76

0.5

22

11

5

NT

96

22

17

16

NT

91

5

21

4

6

17

11

20

19

14

117

86

50

23

2*

8

13

7

29

18

14

62

85

500

15

9*

8

16

7

20

15

17

53

97

5000*

0

2

0

0

4

4

3

2

0

32

 

 Positive controls

 SA

 526 ± 54

-

-

-

-

-

-

-

 502 ± 28

-

 2NF

-

-

-

-

 284 ± 60

-

 275 ± 74

-

 -

-

 9AA

-

-

 810 ± 95

-

-

-

-

-

-

-

 2AA

-

 306 ± 60

-

 269 ± 47

-

 1027 ± 199

-

 1093 ± 227

-

 723 ± 159

 * toxicity - apparent as a thinning of the background lawn

SA - Sodium azide

2NF - 2 -Nitrofluorene

9AA - 9 -Aminoacridine

2AA - 2 -Aminoanthracene

 

Conclusions:
m-cresol was found to be negative in the Ames study when tested up to a concentration of 5000 ug/plate (the maximum recommended concentration in accordance with currenty regulatory guidelines) in S. typhimurium strains (TA1535, TA1537, TA1538, TA98, TA100). Signs of toxicity were evident at the maximum dose tested, manifest by a slight thinning of the background lawn.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese Hamster Ovary (CHO) cells
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy`s 5a culture medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
A 9000x g supernatant prepared from adult male rat liver induced by Aroclor 1254
Test concentrations with justification for top dose:
without: 150, 200, 250, 300 µg/mL
with: 250, 375, 500, 750, 1000 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: Mitomycin C, Cyclophosphamide
Details on test system and experimental conditions:
according to OECD guideline 473
Evaluation criteria:
concentration related increase
Statistics:
Fisher's exact test with an adjustment for multiple comparisons
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: determined in the range-finding study: with metabolic activation: >= 1000 µg/mL; without metabolic activation: >= 300 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
o-cresol showed clastogenic activity when tested for chromosome aberrations in Chinese Hamster Ovary (CHO) cells in vitro according to OECD TG 473 in the presence and in the absense of a metabolic activation system.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
no E.coli or S. typhimurium TA 102 tested
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
yes
Remarks:
only 4 strains used
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his-operon
Species / strain / cell type:
other: salmonella typhimurium TA98, TA100, TA1535, TA1537
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
A 9000 x g supernatant from male Syrian Hamster liver and from male Sprague-Dawley rat liver, induced with Aroclor 1254
Test concentrations with justification for top dose:
0.0, 1.0, 3.3, 10.0, 33.0, 100.0 µg/plate in water as solvent
Vehicle / solvent:
water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 4-nitro-o-phenylene diamine, sodium azide, 9-aminoacridine
Details on test system and experimental conditions:
Preincubation methodology according to Ames, Mutat. Res. 31,347 (1975) and Yahagi, Cancer Lett. 1,91 (1975);
Rationale for test conditions:
DETERMINATION OF DOSE: to select dose-range the chemical was checked for toxicity to S. typh. TA 100 (details not given)
Evaluation criteria:
positive response was indicated by a reproducible, dose-related increase whether it be twofold over the background or not
Statistics:
based on the models of Margolin, Kaplan and Zeiger (1981): Proc.Natl. Acad.Sci (USA) 78, 3779-3783
Key result
Species / strain:
other: TA 98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: from 33.0 µg/plate onwards
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
no data
Conclusions:
o-cresol yielded a negative result when tested in the Ames test according to OECD TG 471 using 4 strains of Salmonella typhimurium (Salmonella typhimurium TA 98, TA100, TA1535, TA1537) with and without a metabolic activation system. The positive controls were functional.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
no E.coli or S. typhimurium TA 102 tested
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
yes
Remarks:
purity of TS is not given
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his-operon
Species / strain / cell type:
other: Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
A 9,000 x g supernatant prepared from sprague-Dawley adult male rat liver induced by Aroclor 1254
Test concentrations with justification for top dose:
0.01, 0.1, 1.0, 5.0, 10.0, 25.0, 50.0 µL/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Sodium azide, 2-Nitrofluorene, 9-Aminoacridine, 2-Anthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation);

Evaluation criteria:
dose-related mutant increase
Statistics:
no data
Key result
Species / strain:
other: TA98, TA100, TA1535, TA1537, TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 10-50 µL/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
o-cresol was tested for genotoxic effects in the Ames test according to OECD TG 471 using Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538 with and without a metabolic activation system and revealed a negative result. The positive controls were functional.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
no information on colony size and no information of purity of TS
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
other: mammalian cell gene mutation assay
Target gene:
tk locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
no further data
Metabolic activation:
with and without
Metabolic activation system:
A 9000 x g supernatant prepared from Fisher 344 adult male rat liver induced by Aroclor 1254
Test concentrations with justification for top dose:
without: 15.6, 31.3, 62.5, 125.0, 250.0 µg/mL
with: 3.91, 7.816, 15.600, 31.300, 62.5 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: Dimethylnitrosamine, Ethylmethane sulfonate
Details on test system and experimental conditions:
no further data
Evaluation criteria:
the minimum criterium for a positive response in this assay was a mutant frequency exceeding 30.6 x 10 [exp-6].
Statistics:
no data
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: without: at and above 500 µg/mL; with: at and above 15.6 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

o-cresol yielded a negative result when tested with the Mouse Lymphoma assay in-vitro according to OECD TG 476 in the presence and in the absence of a metabolic activation system. The positive controls were functional.

Conclusions:
o-cresol yielded a negative result when tested with the Mouse Lymphoma assay in-vitro according to OECD TG 476 in the presence and in the absence of a metabolic activation system. The positive controls were functional.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
no GLP, no E.coli or S. typhimurium TA 102 tested
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
yes
Remarks:
no E.coli or S. typhimurium TA 102 tested
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA1538 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced S9
Test concentrations with justification for top dose:
0, 5, 50, 500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle: DMSO
- Justification for choice of solvent/vehicle: widely used
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Sodium azide ( -S9 TA1535 & TA100); 2-Nitrofluorene (-S9 TA1538; -S9 TA98); 9-Aminoacridine (-S9 TA1537); 2-Aminoanthracene (+S9 all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Plate incoporation method

Evaluation criteria:
A dose-related significantly increased number of revertants was evaluated as a positive result.
Statistics:
Dose rsponse effects were assessed using the Joncheere test
Key result
Species / strain:
other: TA 1535, TA 1537, TA1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Observed in all strains
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

o-cresol

Conc

TA1535

TA1537

TA1538

TA98

TA100

(ug/plate)

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0

22

12

10

14

17

30

21

35

89

108

5

14

11

6

7

12

29

26

35

100

84

50

16

11

9

9

13

16

21

33

107

90

500

30

11

7

4

14

20

22

36

100

90

5000*

0

0

0

0

1

11

0

14

98

30

 

 Positive controls

 SA

 526 ± 54

-

-

-

-

-

-

-

 502 ± 28

-

 2NF

-

-

-

-

 284 ± 60

-

 275 ± 74

-

 -

-

 9AA

-

-

 810 ± 95

-

-

-

-

-

-

-

 2AA

-

 306 ± 60

-

 269 ± 47

-

 1027 ± 199

-

 1093 ± 227

-

 723 ± 159

 * toxicity - apparent as a thinning of the background lawn

SA - Sodium azide

2NF - 2 -Nitrofluorene

9AA - 9 -Aminoacridine

2AA - 2 -Aminoanthracene

 

Conclusions:
o-cresol was found to be negative in the Ames study when tested up to a concentration of 5000 ug/plate (the maximum recommended concentration in accordance with currenty regulatory guidelines) in S. typhimurium strains (TA1535, TA1537, TA1538, TA98, TA100). Signs of toxicity were evident at the maximum dose tested, manifest by a slight thinning of the background lawn.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
18 May 2004 to 27 July 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes, working cell stocks were not used beyond passage 20.
- Periodically "cleansed" against high spontaneous background: not stated
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced S9
Test concentrations with justification for top dose:
In the absence of S9 activation: 50, 100, 200, 400, 600, 800, 1000 and 1200 µg/mL (4 hour treatment) and 5, 10, 20, 40, 60, 80, 100 and 120 µg/mL (20 hour treatment).
In the presence of S9 activation: 50, 100, 200, 400, 600, 800, 1000 and 1200 µg/mL (4 hour treatment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Selected following a solubility test using water and DMSO to determine the highest soluble stock concentration up to 500 mg/mL.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
10 and 20 µg/mL in water for the treatments without S9 activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
1 and 2 mg/mL in water for the treatments with S9 activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 20 h

SPINDLE INHIBITOR (cytogenetic assays): colcemid (2 hours prior to the scheduled harvest)

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): a minimum of 200 metaphase spreads per treatment

DETERMINATION OF CYTOTOXICITY
- Method: 4 h treatment: viability, cell counts, 20 h: mitotic index; cloning efficiency; relative total growth; other:


Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
Positive for the induction of structural and numerical chromosome aberration
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
4 h / +S9: 17% at 250 µg/mL (the highest dose evaluated for aberrations); mitotic index: 52% reduction at 250 µg/mL relative to the solvent control.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

A repeat CHO assay was performed due to the 8.5% aberrant cells in the solvent group of the S9 activate 4 hour treatment being outside the historical solvent control range of 0 - 5.0%.

The toxicity of mixed ethylphenols to CHO cells treated for 4 hours in the presence of S9 activation was 17% at 250 µg/mL (the highest dose evaluated for aberrations). The mitotic index showed a 52% reduction at 250 µg/mL relative to the solvent control. The % of cells with structural aberrations was significantly increased (p<0.01, Fisher's exact test) at all dose levels. The Cochran-Armitage test was also positive for a dose response (p>0.05). The % of cells with numerical aberrations in the test substance treated groups was not significantly increased above that of the solvent control at any dose level (p>0.05, Fisher's exact test). The % of structurally damaged cells in the CP positive control group was statistically significant at 19%.

