Reaction mass of m-cresol and 2-chloro-m-cresol and 6-chloro-m-cresol

Endpoint summary

Additional information

The relevant data on genetic toxicity for Preventol KMX and the read across substance 4-chloro-3-methylphenol are summarized in the table below. The read across justification and additional data for m-cresol are attached to the Chemical Safety Report in Annex I. In comparison to the chlorocresols, m-cresol is only a minor component of Preventol KMX. Therefore these data are not considered for the assessment of the genotoxic potential of Preventol KMX.

Table 1: Comparison of relevant data on genetic toxicity of Preventol KMX and 4-chloro-3-methylphenol

Endpoint	Preventol KMX	4-chloro-3-methylphenol
In vitro gene mutation study in bacteria	Negative	Negative
In vitro gene mutation study in mammalian cells	Read across to 4-chloro-3-methylphenol: negative	Negative
In vitro gene mutation study in mammalian cells	Read across to 4-chloro-3-methylphenol: negative	Negative
In vivo gene mutation	Read across to 4-chloro-3-methylphenol: negative	Negative

 

The mutagenic potential of Preventol KMX was assessed in a reliable and GLP compliant bacterial reverse mutation assay with the tester strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA102 according to OECD Guideline 471 (Nern, 2012). The test was performed as plate incorporation and as pre incubation assay both with and without metabolic activation. No increase of revertants per plate was seen in any tester strain under both conditions. Cytotoxicity was seen at 500 µg/plate in the pre-incubation experiment and 1600 µg/plate in the plate incorporation experiment. The positive controls increased the revertant counts significantly while the vehicle control showed no increase in revertants/plate. Thus, under the conditions of this study Preventol KMX did not show mutagenic potential in bacteria.

No further data on Preventol KMX (reaction mass of 6-chloro-m-cresol, 2-chloro-m-cresol and m-cresol) are available. The possibility of a read-across to Preventol CMK (4-chloro-3-methylphenol) in accordance with Regulation (EC) No 1907/2006 Annex XI 1.5. Grouping of substances and read-across approach was assessed. There it is given that a read-across approach is possible for substances, whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity. Preventol KMX contains of approximately 58% 6-chloro-m-cresol, 30% 2-chloro-m-cresol, 9% m-cresol and smaller amounts of p-cresol (approx. 2.5%), 4-chloro-3-methylphenol (approx. 0.5%) and other impurities (approx. 0.5%). A literature search and QSAR predictions were performed for 2-chloro-m-cresol, 6-chloro-m-cresol using m-cresol and 4-chloro-3-methylphenol as reference substances. Next to physico-chemical properties, environmental fate, ecotoxicity, toxicity, and metabolism of chlorocresols and chlorophenols in various databases, special emphasis was set on determining the effects of different positions of chlorine on aromatic ring structures as this is the determinant difference between the 3 chlorocresol constituents, which may influence outcome in physicochemical and toxicological behaviour. The aim was to evaluate the read-across from 4-chloro-3-methylphenol to Preventol KMX and to ensure a safe and legally valid analogue approach.

The chlorocresols are found to be similar in structure and the available data shows that the substances are similar in physico-chemical properties, environmental fate, metabolism and predicted affected pathways. Literature revealed no evidence of relevant influence of the position of chlorination on ring structures on the toxicological properties of a substance. Thus, the position of the chlorination is not regarded to significantly modulate the toxic effects of the chlorocresols. Since the discussed chlorocresols differ only in the position of the chlorination, the toxicological properties of the substances can be expected to be similar. This assumption is also supported by the fact that results of available studies are in the same range for Preventol KMX compared to results of 4-chloro-3-methylphenol. For instance both, Preventol KMX and 4-chloro-3-methylphenol did not show mutagenic potential in bacteria. Hence, 4-chloro-3-methylphenol is determined as a suitable read-across substance to predict toxicity endpoints in the chemical risk assessment of reaction mass of 6-chloro-m-cresol, 2-chloro-m-cresol and m-cresol (Preventol KMX). A detailed justification for the read-across is provided in the technical dossier (see IUCLID Section 13), as well as in the Chemical Safety Report (see Part B).

 

Available studies of interest for 4-chloro-3-methylphenol:

A reliable GLP compliant in vitro mammalian HPRT assay was performed with 4-chloro-3-methylphenol in Chinese Hamster Ovary cells (CHO-K1-BH4 cell) similar to OECD Guideline 476 with and without metabolic activation (Lehn, 1989). Six concentrations from 50 to 300 µg/mL (vehicle: DMSO) were assessed for the induction of mutagenicity based on a concentration range finding cytotoxicity test. Eight dishes per dose level were investigated and the test was performed as two independent trials. Due to marked cytotoxicity at 250 µg/mL and above only 4 dose levels could be used for mutagenicity evaluation. In the mutagenicity tests, a dose-related decrease of the relative survival with and without metabolic activation was observed. No increase in the mutation frequency was observed at any concentration tested. Thus, under the conditions of this study 4-chloro-3-methylphenol did not show mutagenic potential in mammalian cells.

