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Genetic toxicity in vitro

Description of key information

In absence of reliable data on 2,5-Xylenol (CAS 95-87-4) to fulfil the standard data requirements defiined in Regulation (EC) No 1907/2006, Annex VII and VIII, 8.4, an analogue read-across approach was conducted:

Gene mutation in bacteria:

WoE - 2,5-Xylenol (RL4; similar to OECD 471) negative with and without S9-mix in S. typhimurium strains TA 98 and TA 100;

WoE - 2,4-Xylenol (RL2; similar to OECD 471) negative with and without S9-mix in S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and 1538;

WoE - mixed xylenols (RL1; according to OECD 471) negative with and without S9-mix in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A

WoE, QSAR prediction with QSAR Toolbox database (v.4.1) on gene mutations in the bacterial reverse muation assay, results: negative

Mammalian chromosome aberration (in vitro):

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

Mammalian gene mutation (in vitro):

Key - mixed xylenols (RL1; according 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:
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 for cross-reference:
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
Conclusions:
The results of the assay indicate that, under the conditions of the study, the source substance mixed xylenols caused a positive response in chromosome aberrations in both the presence and absence of metabolic activation. As detailed in the analogue justification, it is considered that the target and the source substances are unlikely to lead to differences in genetic toxicity potential. Therefore, the results can be taken for the hazard assessment of the target substance 2,5-Xylenol (CAS 95-87-4).
Endpoint:
in vitro gene mutation study in mammalian cells
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 for cross-reference:
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
Conclusions:
It is concluded that the source substance 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. As detailed in the analogue justification, it is considered that the target and the source substance are unlikely to lead to differences in genetic toxicity potential. Therefore, the results can be taken for the hazard assessment of the target substance 2,5-Xylenol (CAS 95-87-4).
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
Please refer to "Attached justification".
Qualifier:
no guideline followed
Principles of method if other than guideline:
QSAR Toolbox prediction for single chemical:
Predicted endpoint: Gene mutation; No effect specified; Escherichia coli, S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, Salmonella typhimurium, TA 98 and TA 100; No duration specified; No guideline specified
Data gap filling method: Read-across analysis
Calculation approach (OECD principle 2 - Unambiguous algorithm): takes the highest mode value from the nearest 7 neighbours
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 97
Metabolic activation:
with and without
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)

The experimental values for the following 7 nearest neighbours in the database were negative: 3,4-xylenol (CAS No 95-65-8), 2,3-xylenol (CAS No 526-75-0), 2,6-xylenol (CAS No 576-26-1), 2,4-xylenol (CAS No. 105-67-9), 4-ethylphenol (CAS No 123-07-9), trimethylhydroquinone (CAS No 700-13-0), and 2-ethylphenol (CAS No 90-00-6)

Uncertainty of the prediction (OECD principle 4 - Uncertainty of the prediction):

The prediction is based on 68 values, 68 of them (100%) equal to predicted value

Prediction confidence is measured by the p-value: 3,6E-33

Conclusions:
2,5-xylenol is predicted by read-across analysis, which is supported by QSAR Toolbox v.4.1, to be non-mutagenic in the bacterial reverse mutation assay. Regarding the high confidence of the prediction (p < 3,6E-33), the result is considered to be reliable.
Executive summary:

Based on the QSAR Toolbox database (v.4.1), 2,5-xylenol was predicted not to induce gene mutations in the bacterial reverse mutation assay. The read-across analysis refers to the highest mode value from the following 7 nearest neighbours with test results obtained in different Salmonella typhimurium strains: 3,4-xylenol (CAS No 95-65-8), 2,3-xylenol (CAS No 526-75-0), 2,6-xylenol (CAS No 576-26-1), 2,4-xylenol (CAS No. 105-67-9), 4-ethylphenol (CAS No 123-07-9), trimethylhydroquinone (CAS No 700-13-0), and 2-ethylphenol (CAS No 90-00-6). In total, the prediction relies on 68 values, with all of them equal to the predicted value (i.e. negative). The p-value of 3,6E-33 reflects the high confidence of the prediction and indicates that the prediction is reliable.

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:
weight of evidence
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
S. typhimurium, 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 values of the weight of evidence approach (source: mixed xylenols) are given. In the following, the results are shown for the other source substances of this weight of evidence approach:
Source 2,4-Xylenol (CAS 105-67-9): negative with and without metabolic activation in S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538; Pool and Lin, 1982
Conclusions:
The source substance 2,4-xylenol did not show mutagenic effects, both in the presence and absence of a metabolic activation system, when tested in S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100. In addition, also the source substance mixed xylenols did not cause a positive response either in the presence or absence of metabolic activation by Aroclor-induced rat liver S9. in the tested strains (S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A). The source substances were therefore concluded to be negative for genotoxicity in bacteria. As detailed in the analogue justification, it is considered that the target and the source substances are unlikely to lead to differences in genetic toxicity potential. Therefore, the results can be taken for the hazard assessment of the target substance 2,5-Xylenol (CAS 95-87-4).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In absence of data on 2,5-Xylenol (CAS 95-87-4) 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;

Link to relevant study records
Reference
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:
weight of evidence
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
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
Conclusions:
Based on all available information (weight-of-evidence), following an analogue read-across approach, 2,5-Xylenol (CAS 95-87-4) is not considered to have cytogenic potential in vivo.
Executive summary:

Available experimental data on source substances (2,4-Xylenol, 2,6-Xylenol and 3,5-Xylenol) give negative results in several micronucleus assays conducted according 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 2,5-Xylenol (CAS 95-87-4) a weight-of-evidence approach was conducted taking into account all available data on the source substances.