Table 1: Summary of results from the repeat chromosome aberration assay with mixed ethylphenols

Treatment (µg/mL)

Treatment time (h)

Mean mitotic index

Cells scored

Aberrations per cell (mean ± SD)

Cells with aberrations

Numerical

Structural

Numerical

Structural

Without S9 activation

DMSO

4

10.1

200

200

0.030 ± 0.171

1.5

3.0

Mixed xylenols

 

100

4

8.3

200

200

0.195 ± 0.866

3.5

10.0**

200

4

6.9

200

200

0.130 ± 0.752

3.5

8.5**

400

4

4.8

200

200

0.075 ± 0.300

2.0

6.5

MMC, 0.2

4

6.6

200

200

0.660 ± 1.507

2.0

40.0**

DMSO

20

9.6

200

200

0.045 ± 0.271

1.5

3.5

Mixed xylenols

 

100

20

6.7

200

200

0.025 ± 0.186

2.0

2.0

200

20

5.9

200

200

0.085 ±0.297

1.5

8.0*

400

20

4.7

200

200

0.145 ± 0.393

3.0

13.0**

MMC, 0.1

20

6.5

200

200

0.115 ± 0.335

3.0

11.0**

With S9 activation

DMSO

4

10.1

200

200

0.005 ± 0.340

4.0

0.5

Mixed xylenols

 

100

4

6.8

200

200

0.070 ± 0.340

4.0

5.0**

200

4

5.7

200

200

0.680 ± 2.108

0.0

19.0**

250

4

4.8

200

200

0.790± 2.263

0.0

29.0**

CP, 10

4

5.0

200

200

0.560 ± 1.976

0.0

19.0**

SD = standard deviation
Cells were harvested 20 hours after the initiation of the treatments.
Severely damaged cells were counted as 10 aberrations.
* = Significant at p<0.05 using Fisher’s exact test.
** = Significant at p<0.01 using Fisher’s exact test.

Conclusions:
The results of the assay indicate that under the conditions of the study, mixed ethylphenols caused a positive response in chromosome aberrations in both the presence and absence of metabolic activation.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his and trp operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 derived from Aroclor 1254 induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 µg/plate for strains TA98, TA100, TA1535, TA1537 and WP2 uvrA for the preliminary toxicity study (with and without metabolic activation).
75, 200, 600, 1800 and 5000 µg/plate for TA98, TA100, TA1535, TA1537 and WP2 uvrA for the bacterial mutation assay (with and without metabolic activation).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO) (and water for sodium azide dilution in the positive control)
- Justification for choice of solvent/vehicle: Not stated, commonly used solvent
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Remarks:
2-aminoanthracene for WP2 uvrA in the presence of metabolic activation. 2-nitroluorene for TA98; sodium azide for TA100 and TA1535; 9-aminoacridine for TA1537; methyl methanesulfonate for WP2 uvrA in the absence of metabolic activation.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 12 hours
- Exposure duration: 48 - 72 hours
Evaluation criteria:
All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.The mean of each positive control must exhibit at least a 3.0 fold increase in the number of revertants over the mean value of the respective control. A minimum of three non toxic dose levels is required for evaluation. A dose level is considered to be toxic if there is a > 50% reduction in the mean number of revertants per plate compared to the mean vehicle control value and at least a moderate reduction in the background lawn.
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: Historical control data were found to support the study outcome.

The results of the genotoxicity study found that mixed xylenols were not genotoxic at any dose level tested. A summary of the results is presented below.

Table 1: Summary of results of the mutagenicity assay

Dose (µg/plate)

Average revertants per plate ± standard deviation

TA98

TA100

TA1535

TA1537

WP2 uvrA

In the absence of metabolic activation

Vehicle control

17 ± 2

153 ± 13

16 ± 2

7 ± 2

24 ± 3

75

14 ± 5

126 ± 3

23 ± 5

7 ± 1

24 ± 2

200

17 ± 2

126 ± 31

19 ± 4

6 ± 2

20 ± 5

600

14 ± 5

133 ± 26

19 ± 2

6 ± 1

15 ± 3

1800

16 ± 3

125 ± 11

21 ± 7

4 ± 1

13 ± 3

5000

3 ± 1

0 ± 0

3 ± 1

0 ± 0

1 ± 2

Positive control

115 ± 12

551 ± 8

272 ± 6

644 ± 121

117 ± 7

In the presence of metabolic activation

Vehicle control

20 ± 2

151 ± 15

17 ± 5

6 ± 2

20 ± 6

75

22 ± 5

163 ± 20

17 ± 2

5 ± 1

19 ± 6

200

18 ± 5

168 ± 6

19 ± 2

6 ± 2

16 ± 2

600

22 ± 3

154 ± 6

21 ± 4

6 ± 3

16 ± 3

1800

22 ± 2

144 ± 11

23 ± 2

6 ± 2

9 ± 3

5000

4 ± 2

0 ± 0

6 ± 2

0 ± 0

0 ± 0

Positive control

1000 ± 147

74 ± 74

108 ± 9

128 ± 17

794 ± 95

 

Conclusions:
All criteria for a valid study were met. The results indicate that mixed xylenols did not cause a positive response either in the presence or absence of metabolic activation by Aroclor-induced rat liver S9. Mixed xylenols were therefore concluded to be negative for genotoxicity.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
18 May 2004 to 20 July 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes, working cell stocks were not used beyond passage 20.
- Periodically "cleansed" against high spontaneous background: not stated
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced S9
Test concentrations with justification for top dose:
In the absence of S9 activation: 37.5, 75, 150, 300, 600, 800, 1000 and 1200 µg/mL (4 hour treatment) and 12.5, 25, 50, 100, 200, 300, 400, 500 and 600 µg/mL (20 hour treatment).
In the presence of S9 activation: 37.5, 75, 150, 300, 600, 800, 1000 and 1200 µg/mL (4 hour treatment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Selected following a solubility test using water and DMSO to determine the highest soluble stock concentration up to 500 mg/mL.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
10 and 20 µg/mL in water for the treatments without S9 activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
1 and 2 mg/mL in water for the treatments with S9 activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 20 h

SPINDLE INHIBITOR (cytogenetic assays): colcemid (2 hours prior to the scheduled harvest)

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): a minimum of 200 metaphase spreads per treatment

DETERMINATION OF CYTOTOXICITY
- Method: 4 h treatment: viability, cell counts, 20 h: mitotic index; cloning efficiency; relative total growth; other:


Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
Positive for the induction of structural and numerical chromosome aberrations.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
4 h / -S9: 47% at 550 µg/mL (the highest dose evaluated for aberrations); mitotic index: 53% reduction at 550 µg/mL relative to the solvent control.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

A repeat CHO assay was performed due to lack of sufficient scorable cells in the highest doses and a lack of at least 50% reduction in mitotic index at the lower doses in the first assay.

The toxicity of mixed xylenols to CHO cells treated for 4 hours in the absence of S9 activation was 47% at 550 µg/mL (the highest dose evaluated for aberrations). The mitotic index showed a 53% reduction at 550 µg/mL relative to the solvent control. The % of cells with structural aberrations was significantly increased (p<0.05, Fisher's exact test) at 300 and 500 µg/mL. The % of structurally damaged cells in the MMC positive control group was statistically significant at 22%.

Treatment in the presence of S9 activation showed 54% toxicity at 550 µg/mL. The mitotic index at 550 µg/mL showed a 63% reduction relative to the solvent control.

The % of cells with structural aberrations was significantly increased (p<0.05, p<0.01 Fisher's exact test) at 400 and 500 µg/mL, respectively. The Cochran-Armitage test was also positive for a dose response (p<0.05). The % of structurally damaged cells in the CP positive control group was statistically significant at 17.5%.

Table 1: Summary of results from the repeat chromosome aberration assay with mixed xylenols

Treatment (µg/mL)

Treatment time (h)

Mean mitotic index

Cells scored

Aberrations per cell (mean ± SD)

Cells with aberrations

Numerical

Structural

Numerical

Structural

Without S9 activation

DMSO

4

11.2

200

200

0.000 ± 0.000

3.0

0.0

Mixed xylenols

 

300

4

12.6

200

200

0.035 ± 0.210

11.0**

3.0*

400

4

11.2

200

200

0.045 ± 0.322

10.0**

2.0

550

4

5.3

200

200

0.040 ± 0.262

7.5*

3.0*

MMC, 0.2

4

4.7

200

200

0.300 ± 0.704

3.0

22.0**

With S9 activation

DMSO

4

13.8

200

200

0.015 ± 0.158

7.0

1.0

Mixed xylenols

 

300

4

14.0

200

200

0.015 ± 0.122

8.5

1.5

400

4

12.6

200

200

0.070 ± 0.395

8.5

4.0*

550

4

5.1

200

200

0.185 ± 0.635

6.0

11.5**

CP, 10

4

6.1

200

200

0.255 ± 0.680

5.0

17.5**

SD = standard deviation
Cells were harvested 20 hours after the initiation of the treatments.
Severely damaged cells were counted as 10 aberrations.
* = Significant at p< 0.05 using Fisher’s exact test.
** = Significant at p< 0.01 using Fisher’s exact test.

Conclusions:
The results of the assay indicate that under the conditions of the study, mixed xylenols caused a positive response in chromosome aberrations in both the presence and absence of metabolic activation.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
17 June 2010 to 5 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: mammalian cell gene mutation assay
Target gene:
HPRT gene
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
The master stock of V79 (Lot No. 05F013) was originally obtained by Ford and Yerganian in 1958. The cells used in this study were obtained from ECACC (European Collection of Cell Cultures), UK. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in DMEM and incubated in a humidified atmosphere of 5% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks. Subculturing was performed 2-3 times a week with the aid of trypsin (0.025%)/EDTA solution and counted in suspension using Bürker’s chamber.
Dulbecco’s Modified Eagle’s medium (DMEM) with 4.5 g/L of glucose supplemented with L-glutamine
Metabolic activation:
with and without
Metabolic activation system:
S9 mix was prepared from Sprague Dawley male rats (ANLAB, Czech Republic) weighing approximately 200 g. Animals were pretreated with Aroclor 1254 (ip) at 500 mg/kg 5 days prior to sacrifice.
Test concentrations with justification for top dose:
Preliminary cytotoxicity test:
Range Finder 1: -S9: 0, 6.25, 12.5, 25, 50, 100, 200, 400 µg/mL; +S9: 0, 200, 400, 600, 800 µg/mL
Range Finder 2: -S9: 0, 300, 400, 500, 600 µg/mL; +S9: 0, 360, 400, 420, 440, 480, 500, 520 µg/mL
Range Finder 3: -S9 (only): 0, 500, 510, 525, 550, 575 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethyl methanesulphonate (-S9, 400 µg/mL); 7,12 dimethyl benz[a]antracene (+S9; 3 µg/mL)
Details on test system and experimental conditions:
see "Any other information on materials and methods"
Evaluation criteria:
see "Any other information on materials and methods"
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Toxicity

In the initial cytotoxicity Range-Finder experiment up seven concentrations were tested in the absence and presence of S-9 ranging from 6.25 to 400 µg/mL and 6.25 to 800 µg/mL, respectively. At these concentrations relative plating efficiency (RPE) was reduced to ~33% and 2% in the absence and presence of S-9 respectively. As an inadequate level of cytotoxicity (% plating efficiency) was not obtained (in the case of the –S9 data) or insufficient doses selected to provided adequate information about the dose response (in the case of the +S9 data), further cytotoxicity range finder experiments were performed.