A second reliable mutagenicity test with 4-chloro-3-methylphenol was performed in compliance with the principles of GLP using the unscheduled DNA synthesis test similar to OECD Guideline 482 with primary rat hepatocytes (Cifone, 1988). The primary rat hepatocytes were received from an adult male F344 rat (150-300 g bw). For each trial a single animal was used. Freshly prepared rat hepatocytes were exposed to 6 (trial 02) and 7 (trial 03) concentrations of test substance ranging from 0.25 µg/mL to 30.3 µg/mL in the presence of 5 µCi/mL ³H-thymidine for 18-19 hours in medium with reduced serum content (1% FCS). The test substance was not soluble in medium and was dissolved in DMSO at a concentration of 502 mg/mL forming a clear, colourless solution. With DMSO as vehicle, CMK was soluble in the medium at concentrations up to 2010 µg/mL. The cells were fixed, washed and air-dried prior to coating in the dark with photographic emulsion. The coated slides were stored at 4°C for 4-10 days and then developed. The slides were rinsed and stained with haematoxylin/eosin. Grain counting was done manually using a microscope with 1500x magnification and oil immersion. Each slide was examined for 3H-incorporation (silver grain formation) by counting 50 cells per slide, normally with 3 slides per dose group. The cytotoxicity was assessed using (trypan blue exclusion). Trial 01 was discarded due to excessive cytotoxicity. None of the criteria applied to indicate UDS were met or exceeded by the treatment with 4-chloro-3methylphenol and no dose response was observed. The positive control, 2-acetylaminofluorene, produced a clearly positive effect. Thus, under the conditions of this study 4-chloro-3methylphenol did not show mutagenic potential in mammalian primary hepatocytes.

A GLP compliant in vivo micronucleous test was performed with 4-chloro-3-methylphenol (Herbold, 1991) according to OECD Guideline 474 with some deviations in male and female NMRI mice. The deviations were: only one dose level was tested, the vehicle produced signs of toxicity and only 1000 erythrocytes/animal instead of 2000 were scored for micronuclei. Five mice per sex and group received a single intraperitoneal injection of 125 mg/kg bw. The known clastogen, cyclophosphamide (20 mg/kg bw), served as positive control. Mice of the negative control group were given vehicle (polyethyleneglycol 400). Mice of the control groups were sacrificed 24 hours after treatment. The mice of the test groups were sacrificed at 24, 48 and 72 hours after treatment. All animals were observed for clinical signs of toxicity until sacrifice. Femoral bone marrow cells were prepared 24, 48, and 72 h after administration and smears were produced. The ratio of polychromatic to normochromatic erythrocytes was determined. Animals treated with 125 mg/kg bw 4-chloro-3-methylphenol showed symptoms of toxicity which comprise apathy, roughened fur, staggering gait, prone position, spasm, twitching and diarrhoea. The symptoms lasted until sacrifice. Four mice died during the study period.

The positive control cyclophosphamide caused a clear clastogenic effect which is shown by a significant increase of polychromatic erythrocytes with micronuclei. The ratio of polychromatic to normochromatic erythrocytes was not altered.

The ratio of polychromatic to normochromatic erythrocytes was not altered in the group which received the test substance and no increased polychromatic erythrocytes with micronuclei were seen in treated animals. Thus, no indications of clastogenic effects of 4-chloro-3-methylphenol were found after treatment.

 

Taking all data into consideration, Preventol KMX is considered to be non-genotoxic.

Short description of key information:
A reliable and GLP compliant bacterial reverse mutation assay with the tester strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA102 according to OECD Guideline 471 was performed with Preventol KMX. Preventol KMX did not show mutagenic potential in bacteria.
No further data for the genotoxic potential of Preventol KMX is available. However, a read-across approach to 4-chloro-3-methylphenol was judged to be scientifically reliable (for details see discussion).
4-chloro-3-methylphenol was assessed for its genotoxic potential within a reliable GLP compliant in vitro mammalian HPRT assay in Chinese Hamster Ovary cells (CHO-K1-BH4 cell) similar to OECD Guideline 476 with and without metabolic activation, within a reliable GLP compliant unscheduled DNA synthesis test similar to OECD Guideline 482 with primary rat hepatocytes and within a GLP compliant in vivo micronucleous test according to OECD Guideline 474.
The structural analogue 4-chloro-3-methylphenol was not tested positive in any of the tests. Preventol KMX itself was not tested positive in the bacterial reverse mutation assay and therefore has not to be classified according to Dangerous substance directive 67/548/EEC and according to CLP (1272/2008/EC).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Preventol KMX has not to be classified according to Dangerous substance directive 67/548/EEC and according to CLP (1272/2008/EC).