As detailed in the analogue justification, it is considered that the target and the source substances are unlikely to lead to differences in genetic toxicity potential. Therefore, the results can be taken for the hazard assessment of the target substance 2,5-Xylenol (CAS 95-87 -4). 2,5-Xylenol (CAS 95-87-4) 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 data for genetic toxicity available for 2,5-Xylenol (CAS 95-87-4) to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VII and VIII, 8.4. Therefore, read-across from appropriate source 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”.

2,5-Xylenol (CAS 95-87-4), 3,5-Xylenol (CAS 108-68-9), 2,6-Xylenol (CAS 576-26-1), and 2,4-Xylenol (CAS 105-67-9) are considered to be similar on the basis of the structural similar properties and/or activities. The source substance mixed xylenols is a mixture of the target substance 2,5-Xylenol and several other xylenols (3,4-Xylenol, 2,4-Xylenol, 3,5-Xylenol, 2,3-Xylenol and 2,6-Xylenol), which are also considered to be similar on the basis of the structural similar properties and/or activities. The available endpoint information is thus used to predict the same results for 2,5-Xylenol (CAS 95-87-4). Therefore, the requirements defined in Regulation (EC) No 1907/2006, Annex VII - VIII, 8.4 are fulfiled. A detailed analogue justification is provided in the technical dossier (see IUCLID Section 13).

Bacterial reverse mutation assay

Shale oil fractions and known constituents such as 2,5-Xylenol were examined by Epler and coworkers (1979) in the in vitro bacterial reversion mutation assay (Ames test) using Salmonella typhimurium strains TA 98 and TA 100, both with and without a rat liver enzyme preparation for metabolic activation (S-9 mix). The test was well documented and the procedure was similar to OECD Guideline 471, however only two Salmonella strains were included in the study. Over a 1000-fold concentration range of the test material, no increase of revertant colonies was noted. Thus, 2,5-Xylenol showed no mutagenic potential in this test.

In a read-across approach the genotoxic potential of the isomer analogue 2,4-Xylenol (CAS 105-67-9) was assessed in the in vitro bacterial reversion mutation assay (Ames test) using standard Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and in addition TA 1538 (Pool and Lin, 1982). The study was well documented and similar to OECD Guideline 471. 0, 0.5, 5, 50, 500, or 5000 µg of the test material, which had been dissolved in DMSO, was added to the culture medium of a test plate. Each concentration was tested in quadruplicate, with and without addition of S9-mix. The maximum concentration resulted in toxicity, which was apparent as a thinning of the background lawn and a reduced number of spontaneous revertants. Lower test concentrations did not induce a significant increase in the number of histidine revertant colonies. In conclusion, 2,4-xylenol did not show mutagenic effects, both in the presence and absence of a metabolic activation system, when tested in S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100.

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, 2004).  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.

Based on the QSAR Toolbox database (v.4.1), 2,5-xylenol was predicted not to induce gene mutations in the bacterial reverse muation assay (Chemservice, 2017). The read-across analysis refers to the highest mode value from the following 7 nearest neighbours with test results obtained in different Salmonella typhimurium strains: 3,4-xylenol (CAS No 95-65-8), 2,3-xylenol (CAS No 526-75-0), 2,6-xylenol (CAS No 576-26-1), 2,4-xylenol (CAS No. 105-67-9), 4-ethylphenol (CAS No 123-07-9), trimethylhydroquinone (CAS No 700-13-0), and 2-ethylphenol (CAS No 90-00-6). In total, the prediction relies on 68 values, with all of them equal to the predicted value (i.e. negative). The p-value of 3,6E-33 reflects the high confidence of the prediction and indicates that the prediction is reliable.

Conclusion:

Based on all available information (weight-of-evidence), following an analogue read-across approach, 2,5-Xylenol (CAS 95-87-4) is not considered to induce gene mutation in bacteria.

Mammalian mutagenicity

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 the available information (key study of an adequate source substance), following an analogue read-across approach, 2,5-Xylenol (CAS 95-87-4) is not considered to induce gene mutation in mammalian cells in vitro.

Cytogenicity

An in vitro mammalian chromosome aberration test was performed according to OECD 473 with mixed xylenols (Merisol, 2004). 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.

Conclusion:

Based on the available information (key study of an adequate source substance), following an analogue read-across approach, 2,5-Xylenol (CAS 95-87-4) is considered to induce chromosome aberrations in vitro.

 

In vivo

2,4-Xylenol (CAS 105-67-9)

An in vivo micronucleus test was performed according to OECD 474 with 2,4-xylenol (BG Chemie, 1998). 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, 1998). 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, 1998). 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 give negative results in micronucleus assays according to OECD 474. 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 2,5-Xylenol (CAS 95-87-4) a weight-of-evidence approach was conducted taking into account all available data on the source substances. 2,5-Xylenol (CAS 95-87-4) was therefore not considered to be genotoxic in vivo.

Justification for classification or non-classification

Based on all available information (key studies from adquate source substances or weight-of-evidence), following an analogue read-across approach, for 2,5-Xylenol (CAS 95-87-4) no classification for genetic toxicity is required according to Regulation (EC) No 1272/2008.