 

Further Range Finder experiments were undertaken in both the absence (300 to 600 µg/mL and 500 to 575 µg/mL) and presence of S-9 (360 to 520 µg/mL). At these concentrations acceptable reductions in RPE (10-20%) were achieved. The RPE efficiencies are shown in Table 1.

 

No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentrations tested as compared to the concurrent vehicle controls (individual data not reported).

 

Table 1: %Relative plating efficiency values – Range Finder Experiments

Treatment
(µg/mL)

3 hour –S9

 

Treatment
(µg/mL)

3 hour +S9

RF Expt 1b

RF Expt 2b

RF Expt 3b

 

RF Expt 1b

RF Expt 2b

0a

100

100

100

 

0a

100

100

6.25

105.32

-

-

 

200

57.75

-

25

102.86

-

-

 

360

-

20.96

50

99.18

-

-

 

400

26.47

18.75

100

110.66

-

-

 

420

-

18.01

200

104.51

-

-

 

440

-

16.54

300

57.37

38.86

-

 

480

-

7.35

400

-

33.96

-

 

500

-

4.04

500

32.78

29.05

21.64

 

520

-

6.62

510

-

-

18.65

 

600

2.21

-

525

-

-

14.17

 

800

0

-

550

-

-

5.22

 

575

-

-

2.97

 

600

-

9.80

-

 

700

-

-

-

 

a.   Negative control DMSO (1%v/v)

b.   5 plates counted/treatment level

 

In Experiment 1 concentrations, ranging from 100 to 600 µg/mL and 75 to 475 µg/mL were tested in the absence and presence of S-9, respectively. No precipitate was observed upon addition of the test article to the cultures or at the end of the 3 hour incubation period. Seven days after treatment all concentrations in the absence and presence of S-9 were selected to determine viability (at the end of treatment and at the point of mutant frequency determination) and 6TG resistance. The highest concentrations plated for viability assessment (%RPE) were 600 µg/mL in the absence of S-9 and 475 µg/mL in the presence of S-9, which gave ~1% and ~9% RPE, respectively (seeTable2). In the absence of S9 almost complete cytotoxicity was observed at the highest concentration tested. Therefore the highest concentration plated which gave an acceptable level of cytotoxicity was 500 µg/mL, where RPE was reduced to ~23%. Whilst this was marginally outside of the required range, this level was cytotoxicity was considered acceptable as no evidence of a dose related increase in mutant frequency was observed at any concentration tested (see Table2).

 

In Experiment 2 concentrations, ranging from 62.5 to 575 µg/mL and 75 to 475 µg/mL were tested in the absence and presence of S‑9, respectively. No precipitate was observed upon addition of the test article to the cultures or at the end of the 3 hour incubation period. Seven days after treatment all concentrations in the absence and presence of S-9 were selected to determine viability (at the end of treatment and at the point of mutant frequency determination) and 6TG resistance. The highest concentrations plated for viability assessment (RPE) were 575 µg/mL and 475 µg/mL in the absence and presence of S‑9 respectively. In the absence of S-9 almost complete cytotoxicity (~3%) was observed at this level, the next highest concentration which gave acceptable RPE was 525 µg/mL (~12%). In the presence of S-9 RPE was reduced to ~9% (seeTable 3).

 

Table2: Summary of mutation data experiment 1

Treatment
(
µg/mL)

3 hour –S9

 

Treatment
(
µg/mL)

3 hour +S9

%RPE

MFa

 

%RPE

MFa

Vehicleb

100

34.1

 

Vehicleb

100

9.1

100

73.43

39.6

 

75

111.21

24.2*

125

71.33

40.9

 

150

63.68

18.0*

200

50.35

38.2

 

300

30.04

21.7*

250

33.21

42.1

 

350

32.29

16.4

300

28.32

43.6

 

375

33.63

14.3

400

28.32

45.2

 

400

26.45

13.5

500

27.22

26.6

 

425

13.89

17.4*

600

1.05c

-

 

450

13.89

37.8*

EMS (400)

77.21

335.1*

 

475

9.42c

36.2*

 

 

 

 

DMBA (3)

10.35

495.7*

a   6TG resistant mutants/106 viable cells 7 days after treatment

b   DMSO (1%v/v)

c   Data disregarded due to unacceptable cytotoxicity

*    Statistically (p<0.01) significant using the Kruskal-Wallis test


Table3: Summary of mutation data experiment 2

Treatment
(
µg/mL)

3 hour –S9

 

Treatment
(
µg/mL)

3 hour +S9

%RPE

MFa

 

%RPE

MFa

Vehicleb

100

28.7

 

Vehicleb

100

8.6

62.5

92.53

40.2

 

75

108.92

19.7

125

88.44

9.6

 

150

76.96

17.5

250

35.82

24.1

 

300

37.17

6.7

450

30.59

10.7

 

350

25.65

27.7*

500

24.99

20.6

 

375

25.27

18.5*

525

11.93

104.8*

 

400

17.85

37.7*

550

5.59b

54.7

 

425

10.03

29.7*

575

3.36c

-

 

450

10.41

24.1*

EMS (400)

98.13

345.0*

 

475

8.54c

30.0*

 

 

 

 

DMBA (3)

79.19

341.4*

a   6TG resistant mutants/106 viable cells 7 days after treatment

b   DMSO (1%v/v)

c   Data disregarded due to unacceptable cytotoxicity

*    Statistically (p<0.01) significant using the Kruskal-Wallis test

 

Mutation

The background mutant frequency in the vehicle controls from all experiments was from acceptance range therefore the data were considered acceptable.

 

In Experiment 1 in the absence of S-9 no statistically significant increases in mean mutant frequency (MMF) were observed following treatment with HPV Xylenols at any concentration tested. Cytotoxicity (expressed in terms of RPE at the end of treatment) was reduced to 22.72% at 500 µg/mL.

 

In the presence of S-9 statistically significant increases in MMF were observed at concentrations of 75, 300, 425, 450 and 475 µg/mL. At these concentrations MMF of 24.2, 21.7, 17.4, 37.8 and 36.2 resistant mutants/106 viable cellswere obtained, respectively compared to a value of 9.1 resistant mutants/106 viable cellsfor the vehicle control. Fold increases greater than 3-fold over the vehicle control were observed at concentrations where the level of cytotoxicity was reduced to ~9-14%. Therefore caution is applied when interpreting these data, with increases in mutant frequency greater than 3-fold only observed in the presence of cytotoxicity.

 

In Experiment 2 in the absence of S-9 a statistically significant increases in MMF was observed following treatment with HPV Xylenols at a concentration of 525 µg/mL only. At this level RPE was reduced to ~12%. Whilst the increase in MMF was greater than 3-fold above that of the concurrent vehicle control, the increase was accompanied by marked cytotoxicity with no evidence of an increase in MMF at any of the other concentrations tested. Therefore caution was applied when interpreting these data.

In the presence of S-9 statistically significant increases in MMF were observed at concentrations of 350, 375, 400, 425, 450 and 475 µg/mL. At these concentrations mean mutant frequencies of 27.7, 18.5, 37.7, 29.7, 24.1 and 30.0 resistant mutants/106 viable cellswere obtained respectively, compared to a value of 8.6 resistant mutants/106 viable cellsfor the vehicle control. Increases of greater than 3-fold over the vehicle control were observed at 350, 400, 450 and 475 µg/mL. At these concentrations the level of cytotoxicity was either approaching or was within the cytotoxic range of 10-20%. Whilst data from this experiment would indicate a weakly positive result, due to lack of reproducibility from Experiment 1 the overall conclusion is considered to be negative result for this treatment condition.

 

In compliance with OECD 476 recommendations linear regression analysis was performed to assess a possible dose dependent increase in mutant frequencies for each experiment undertaken. As the choice of weighting is likely in practice to have little effect on the analysis the regression analysis on outweighed data was used. In all experiments undertaken (with the exception of Experiment 1, -S9) a significant dose dependent linear trend (p<0.01) was obtained, however as all increases in mutant frequency were consistent with the historical control, the significant linear trend tests obtained are not considered biologically relevant.

Conclusions:
It is concluded that HPV Xylenols did not induce mutation at the hprt locus of V79 Chinese Hamster lung cells when tested under the conditions employed in this study. These conditions included treatments up to 525 and 450 µg/mL in the absence and presence of a rat liver metabolic activation system (S9), respectively, in two independent experiments. The maximum concentration tested was limited by toxicity.
Executive summary:

HPV Xylenols was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in Chinese hamster lung V79 cells plated into Petri dishes. The study consisted of a cytotoxicity Range-Finder experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post‑mitochondrial fraction (S‑9). The test article was formulated in DMSO and dosed at 1%v/v.

 

A 3 hour treatment incubation period was used for all experiments.

 

In an initial cytotoxicity Range-Finder Experiment inadequate dose levels were selected in order to provide sufficient information for dose selection for the mutation test. Further range finding was undertaken; the highest concentrations tested which gave acceptable survival (measured by relative plating efficiency [RPE] in the range of 10-20%) were 525 mg/mL in the absence of S-9 and 440 mg/mL in the presence of S-9, which gave ~14% and ~17%.

 

Accordingly, for Experiment 1 nine concentrations, ranging from 100 to 600 µg/mL and 75 to 475 µg/mL were tested in the absence and presence of S‑9 respectively. Seven days after treatment the highest concentrations selected to determine viability and 6TG resistance were 500 µg/mL in the absence of S9 and 475 µg/mL in the presence of S9, which gave ~23% and ~9% RPE, respectively.

 

In Experiment 2, up to nine concentrations, ranging from 62.5 to 575 µg/mL, and 75 to 475 µg/mL were tested in the absence and presence of S‑9 respectively. Seven days after treatment the highest concentrations selected to determine viability and 6TG resistance were 525 µg/mL in the absence of S9 and 475µg/mL in the presence of S9, which gave ~12% and ~9% RPE, respectively.

 

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S9. Mutant frequencies in negative control were consistent with the acceptable range and clear increases in mutant frequency were observed by the positive controls, ethyl methanesulphonate (without S9) and 7,12-dimethyl-benz(a)anthracene (with S9). The assay system was therefore considered to be both sensitive and valid.

 

In Experiment 1 in the absence of S9 no statistically significant increases in mean mutant frequency (MMF) were observed following treatment with HPV Xylenols at any concentration tested. Cytotoxicity (expressed in terms of RPE at the end of treatment) was reduced to ~23% at 500 µg/mL. In the presence of S9, statistically significant increases in MMF were observed at concentrations of 75 to 475 µg/mL; however a 3-fold increase in MMF over the vehicle control was only observed at concentrations where the level of cytotoxicity was reduced to ~9 to 14%. Therefore caution was applied when interpreting these data, with increases in MMF greater than 3-fold only observed in the presence of cytotoxicity.

 

In Experiment 2 in the absence of S-9, a statistically significant increases in MMF were observed following treatment with HPV Xylenols at a concentration of 525 µg/mL only. At this level RPE was reduced to ~12%. Whilst the increase in MMF was greater than 3-fold above that of the concurrent vehicle control, the increase was accompanied by marked cytotoxicity with no evidence of an increase in MMF at any of the other concentrations tested. Therefore caution was applied when interpreting these data.

In the presence of S-9 statistically significant increases in MMF were observed at concentrations ranging from 350 to 475 µg/mL. At these concentrations MMF greater that 3‑fold over the vehicle control, with the level of cytotoxicity approaching (~26%) or within the cytotoxic range of 10-20%. Whilst data from this experiment would indicate a potentially weakly positive result. Due to lack of reproducibility from Experiment 1 the overall conclusion is consider to be that of a negative result for this treatment condition.

 

In all experiments undertaken (with the exception of Experiment 1, -S9) a significant dose dependent linear trend (p<0.01) was obtained, however as all increases in mutant frequency were consistent with the historical control, the significant linear trend tests obtained are not considered biologically relevant.

 

It is concluded that HPV Xylenols did not induce mutation at thehprt locus of V79 Chinese Hamster lung cells when tested under the conditions employed in this study. These conditions included treatments up to 525 and 450 µg/mL in the absence and presence of a rat liver metabolic activation system (S9) respectively in two independent experiments. The maximum concentration tested was limited by toxicity.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the result table above the most critical and relevant value of the weight of evidence approach is given. In the following, the results are shown for the other source substances of this weight of evidence approach:
Source CAS 95-48-7: o-cresol: negative with and without metabolic activation in S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538; Pepper, 1981
Source CAS 95-48-7: o-cresol: negative with and without metabolic activation in S. typhimurium TA 98, TA 100, TA 1535, TA 1537; Haworth, 1993
Source CAS 95-48-7: o-cresol: negative with and without metabolic activation in S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538; Pool & Lin, 1982
Source CAS 106-44-5: p-cresol: negative with and without metabolic activation in S. typhimurium TA 98, TA 100, TA 1535, TA 1537; Haworth, 1993
Source CAS 106-44-5: p-cresol: negative with and without metabolic activation in S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538; Pool & Lin, 1982
Source CAS 108-39-4: m-cresol: negative with and without metabolic activation in S. typhimurium TA 98, TA 100, TA 1535, TA 1537, E. coli WP2 uvr A; MHLW, 2001
Source CAS 108-39-4: m-cresol: negative with and without metabolic activation in S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538; Pool & Lin, 1982
Remarks on result:
other: Source: mixed xylenols, Merisol, 2004
Conclusions:
Based on all available information (weight-of-evidence), following an analogue read-across approach, Tar acids, Xylenol fraction (CAS 84989-06-0) is not considered to induce gene mutation in bacteria.
Executive summary:

Available experimental data on source substances (three creosol isomers and mixed xylenols) all give negative results for bacterial gene mutation as shown in several studies according to OECD 471. All study results were negative with and without metabolic activation. As there are no experimental data available regarding bacterial gene mutation for Tar acids, Xylenol fraction (CAS 84989-06-0) a weight-of-evidence approach was conducted taking into account all available data on the source substances which are surrogate substances for the target substance. Therefore, Tar acids, Xylenol fraction (CAS 84989-06-0) was considered to be not mutagenic in bacteria.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
Positive for the induction of structural and numerical chromosome aberrations.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
4 h / -S9: 47% at 550 µg/mL (the highest dose evaluated for aberrations); mitotic index: 53% reduction at 550 µg/mL relative to the solvent control.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the result table above the most critical and relevant value of the weight of evidence approach is given. In the following, the results are shown for the other source substances of this weight of evidence approach:
Source CAS 95-48-7: o-cresol: positive with and without metabolic activation in CHO cells; CMA, 1988
Source CAS 106-44-5: p-cresol: positive with and without metabolic activation in CHO cells; CMA, 1988
Source CAS 108-39-4: m-cresol: negative without and positive with metabolic activation in CHO cells; CMA, 1988
Source mixed ethylphenols: positive with and without metabolic activation in CHO cells; Merisol, 2004
Remarks on result:
other: Source: mixed xylenols, Merisol, 2004
Conclusions:
Based on all available information (weight-of-evidence), following an analogue read-across approach, Tar acids, Xylenol fraction (CAS 84989-06-0) is considered to induce chromosome aberrations in vitro under the test conditions used.
Executive summary:

Available experimental data on source substances (three creosol isomers, mixed ethylphenols and mixed xylenols) give positive results,i.e. an increase in structural and numerical chromosome aberrations, as shown in several studies according to OECD 473. All study results were positive with and without metabolic activation; except for m-cresol where clear positive results were observed with metabolic activation only. As there are no experimental data available on in vitro cytogenicity for Tar acids, Xylenol fraction (CAS 84989-06-0) a weight-of-evidence approach was conducted taking into account all available data on the source substances. Therefore, Tar acids, Xylenol fraction (CAS 84989-06-0) was considered to be able to induce chromosome aberrations in vitro under the condistions tested.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the result table above the most critical and relevant value of the weight of evidence approach is given. In the following, the results are shown for the other source substances of this weight of evidence approach:
Source CAS 95-48-7: o-cresol: negative with and without metabolic activation in mouse lymphoma L5178Y cells; Pepper, 1981
Source CAS 106-44-5: p-cresol: negative with and without metabolic activation in mouse lymphoma L5178Y cells; CMA, 1988
Source CAS 108-39-4: m-cresol: negative with and without metabolic activation in mouse lymphoma L5178Y cells; CMA, 1988
Remarks on result:
other: Source: mixed xylenols, Merisol, 2010
Conclusions:
Based on all available information (weight-of-evidence), following an analogue read-across approach, Tar acids, Xylenol fraction (CAS 84989-06-0) is not considered to induce gene mutation in mammalian cells in vitro.
Executive summary:

Available experimental data on source substances (o-creosol, p-cresol, m-cresol and mixed xylenols) give negative results for gene mutation in mammalian cells as shown in studies according to OECD 476. All study results were negative with and without metabolic activation. As there are no experimetnal data available regarding in vitro gene mutation in mammalian cells for Tar acids, Xylenol fraction (CAS 84989-06-0) a weight-of-evidence approach was conducted taking into account all available data on the source substances. Tar acids, Xylenol fraction (CAS 84989-06-0) was therefore not considered to be mutagenic in mammalian cells in vitro.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In absence of experimental data on Tar acids, Xylenol fraction (CAS 84989-06-0) an analogue read-across approach was conducted:

Cytogenicity (in vivo):
WoE - 3,5 xylenol (RL1; according to OECD 474): negative;
WoE - 2,4 xylenol (RL1, according to OECD 474): negative;
WoE - 2,6 xylenol (RL1, according to OECD 474): negative;
WoE - m-cresol (RL2, according to OECD 475): negative;

WoE - o-cresol (RL1; similar to OECD 474): negative;

WoE - o-cresol (RL1; according to OECD 478): negative;

WoE - p-cresol (RL1; according to OECD 478): negative

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
other: micronucleus assay
Species:
mouse
Strain:
NMRI
Details on species / strain selection:
no data
Sex:
male/female
Details on test animals 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

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
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
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).
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.

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.

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.

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
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
other: micronucleus assay
Species:
mouse
Strain:
NMRI
Details on species / strain selection:
no data
Sex:
male/female
Details on test animals 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 26.8 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

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
Dose / conc.:
250 other: mg/kg
Remarks:
24 h
Dose / conc.:
500 other: mg/kg
Remarks:
24 h
Dose / conc.:
1 000 other: mg/kg
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
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).
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. 1000 and 1250 mg/kg bw were administered. Mortality was observed at 1250 mg/kg, with all animals suriving at 1000 mg/kg, with clincial signs of toxicity evident. The MTD was therefore deemed to be 1000 mg/kg by the SD.

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

7290

207290

36.5

0.6

500

20000

7509

27509

37.5

0.7

1000

20000

8650

28650

43.3

1.0

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

7959

27959

40.0

0.7

Deaths (1 male) at 1000mg/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 2,4-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. Indiviudal 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.

Conclusions:
It is concluded that 2,4-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 bw. 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 2,4-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 1000 and 1250 mg/kg bw were administered. Mortality was observed at 1250 mg/kg, with all animals suriving at 1000 mg/kg, with clincial signs of toxicity 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 2,4-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) at 1000mg/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 2,4-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. Indiviudal 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 2,4 -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.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
GLP compliance:
yes
Type of assay:
rodent dominant lethal assay
Species:
mouse
Strain:
ICR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: males: 9.5 weeks
- Weight at study initiation: 26.7 - 36.7 g
- Housing: males individually and females in groups
- Diet ad libitum
- Water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12 / 12


Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
all dosing solutions were prepared immediately prior to dosing

Duration of treatment / exposure:
Single dose
Frequency of treatment:
once
Post exposure period:
6 weeks
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
275 mg/kg bw/day (actual dose received)
Dose / conc.:
550 mg/kg bw/day (actual dose received)
Remarks:
Due to high mortality and toxicity in the 650 mg/kg bw-group during the first week mice were removed from the study. Two weeks after the initiation of the assay another group of males dosed with 550 mg/kg bw was assigned as the new high dose to be evaluated.
No. of animals per sex per dose:
25 males/group / 50 females/group were treated
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine
- Route of administration: i.p.
- Doses / concentrations: 0.3 mg/kg bw
Tissues and cell types examined:
All females were examined for the number of live and dead implants within the uterine horn and whether the dead implants had occurred early or late in gestation. Live fetuses were identified as those which appeared to have a functional circulatory capacity.
Evaluation criteria:
Statistically significant dose-related increase in the number of dominant lethals is considered as mutagenic in this test system.
Statistics:
Chi-square test, ANOVA, Dunnett's one-tailed t-test
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
see section "remarks on results"
Vehicle controls validity:
valid
Negative controls validity:
other: vehicle control is negative control
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY Dose selection based upon the results of a dose range-finding assay.

Mortality: 
650 mg/kg bw: 10/25 males within the first week; as signs of toxicity mice exhibited rapid breathing, several became languid with mild clonic convulsions and squinted eyes and were 

prostrate and had scruffy coats 550 mg/kg bw: 6/25 males died during the test

Body weight:
No significant reduction in body weight were observed in any of the males  in any of the dose groups. The statistical evaluation of the parameters indicated that no significant effects 

of p-cresol were induced at any dose levels. The treatment had no adverse effects with respect to number of early and late resorptions, and live implants, indicating that the test 

compound did not induce dominant lethal mutations in male germ cells of mice under the conditions of this assay.
The concurrent positive control substance TEM induced  a significant  increase in the number of dead  implantations, in the portion of females with either one or more dead implantations, the frequency of dead implants relative to the total number of implants in each

 female during mating weeks 1 through 3 TEM induced a significant reduction  in total implants relative to the  vehicle control group.

Conclusions:
p-cresol did not induce dominant lethal mutations in male germ cells of mice under the conditions of this assay when tested according to OECD TG 478 (Rodent Dominant Lethal Assay).
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
yes
Remarks:
first sample time should be 12-18 hours after treatment
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:
other: mammalian bone marrow chromosome aberration
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals 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:
6, 24 and 48 hours
Dose / conc.:
96 mg/kg bw/day (actual dose received)
Dose / conc.:
320 mg/kg bw/day (actual dose received)
Dose / conc.:
960 mg/kg bw/day (actual dose received)
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
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:

RESULTS OF RANGE-FINDING STUDY: m-cresol was solubilized in corn oil and dosed once by oral gavage to 3 ICR mice/sex/group at 400, 800, 1200, 1600 and 2000 mg/kg b. The survivors were killed 2 days later. Toxicity and mortality was assessed in mice. Based on the result of this assay the dose levels were selected.

RESULTS OF DEFINITIVE STUDY
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

Conclusions:
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.
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
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
other: micronucleus assay
Species:
mouse
Strain:
NMRI
Details on species / strain selection:
no data
Sex:
male/female
Details on test animals 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 26.6 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

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
Dose / conc.:
375 other: mg/kg
Remarks:
24 h
Dose / conc.:
750 other: mg/kg
Remarks:
24 h
Dose / conc.:
1 500 other: mg/kg
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
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).
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Clincial signs of toxicity, with mortality
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. 1500 and 1750 mg/kg bw were administered. Mortality was observed at 1750 mg/kg and evident signs of toxicity were observed at 1500 mg/kg. The MTD was therefore deemed to be 1500 mg/kg.

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

Dose (mg/kg)

PCE

NCE

Total

%PCE

MN PCE

0

20000

7942

27944

39.7

1.5

75

20000

7225

27228

36.1

1.0

750

20000

6986

26991

34.9

0.7

1500

20000

8175

28182

40.2

1.0

CPA 20

10000

4610

14612

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

7968

27968

39.8

0.8

1500

16000

9474

25484

59.2

0.8

Deaths (1 male and 1 female) at 1500mg/kg, 48 hr sample point were observed. Clinical signs of toxicity included irregular breathing (375 mg/kg), squatting posture (750 mg/kg) and piloerection and squatting (1500 mg/kg). There were no marked decreases in mean PCE/total erythrocyte ratio observed for any of the 3,5-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.

Conclusions:
It is concluded that 3,5-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 1500 mg/kg (deemed a maximum tolerated dose) under the conditions of the assay described.
Executive summary:

In a bone marrow micronucleus assay using NMRI mice, a single oral gavage of 3,5-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 1500 and 1750 mg/kg/bw were administered. Mortality was observed at 1750mg/kg and evident signs of toxicity were observed at 1500mg/kg. The MTD was therefore deemed to be 1500 mg/kg.

 

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 3,5 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 1500mg/kg, 48 hr sample point were observed. Clinical signs of toxicity included irregular breathing (375mg/kg). squatting posture (750mg/kg) and piloerection and squatting (1500mg/kg). There were no marked decreases in mean PCE/total erythrocyte ratio observed for any of the 3,5 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 3,5-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 1500 mg/kg (deemed a maximum tolerated dose) under the conditions of the assay described.

 

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
GLP compliance:
yes
Type of assay:
rodent dominant lethal assay
Species:
mouse
Strain:
ICR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMAL
- Age at study initiation: 8 weeks
- Assigned to test groups randomly: yes
- Housing: males individually, females in groups
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 4 days
- body weights at initiation and at termination of the assay

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
no further data
Duration of treatment / exposure:
once
Frequency of treatment:
once
Post exposure period:
6 weeks
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 males per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine
- Route of administration: single i.p. injection
- Doses / concentrations: 0.3 mg/kg as a volume of 10 mL/kg
Tissues and cell types examined:
All females were examined for the number of live and dead implants within the uterine horn and whether the dead implants had occurred early or late in gestation. Live fetuses were identified as those which appeared to have a functional circulatory capacity.
Details of tissue and slide preparation:
no data
Evaluation criteria:
Statistically significant dose-related increase in the number of dominant leathals is considered as mutagenic in this system.
Statistics:
Chi-square test, ANOVA, Dunnett's one-tailed t test
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Remarks:
concentrations were chosen based on dose-range toxicity study
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY: dose selection was based upon a previously conducted range finding assay
high dose males:
Mortality: 8/25 within 6 days post dosing
Signs of intoxication: lethary, rough haircoat, languid, tremor, distended abdomen, squinted eyes
All surviving males recovered from these effects within 6 days after dosing.
No effects were observed on fertility index, total implants, mean implants per pregnant female, total live implants, total dead implants, and body weight gain of males.
The test article was considered negative for inducing dominant lethal mutations in germ cells of male mice under conditions of this assay.
Conclusions:
o-cresol did not induce dominant lethal mutations in male germ cells of mice under the conditions of this assay when tested according to OECD TG 478 (Rodent Dominant Lethal Assay).
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
sample time post final dose insufficient
Principles of method if other than guideline:
As described by Kong Z, Xu Ch (1995). Environmental Toxicology, Nanjing University Press (5 mice/group, solvent control, positive control: Mitomycin C, bone marrow cells)
GLP compliance:
not specified
Type of assay:
other: micronucleus assay
Species:
mouse
Strain:
not specified
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Animal Breeding Center of Nanjing University
- Weight at study initiation: 18 - 22 g
- Assigned to test groups randomly: yes
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25
- Photoperiod (hrs dark / hrs light): natural light /dark
Route of administration:
intraperitoneal
Vehicle:
DMSO
Details on exposure:
no futrther data
Duration of treatment / exposure:
twice at 24 hours interval
Frequency of treatment:
twice at 24 hour interval
Post exposure period:
6 hours after the last injection
Dose / conc.:
20 mg/kg bw/day
Remarks:
analytical conc.
Dose / conc.:
40 mg/kg bw/day
Remarks:
analytical conc.
Dose / conc.:
80 mg/kg bw/day
Remarks:
analytical conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C
Tissues and cell types examined:
polychromtic erythrocytes of the mouse bone marrow
Details of tissue and slide preparation:
Smears slides of bone marrow cells were prepared and fixed with methanol and stained with 2.5% Giemsa solution. In each experiment 1000 polychromatic erythrocytes (PCEs) were scored per slide/animal and among the 1000 PCEs micronucleated polychromatic erythrocytes (MNPCEs) were counted to estimate the MNPCE frequency (MNPCE%) among the PCEs. The ratio of PCEs to normochromatic erythrocytes (NE) based on 1000 PCEs and NE assessed per slide/animal, was determined as well.
Evaluation criteria:
statistical significant increase in micronuclei over the control was evaluated as a positive result
Statistics:
Student's t-test
Key result
Sex:
male
Genotoxicity:
positive
Toxicity:
yes
Remarks:
80 mg/kg bw (statistically significant decrease of PCE%)
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Examination of bone marrow cells:
o-Cresol causes a significant increase in the MN frequencies as compared to DMSO (solvent control). These increases are not dose-related because the dose of 40 mg/kg bw showed the

highest frequencies on micronuclei. At the concentration of 80 mg/kg bw of o-cresol the MN frequency decreased which does not refer to severe cytotoxicity because 40% PCE were observed at that dose.

Conclusions:
The results of the study are inconclusive and do not allow for assessment due to the poor experimental protocol e.g. strain unknown, no historical data given, sacrifice 6 hours after the last treatment is too early to show effects on bone marrow (respective guideline: 18 to 24 hours after the last treatment).
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
no data on the ratio of NCE:PCE, no positive control
Reason / purpose:
reference to same study
Principles of method if other than guideline:
Smears were prepared from peripheral blood samples obtained by cardiac punction of dosed and control animals at the termination of the 13 week study. Polychromatic and normochromatic erythrocytes from each animal were scored for micronuclei.
GLP compliance:
yes
Type of assay:
other: micronucleus assay
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
see the respective record in the chapter 7.5.1
Route of administration:
oral: feed
Vehicle:
see the respective record in the chapter 7.5.1
Details on exposure:
see the respective record in the chapter 7.5.1
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Post exposure period:
no
Dose / conc.:
5 000 ppm (nominal)
Dose / conc.:
10 000 ppm (nominal)
Dose / conc.:
20 000 ppm (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Positive control(s):
no data
Tissues and cell types examined:
erythrocytes from peripheral blood samples
Details of tissue and slide preparation:
Slides were stained with Hoechst 33258/pyrcein Y according to MacGregor et al (1983, no further data). At least 2000 polychromnatic erythrocytes (PCE) and 10000 normochromatic erythrocytes (NCE) from each animal were scored for micronuclei.
Evaluation criteria:
Positive if a significant elevation in the frequenciy of micronucleated erythrocytes was observed either in males or in females.
Statistics:
yes, but method not mentioned
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not examined

No significant elevation in the frequency of micronucleated erythrocytes was observed in either male or female mice. NOAEL for systemic toxicity is 1250 ppm (199 and 237 mg/kg bw/d,

respectively) based on increased relative and absolute liver and kidney weights without histopathological correlate from 2500 ppm onwards.

Conclusions:
Following repeated oral dosing of mice over a period of 13 weeks with o-cresol, the MNT with peripheral erythrocytes yielded a negative result.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose:
read-across source
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Source: CAS 108-39-4, m-cresol, CMA, 1988
Conclusions:
Based on all available information (weight-of-evidence), following an analogue read-across approach and in the absence of data on in vivo cytogenicity of Tar acids, Xylenol fraction (CAS 84989-06-0), Tar acids, Xylenol fraction (CAS 84989-06-0) is not considered to have cytogenic potential.
Executive summary:

Available data on the source substance m-creosol show negative results in mammalian bone marrow chromosome aberration test according to OECD 475. The study result was negative. However, as there are no data available on in vivo chromosome aberration for Tar acids, Xylenol fraction (CAS 84989-06-0) a weight-of-evidence approach was conducted taken into account all available data on the source substances and Tar acids, Xylenol fraction (CAS 84989-06-0) was therefore not considered to induce chromosome aberrations in vivo.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Remarks:
concentrations were chosen based on dose-range toxicity study
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Source: CAS 95-48-7, o-cresol, CMA, 1989
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
other: vehicle control is negative control
Positive controls validity:
valid
Remarks on result:
other: Source: CAS 106-44-5, p-cresol, CMA, 1989
Conclusions:
Based on all available information (weight-of-evidence), following an analogue read-across approach, Tar acids, Xylenol fraction (CAS 84989-06-0) is not considered to induce dominant lethal mutations in male germ cells.
Executive summary:

Available experimental data on the source substances o-creosol and p-cresol give negative results in the rodent dominant lethal assay according to OECD 478. As there are no data available on in vivo germ cell mutations for Tar acids, Xylenol fraction (CAS 84989-06-0) a weight-of-evidence approach was conducted taking into account all available data on the source substances. Tar acids, Xylenol fraction (CAS 84989-06-0) was therefore not considered to induce dominant lethal mutations.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
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
Remarks on result:
other: Source: CAS 105-67-9, 2,4-xylenol / CAS 576-26-1, 2,6-xylenol / CAS 108-68-9, 3,5-xylenol; BG Chemie, 1998
Additional information on results:
In the result table above the most critical and relevant value of the weight of evidence approach is given. In the following, the results are shown for the other source substances of this weight of evidence approach:
Source CAS 95-48-7: o-cresol: negative in male and female mice; NTP, 1991
Source CAS 95-48-7: o-cresol: positive in male mice; Li, 2005 (RL3)
Conclusions:
Based on all available information (weight-of-evidence), following an analogue read-across approach, Tar acids, Xylenol fraction (CAS 84989-06-0) is not considered to have cytogenic potential in vivo.
Executive summary:

Available experimental data on source substances (o-creosol, 2,4 -xylenol, 2,6 -xylenol and 3,5 -xylenol) give negative results in several micronucleus assays conducted according or similar to OECD 474. All reliable studies provided negative,i.e. non-cytogenetic, results. As there are no data available on in vivo micronucleus tests for Tar acids, Xylenol fraction (CAS 84989-06-0) a weight-of-evidence approach was conducted taking into account all available data on the source substances. Tar acids, Xylenol fraction (CAS 84989-06-0) was therefore not considered to induce micronuclei in vivo.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for read-across

There are no experimental data on genetic toxicity available for Tar acids, Xylenol fraction (CAS 84989-06-0). In order to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VII and VIII, 8.4, read-across from appropriate substances is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5.

According to Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met”. In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across) “to avoid the need to test every substance for every endpoint”.

Tar acids, Xylenol fraction (CAS 84989-06-0) is an UVCB substance comprised of the main constituents xylenols (all isomers in total > 60%; 2,4- and 2,5 -xylenol > 40%), ethyl phenols (< 30%) and cresols (< 25%). The read-across approach is therefore based on the main constituents of Tar acids, Xylenol fraction (CAS 84989-06-0), given common functional groups, common precursors and the likelihood of common breakdown products via biological processes. For further details on the read-across approach, please refer to the analogue justification in section 13 of the technical dossier.

For bacterial mutagenicity read-across from reliable data on the surrugate substances mixed xylenols, as well as to the cresol isomers (m- (CAS 108-39-4), p- (CAS 106-44 5) and o-cresol (CAS 95-48-7)) was conducted. For mammalian mutagenicity in vitro read-across from reliable data on the surrogate substances mixed xylenols, mixed ethyl phenols as well as to the cresol isomers (m- (CAS 108-39-4), p- (CAS 106-44 5) and o-cresol (CAS 95-48-7)) was conducted. For cytogenicity in vitro read-across from reliable data on the surrogate substances mixed xylenols as well as to the cresol isomers (m- (CAS 108-39-4), p- (CAS 106-44 5) and o-cresol (CAS 95-48-7)) was conducted. As the source substances were shown to be positive in the chromosome aberration assays further in vivo tests were conducted. For cytogenicity in vivo read-across from reliable data on the surrogate substances 2,4-xylenol (CAS 100-67-9), 2,6-xylenol (CAS 576-26-1) and 3,5-xylenol (CAS 108-68-9) as well as to the cresol isomers (m- (CAS 108-39-4), p- (CAS 106-44 5) and o-cresol (CAS 95-48-7)) was conducted in order to fulfill the requirements defined in Regulation (EC) No 1907/2006, Annex VIII, 8.4.

In vitro

Bacterial mutagenicity

o-cresol (CAS 95-48-7)

o-cresol was tested for genotoxic effects in the Ames test according to OECD TG 471 using Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 1538 at concentrations ranging from 0.01 to 50.0 µL/plate (Pepper, 1981).  The tests were conducted, using the pre-incubation method, on agar plates in the presence and absence of an Aroclor 1254 induced rat liver preparation and co-factors (S9 mix). Positive control compounds demonstrated the sensitivity of the assay and the metabolising potential of the S9 mix. No mutagenic activity was observed in any of the 5 bacterial strains, in either activation condition. Toxicity was noted with all the strains from 10 µL/plate onwards.

In a second test, o-cresol was tested for genotoxic effects in the Ames test according to OECD TG 471 using Salmonella typhimurium TA 98, TA 100, TA 1535, and TA 1537 at concentrations ranging from 1.0 to 100.0 µg/plate (Haworth, 1993).  The tests were conducted, using the pre-incubation method, on agar plates in the presence and absence of an Aroclor 1254 induced rat and hamster liver preparation and co-factors (S9 mix). Positive control compounds demonstrated the sensitivity of the assay and the metabolising potential of the S9 mix. No mutagenic activity was observed in any of the 4 bacterial strains, in either activation condition. Toxicity was noted with all the strains from 33 µg/plate onwards.

In a third test, o-cresol was tested for genotoxic effects in the Ames test similar to OECD TG 471 using Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 1538 at concentrations ranging from 5.0 to 5000.0 µg/plate (Poor & Lin, 1982).  The tests were conducted using the plate incorporation method, on agar plates in the presence and absence of an Aroclor 1254 induced preparation and co-factors (S9 mix). Positive control compounds demonstrated the sensitivity of the assay and the metabolising potential of the S9 mix. No mutagenic activity was observed in any of the 5 bacterial strains, in either activation condition. Toxicity was evident at the maximum dose tested, manifest by a slight thinning of the background lawn.

p-cresol (CAS 106-44 5)

p-cresol was tested for genotoxic effects in the Ames test according to OECD TG 471 using Salmonella typhimurium TA 98, TA 100, TA 1535, and TA 1537 at concentrations ranging from 3.3 to 333.0 µg/plate (Haworth, 1993).  The tests were conducted, using the pre-incubation method, on agar plates in the presence and absence of an Aroclor 1254 induced rat and hamster liver preparation and co-factors (S9 mix). Positive control compounds demonstrated the sensitivity of the assay and the metabolising potential of the S9 mix. No mutagenic activity was observed in any of the 4 bacterial strains, in either activation condition.

In a second test, p-cresol was tested for genotoxic effects in the Ames test similar to OECD TG 471 using Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 1538 at concentrations ranging from 0.5 to 5000.0 µg/plate (Poor & Lin, 1982).  The tests were conducted using the plate incorporation method, on agar plates in the presence and absence of an Aroclor 1254 induced preparation and co-factors (S9 mix). Positive control compounds demonstrated the sensitivity of the assay and the metabolising potential of the S9 mix. No mutagenic activity was observed in any of the 5 bacterial strains, in either activation condition. Toxicity was evident at the maximum dose tested, manifest by a slight thinning of the background lawn.

m-cresol (CAS 108-39-4)

m-cresol was tested for genotoxic effects in the Ames test according to OECD TG 471 using Salmonella typhimurium TA 98, TA 100, TA 1535, and TA 1537 and E. coli WP2 uvrA at concentrations ranging from 156 to 5000 µg/plate (MHLW, 2001b).  The tests were conducted, using the pre-incubation method, on agar plates in the presence and absence of a phenobarbital and 5,6-benzoflavone induced rat liver preparation and co-factors (S9 mix). Positive control compounds demonstrated the sensitivity of the assay and the metabolising potential of the S9 mix. No mutagenic activity was observed in any of the 5 bacterial strains, in either activation condition. Toxicity was noted with all the strains from 2500 µg/plate onwards.

In a second test, m-cresol was tested for genotoxic effects in the Ames test similar to OECD TG 471 using Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 1538 at concentrations ranging from 0.5 to 5000.0 µg/plate (Poor & Lin, 1982).  The tests were conducted using the plate incorporation method, on agar plates in the presence and absence of an Aroclor 1254 induced preparation and co-factors (S9 mix). Positive control compounds demonstrated the sensitivity of the assay and the metabolising potential of the S9 mix. No mutagenic activity was observed in any of the 5 bacterial strains, in either activation condition. Toxicity was evident at the maximum dose tested, manifest by a slight thinning of the background lawn.

Mixed xylenols

Mixed xylenols were tested for genotoxic effects in the Ames test according to OECD TG 471 using Salmonella typhimurium TA 98, TA 100, TA 1535, and TA 1537 and E. coli WP2 uvrA at concentrations up to 5000 µg/plate (Merisol, 2004a).  The tests were conducted, using the plate incorporation method, on agar plates in the presence and absence of an Aroclor 1254 induced rat liver preparation and co-factors (S9 mix). Positive control compounds demonstrated the sensitivity of the assay and the metabolising potential of the S9 mix. No mutagenic activity was observed in any of the 5 bacterial strains, in either activation condition. Toxicity was noted with all the strains at 5000 µg/plate.

Conclusion:

Based on all available information (weight-of-evidence), following an analogue read-across approach, Tar acids, Xylenol fraction (CAS 84989-06-0) is not considered to induce gene mutation in bacteria.

Mammalian mutagenicity

o-cresol (CAS 95-48-7)

o-cresol was tested for genotoxic effects in the mouse lymphoma forward mutation assay according to OECD 476 targeting the tk-locus at concentrations ranging from 3.91 to 250.0 µg/mL (Pepper, 1981). The tests were conducted in the presence and absence of an Aroclor 1254 induced rat liver preparation and co-factors (S9 mix). Positive control compounds demonstrated the sensitivity of the assay and the metabolising potential of the S9 mix. No mutagenic activity was observed in either activation condition. Cytotoxicity was observed with metabolic activation from 15.6 µg/mL onwards and without metabolic activation above 500 µg/mL.

p-cresol (CAS 106-44 5)

p-cresol was tested for genotoxic effects in the mouse lymphoma forward mutation assay according to OECD TG 476 targeting the tk-locus at concentrations ranging from 0.256 to 409 µg/mL (CMA, 1988a).  The tests were conducted in the presence and absence of a rat liver preparation and co-factors (S9 mix). Positive control compounds demonstrated the sensitivity of the assay and the metabolising potential of the S9 mix. No mutagenic activity was observed in either activation condition. Cytotoxicity at the highest concentration tested.

m-cresol (CAS 108-39-4)

m-cresol was tested for genotoxic effects in the mouse lymphoma forward mutation assay similar to OECD TG 476 targeting the tk-locus at concentrations ranging from 52.0 to 520 µg/mL (CMA, 1988b).  The tests were conducted in the presence and absence of an Aroclor 1254 induced rat liver preparation and co-factors (S9 mix). Positive control compounds demonstrated the sensitivity of the assay and the metabolising potential of the S9 mix. No mutagenic activity was observed in either activation condition. Cytotoxicity at the highest concentration tested.

Mixed xylenols

Mixed xylenols were tested for genotoxic effects in the mammalian cell gene mutation assay according to OECD 476 targeting the hprt-locus in Chinese hamster lung V79 cells (Merisol, 2010). The study consisted of a cytotoxicity range-finder experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post‑mitochondrial fraction (S9). A 3 hour treatment incubation period was used for all experiments. Accordingly, for Experiment 1 nine concentrations, ranging from 100 to 600 µg/mL and 75 to 475 µg/mL were tested in the absence and presence of S9, respectively. Seven days after treatment the highest concentrations selected to determine viability and 6TG resistance were 500 µg/mL in the absence of S9 and 475 µg/mL in the presence of S9, which gave 23% and 9% relative plating efficiency (RPE), respectively. In Experiment 2, up to nine concentrations, ranging from 62.5 to 575 µg/mL, and 75 to 475 µg/mL were tested in the absence and presence of S9, respectively. Seven days after treatment the highest concentrations selected to determine viability and 6TG resistance were 525 µg/mL in the absence of S9 and 475µg/mL in the presence of S9, which gave 12% and 9% RPE, respectively. Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S9. Mutant frequencies in negative control were consistent with the acceptable range and clear increases in mutant frequency were observed by the positive controls, ethyl methanesulphonate (without S9) and 7,12-dimethyl-benz(a)anthracene (with S9). The assay system was therefore considered to be both sensitive and valid. In Experiment 1 in the absence of S9 no statistically significant increases in mean mutant frequency (MMF) were observed following treatment with mixed xylenols at any concentration tested. Cytotoxicity (expressed in terms of RPE at the end of treatment) was reduced to 23% at 500 µg/mL. In the presence of S9, statistically significant increases in MMF were observed at concentrations of 75 to 475 µg/mL; however a 3-fold increase in MMF over the vehicle control was only observed at concentrations where the level of cytotoxicity was reduced to 9 to 14%. Therefore caution was applied when interpreting these data, with increases in MMF greater than 3-fold only observed in the presence of cytotoxicity. In Experiment 2 in the absence of S-9, a statistically significant increases in MMF were observed following treatment with mixed xylenols at a concentration of 525 µg/mL only. At this level RPE was reduced to 12%. Whilst the increase in MMF was greater than 3-fold above that of the concurrent vehicle control, the increase was accompanied by marked cytotoxicity with no evidence of an increase in MMF at any of the other concentrations tested. Therefore caution was applied when interpreting these data.

In the presence of S9 statistically significant increases in MMF were observed at concentrations ranging from 350 to 475 µg/mL. At these concentrations MMF greater that 3-fold over the vehicle control, with the level of cytotoxicity approaching (26%) or within the cytotoxic range of 10-20%. Whilst data from this experiment would indicate a potentially weakly positive result. Due to lack of reproducibility from Experiment 1 the overall conclusion is consider to be that of a negative result for this treatment condition. In all experiments undertaken (with the exception of Experiment 1, -S9) a significant dose dependent linear trend (p<0.01) was obtained, however as all increases in mutant frequency were consistent with the historical control, the significant linear trend tests obtained are not considered biologically relevant.

It is concluded that mixed xylenols did not induce mutation at the hprt locus of V79 Chinese Hamster lung cells when tested under the conditions employed in this study. These conditions included treatments up to 525 and 450 µg/mL in the absence and presence of a rat liver metabolic activation system (S9) respectively in two independent experiments. The maximum concentration tested was limited by toxicity.

Conclusion:

Based on all available information (weight-of-evidence), following an analogue read-across approach, Tar acids, Xylenol fraction (CAS 84989-06-0) is not considered to induce gene mutation in mammalian cells in vitro.

Cytogenicity

o-cresol (CAS 95-48-7)

An in vitro mammalian chromosome aberration test was performed according to OECD 473 with o-cresol (CMA, 1988c). Chinese hamster ovary (CHO) cells were treated with o-cresol at concentrations ranging from 150 to 1000 µg/mL with and without metabolic activation (S9 mix). o-cresol showed clastogenic activity in the presence and in the absence of metabolic activation. No concurrent measure of cytotoxicity was performed. Cytotoxicity conducted in a range-finder experiment was observed with metabolic activation at 1000 µg/mL and without metabolic activation at 300 µg/mL onwards.

p-cresol (CAS 106-44 5)

An in vitro mammalian chromosome aberration test was performed according to OECD 473 with p-cresol (CMA, 1988d). Chinese hamster ovary (CHO) cells were treated with o-cresol at concentrations ranging from 100 to 902 µg/mL with and without metabolic activation (S9 mix). Without metabolic activation and a 20-hour incubation increases in cells ranging between 6.5% and 11% cells with chromosome aberrations (versus 1.0% of solvent control) or between 4% and 14 % cells with aberrations (versus 2.0% of solvent control) were observed, respectively. Incubation for 20 hours with metabolic activation resulted in increases in the chromosome-aberrant cells ranging between 18% and 40.5% cells with aberrations (902 µg/mL was toxic; versus 1.5% of solvent control) and between 17% and 43% cells with aberrations (902 µg/mL was toxic, versus 3.0% of solvent control), respectively. In the test with an incubation for 10 hours in the presence of S9 mix no significant difference to the solvent controls were observed. Positive controls were valid in all tests. Thus, p-cresol showed clastogenic activity in the presence and in the absence of metabolic activation.

m-cresol (CAS 108-39-4)

An in vitro mammalian chromosome aberration test was performed similar to OECD 473 with m-cresol (CMA, 1988e). Chinese hamster ovary (CHO) cells were treated with m-cresol at concentrations ranging from 198 to 1100 µg/mL with and without metabolic activation (S9 mix). m-cresol was considered negative for inducing chromosomal aberrations when tested in CHO cells in vitro without a metabolic activation system after 2 hour treatment. When the test was performed in the presence of a metabolic activation system m-cresol induced a significant increase in aberrations at the highest doses tested despite the short treatment time of 2 hours.

The positive controls were all valid. No concurrent measure of cytotoxicity was performed. Cytotoxicity conducted in a range-finder experiment was observed with and without metabolic activation at 898 µg/mL onwards.

Mixed xylenols

An in vitro mammalian chromosome aberration test was performed according to OECD 473 with mixed xylenols (Merisol, 2004b). Chinese hamster ovary (CHO) cells were treated with mixed xylenols for 4 and 20 hours with and without metabolic activation (S9 mix). Concentrations of mixed xylenols ranged from 12.5 to 1200 µg/mL. A repeat CHO assay was performed due to lack of sufficient scorable cells in the highest doses and a lack of at least 50% reduction in mitotic index at the lower doses in the first assay. The toxicity of mixed xylenols to CHO cells treated for 4 hours in the absence of S9 activation was 47% at 550 µg/mL (the highest dose evaluated for aberrations). The mitotic index showed a 53% reduction at 550 µg/mL relative to the solvent control. The % of cells with structural aberrations was significantly increased (p<0.05, Fisher's exact test) at 300 and 500 µg/mL. The % of structurally damaged cells in the MMC positive control group was statistically significant at 22%. Treatment in the presence of S9 activation showed 54% toxicity at 550 µg/mL. The mitotic index at 550 µg/mL showed a 63% reduction relative to the solvent control. The % of cells with structural aberrations was significantly increased (p<0.05, p<0.01 Fisher's exact test) at 400 and 500 µg/mL, respectively. The Cochran-Armitage test was also positive for a dose response (p<0.05). The % of structurally damaged cells in the CP positive control group was statistically significant at 17.5%. Thus, the results of the assay indicate that under the conditions of the study, mixed xylenols caused a positive response in chromosome aberrations in both the presence and absence of metabolic activation.

Mixed ethylphenols

An in vitro mammalian chromosome aberration test was performed according to OECD 473 with mixed ethylpenols (Merisol, 2004b). Chinese hamster ovary (CHO) cells were treated with mixed ethylphenols for 4 and 20 hours with and without metabolic activation (S9 mix). Concentrations of mixed ethylphenols ranged from 50 to 1200 µg/mL. A repeat CHO assay was performed due to the 8.5% aberrant cells in the solvent group of the S9-activated 4 hour treatment being outside the historical solvent control range of 0 - 5.0%. The toxicity of mixed ethylphenols to CHO cells treated for 4 hours in the presence of S9 activation was 17% at 250 µg/mL (the highest dose evaluated for aberrations). The mitotic index showed a 52% reduction at 250 µg/mL relative to the solvent control. The % of cells with structural aberrations was significantly increased (p<0.01, Fisher's exact test) at all dose levels. The Cochran-Armitage test was also positive for a dose response (p>0.05). The % of cells with numerical aberrations in the test substance treated groups was not significantly increased above that of the solvent control at any dose level (p>0.05, Fisher's exact test). The % of structurally damaged cells in the CP positive control group was statistically significant at 19%. Thus, the results of the assay indicate that under the conditions of the study, mixed ethylphenols caused an increase in chromosome aberrations in both the presence and absence of metabolic activation.

Conclusion:

Based on all available information (weight-of-evidence), following an analogue read-across approach, Tar acids, Xylenol fraction (CAS 84989-06-0) is considered to induce chromosome aberrations in vitro.

In vivo

o-cresol (CAS 95-48-7)

An in vivo micronucleus test was performed with o-cresol (NTP, 1991). Smears were prepared from peripheral blood samples obtained by cardiac puncture of dosed and control mice (B6C3F1) at the termination of the 13-week repeated dose feeding study with a NOAEL for systemic toxicity of

1250 ppm (199 and 237 mg/kg bw/d, males and females, respectively) based on increased relative and absolute liver and kidney weights without histopathological correlate from 2500 ppm onwards.

Mice received 5000, 10000 and 20000 ppm of o-cresol. Polychromatic and normochromatic erythrocytes from each animal were scored for micronuclei. No data on the ratio of NCE:PCE and no positive controls were included. No significant elevation in the frequency of micronucleated erythrocytes was observed in either male or female mice.

Further, a micronucleus assay with significant methodological deficiencies was performed with mice (Li, 2005). Male mice received intraperitoneal o-cresol at 20, 40, or 80 mg/kg bw twice at a 24 hour interval. 6 hours after the last injections polychromatic erythrocytes of the bone marrow were examined. o-cresol caused a significant increase in the micronucleus frequencies as compared to DMSO (solvent control). These increases are not dose-related because the dose of 40 mg/kg bw showed the highest frequencies of micronuclei. At the concentration of 80 mg/kg bw of o-cresol the micronucleus frequency decreased which does not refer to severe cytotoxicity because 40% polychromatic erythrocytes were observed at that dose. The results of the study are inconclusive and do not allow an assessment due to the poor experimental protocol e.g. strain unknown, no historical data given, sacrifice 6 hours after the last treatment which is too early to show effects on bone marrow.

A rodent dominant lethal assay was performed according to OECD 478 with o-cresol (CMA, 1989a). Male mice were treated once via oral gavage with 75, 250 and 750 mg/kg bw o-cresol. One male was mated with 2 untreated females for 5 days. 6 mating periods were performed within 6 weeks in order to evaluate all stages of male germ cell developments. All females were examined for the number of live and dead implants within the uterine horn and whether the dead implants had occurred early or late in gestation. Live fetuses were identified as those which appeared to have a functional circulatory capacity. In the high dose group 8 of 25 males died within 6 days post dosing. Signs of intoxication were lethary, rough hair coat, languid, tremor, distended abdomen, and squinted eyes. All surviving males recovered from these effects within 6 days after dosing. No effects were observed on fertility index, total implants, mean implants per pregnant female, total live implants, total dead implants, and body weight gain of males. A positive control with triethyelenemelamine showed that the test system was valid. o-cresol was considered negative for inducing dominant lethal mutations in germ cells of male mice under conditions of this assay.

p-cresol (CAS 106-44 5)

A rodent dominant lethal assay was performed according to OECD 478 with p-cresol (CMA, 1989b). Male mice were treated once via oral gavage with 100, 275 and 550 (initially 650 mg/kg bw) mg/kg bw p-cresol. Due to high mortality and toxicity in the 650 mg/kg bw group during the first week, these mice were removed from the study. Two weeks after the initiation of the assay another group of males dosed with 550 mg/kg bw was assigned as the new high dose group. 1 male was mated with 2 untreated females for 5 days. 6 mating periods were performed within 6 weeks in order to evaluate all stages of male germ cell developments. All females were examined for the number of live and dead implants within the uterine horn and whether the dead implants had occurred early or late in gestation. Live fetuses were identified as those which appeared to have a functional circulatory capacity. After dosing of 650 mg/kg bw 10 of 25 males died within the first week. As signs of toxicity mice exhibited rapid breathing, several became languid with mild clonic convulsions and squinted eyes and were prostrate and had scruffy coats. After dosing of 550 mg/kg bw 6 of 25 males died during the test. No significant reduction in body weight was observed in any of the males in any of the dose groups. The statistical evaluation of the parameters indicated that no significant effects of p-cresol were induced at any dose levels. The treatment had no adverse effects with respect to the number of early and late resorptions, and live implants, indicating that the test compound did not induce dominant lethal mutations in male germ cells of mice under the conditions of this assay. The concurrent positive control substance triethyelenemelamine induced a significant increase in the number of dead implantations, in the portion of females with either one or more dead implantations, the frequency of dead implants relative to the total number of implants in each female during mating weeks 1 through 3 triethyelenemelamine induced a significant reduction in total implants relative to the vehicle control group. p-cresol was considered negative for inducing dominant lethal mutations in germ cells of male mice under conditions of this assay.

m-cresol (CAS 108-39-4)

An in vivo chromosome aberration test was performed according to OECD 475 with m-cresol (CMA, 1988f). Male and female ICR mice were treated with 96, 320 and 960 mg/kg bw via single oral gavage of m-cresol. Those doses were based on results from a range-finding study. Bone marrow was examined from each group 6, 24 and 48 hours post-treatment. 3 of 40 mice died in the high dose group. At the high dose group within 10 min after dosing squinty eyes, scruffy coats, mild tonic convulsions and rapid breathing which ceased after 30 min. and breathing difficulties were observed. Mice from the 320 mg/kg bw group showed slightly scruffy coats within 22 hours after dosing. In the low dose group no signs of toxicity were observed. The mitotic index in bone marrow cells of treated animals was similar to those of controls indicating that the test substance might not have reached the bone marrow. 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 with CP showed expected results.

2,4-xylenol (CAS 100-67-9)

An in vivo micronucleus test was performed according to OECD 474 with 2,4-xylenol (BG Chemie, 1998b). Male and female NMRI mice were treated with 250, 500 and 1000 mg/kg bw via single oral gavage of 2,4-xylenol. Doses were selected from a pilot toxicity study in which doses of 1000 and 1250 mg/kg bw were administered. Mortality was observed at 1250 mg/kg, with all animals suriving at 1000 mg/kg, with clincial signs of toxicity evident. The MTD was therefore deemed to be 1000 mg/kg bw. 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 2,4-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) at 1000 mg/kg bw, 48 hours 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 2,4-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. It is concluded that 2,4 -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 bw. 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.

2,6-xylenol (CAS 576-26-1)

An in vivo micronucleus test was performed according to OECD 474 with 2,6-xylenol (BG Chemie, 1998c). Male and female NMRI mice were treated with 250, 500 and 1000 mg/kg bw via single oral gavage of 2,6-xylenol. Doses were selected from a pilot toxicity study in which 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 considered to be 1000 mg/kg bw. 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 bw, 48 hours 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. 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 bw. Whilst the high dose was deemed a maximum tolerated dose by the study director under the conditions of the assay described, due to the mortality observed this would confirm that the MTD had been exceeded.

3,5-xylenol (CAS 108-68-9)

An in vivo micronucleus test was performed according to OECD 474 with 3,5-xylenol (BG Chemie, 1998d). Male and female NMRI mice were treated with 375, 750 and 1500 mg/kg bw via single oral gavage of 3,5-xylenol. Doses were selected from a pilot toxicity study and doses of 1500 and 1750 mg/kg/bw were administered. Mortality was observed at 1750 mg/kg bw and evident signs of toxicity were observed at 1500 mg/kg bw. The MTD was therefore deemed to be 1500 mg/kg. 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 bw). Mouse bone marrow was sampled at 24 and 48 hours after dosing for the vehicle and 3,5 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) occured at 1500mg/kg bw. Clinical signs of toxicity included irregular breathing (375 mg/kg bw), squatting posture (750 mg/kg bw) and piloerection and squatting (1500 mg/kg bw). There were no marked decreases in mean PCE/total erythrocyte ratio observed for any of the 3,5 xylenol-treated groups compared to the vehicle control group for either time point. Analysis of the mean MN PCE group data indicated that there were no statistically significant increases in 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. It is concluded, that 3,5-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 1500 mg/kg bw (deemed a maximum tolerated dose) under the conditions of the assay described.

Overall conclusion:

Available in vivo data on source substances 2,4-xylenol, 2,6-xylenol and 3,5-xylenol and the three cresol isomers o-, p-, and m-cresol give negative results in micronucleus assays, rodent dominant lethal assays or chromosome aberration assays according or similar to OECD 474, 478 and 475, respectively. All study results were consistently negative and did not confirm the positive effects from the in vitro studies. As there are no experimental data available on in vivo genotoxicity for Tar acids, Xylenol fraction (CAS 84989-06-0) a weight-of-evidence approach was conducted taking into account all available data on the source substances. Tar acids, Xylenol fraction (CAS 84989-06-0) was therefore not considered to be genotoxic in vivo.

Justification for classification or non-classification

Based on all available information (weight-of-evidence), following an analogue read-across approach, for Tar acids, Xylenol fraction (CAS 84989-06-0) no classification for genetic toxicity is required according to Regulation (EC) No 1272/2008.

However, Tar acids, Xylenol fraction (CAS 84989-06-0) has a harmonized classification for Mutagenicity (Muta. 1B, H340) according to Annex VI of Regulation (EC) No 1272/2008 under the following criteria: The classification as a carcinogen or mutagen need not apply if it can be shown that the substance contains less than 0.1% benzene and/or 0.005% benzo[a]-pyrene.