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EC number: 272-940-1 | CAS number: 68921-45-9
Original posting date for substituted diphenylamine documents: January 15, 2002 Posting date for revised documents for substituted diphenylamines: October 24, 2003 and January 19, 2007.
No toxicity was observed to any of the strains. Precipitates were observed at 1500 ug/plate and 5000 ug/plate but did not interfere with scoring. No significant increase in the frequency of revetant colonies was recorded in any strain with or without activation, and the responses of the positive controls were satifactory.
Chromosome aberrations in human lymphocyte cultures treated with Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene
In the absence of S9-mix in the second cytogentic assay (24h exposure time, 24 h fixation time)
Mitotic Index (%)
No. of Cells scored
No. of Cells with aberrations (+ gaps)a)
No. of Cells with aberrations (- gaps)
total aberr (+ gaps)
In the absence of S9-mix in the second cytogenetic assay (48h exposure time, 48h fixation time)
total aberr (- gaps)
The numerical variation polyploidy (poly) was not counted as an aberration.
In the presence of S9-mix in the second cytogenetic assay (3h exposure time, 48h fixation time)
a)Abbreviations use for various types of aberrations are listed in APPENDIX 2 (detailed under Overall Remarks). Misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.
b)CP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of control.
*) Significantly different from control group (Chi-square test), *P < 0.05, **P < 0.01 or ***P < 0.001.
Evaluation of the ability of Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat experiment).
This report describes the effect of Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of the test substance was tested in two independent experiments.
The study procedures described in this report were based on the most recent OECD and EC guidelines.
Batch EL2B27G336 of Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene was a clear red-brown viscous liquid. The test substance was dissolved in dimethyl sulfoxide.
The concentrations analysed in the samples prepared for use were in agreement with the nominal concentrations (i.e. mean accuracies 88 and 93%). Analysis of low and high formulations after storage yielded a relative difference of ≤ 10%. Based on this, the formulations were found to be stable when stored at room temperature under normal laboratory light conditions for at least 4 hours.
In the first cytogenetic assay, Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene was tested up to 100 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. The test substance precipitated in the culture medium at this dose level.
In the second cytogenetic assay, the test substance was tested up to 200 µg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 100 µg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. In the presence of S9-the test substance was tested up to 50 µg/ml for a 3 h exposure time with a 48 h fixation time. The test substance precipitated in the culture medium at this dose level.
The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.
No effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.
Finally, it is concluded that the test is valid and that Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4 -trimethylpentene is not clastogenic in human lymphocytes under the experimental conditions described in the report.
Study conducted to OECD and EU test guidelines in compliance with GLP.
Results: Cytotoxicity conc: With and without metabolic activation: None toxic
Precipitation conc: 1500 and 5000 ug/plate
Genotoxic effects: With and without metabolic activation: negative
Mutagenicity, in vivo and in vitro
Study conducted to OECD test guidelines in compliance with GLP. Data included for OECD SIDS dossier. The test substance did not produce any signs of clinical toxicity. Statistically lower PCE:NCE ratios, while not dose related, did strongly indicate that the test substance was cytotoxic to the bone marrow. The test substance did not produce any statistically significant increase in micronucleated PCEs relative to the vehicle control at the 24-hour and 48-hour harvest interval. The positive control induced a statistically significant increase in micronucleated PCEs compared to the vehical control. Result: The test substance was tested up to the limit dose (2000 mg/kg) and did not cause chromosome damage in the mouse bone marrow micronucleus assay under the conditions of this test.
Read across to supporting substance, CAS No. 68442 -68 -2, by structural analogue. This substance has been supported under Environmental Protection Agency’s (EPA’s) High Production Volume (HPV) Challenge Program. The American Chemical Councils RAPA Panel, has derived a “Substituted Diphenylamines” category of chemicals for this substance, please refer to EPA reference 201-14700A located at
Relying on several factors specified in EPA’s guidance document on “Development of Chemical Categories in the HPV Challenge Program,” in which use of chemical categories is encouraged, the following closely related chemicals constitute a chemical category:
Structural Similarity. A key factor supporting the classification of these chemicals as a category is their structural similarity (see Figure 1). All share a common starting material; Diphenylamine (Benzenamine, N-phenyl-, CAS# 122-39-4), a common synthetic pathway, and all compounds in this category are diamines with various substitutions.
Similarity of Physicochemical Properties. The similarity of the physicochemical properties of these materials parallels their structural similarity. All are off-white to light brown solids or viscous liquids intended for use as antioxidants in finished rubber articles or as antidegradant additives that extend the useful life of heavy-duty industrial functional fluids used in high-speed, high-temperature and/or high-load applications. As a class, these amine-based antidegradant compounds are less migratory (more polymer-bound) and less staining than the Substituted p-Phenylenediamine antidegradants. The use of these materials requires that they be stable under high temperatures. Their low volatility is due to their low vapor pressure, highly viscous or solid form. The existing information for these materials indicates that they have low water solubility and high flash points.
Toxicological Similarity. Review of existing published and unpublished test data for Substituted Diphenylamines shows the aquatic and mammalian toxicity among the materials within this category are similar.
Mammalian Toxicology - Mutagenicity. Data from bacterial reverse mutation assays, in vitro and in vivo chromosome aberration studies, as well as additional supporting in vitro and in vivo genetic toxicity studies were reviewed, and the findings indicate a low concern for mutagenicity either for aryl or alkyl substituted materials. Similarly, the data for a mixed aryl/alkyl substituted molecule also indicates a lack of mutagenicity. Data are available for several members of the category or close structural analogs, and these data can be bridged to the other members of the category. Therefore, for the purposes of the HPV Program, the category has been adequately tested for mutagenicity, and no additional mutagenicity testing is proposed.
Conclusion. Based upon the data reviewed in “Substituted Diphenylamines” category of chemicals, the physicochemical and toxicological properties of the Substituted Diphenylamine category members are similar and follow a regular pattern as a result of that structural similarity. Therefore, the definition of a chemical category has been met, and read across is considered appropriate for the category of chemical.
Additional information from genetic toxicity in vivo:
The substance was negative in the following tests:
1) Ames Test x 2- salmonella/ e.coli
2) Chromosome aberration test - human lymphocytes.
3) In-vivo Micronucleus Test – mice (read-across)
Two separate bacterial reverse mutation tests showed that the substance does not have any mutagenic properties in different Salmonella strains. In addition, no genotoxic properties have been seen in in-vitro chromosome aberration nor in an in-vivo Micronucleus Test in mice with a structurally very similar compound. Repeat-dose studies with oral treatment in rats did not reveal any tumorigenic properties which could be related to the administration of the test substance. Consequently, additional testing of the test substance in an in vitro gene mutation study in mammalian cells does not appear scientifically necessary and this test has been waived.
The following information is taken into account for any hazard / risk assessment:
Genetic toxicity "in vitro" is discussed below.
Value used for CSA:Genetic toxicity: negative
This substance has been supported under Environmental Protection Agency’s (EPA’s) High Production Volume (HPV) Challenge Program. The American Chemical Councils RAPA Panel, has derived a “Substituted Diphenylamines” category of chemicals for this substance, please refer to EPA reference 201-14700A located at
Relying on several factors specified in EPA’s guidance document on “Development of Chemical Categories in the HPV Challenge Program,” in which use of chemical categories is encouraged, the chemicals constitute a chemical category on the following basis:
Justification for selection of genetic toxicity endpoint All studies are negative; hence the in vivo study is selected for completeness purposes.
The above studies have all been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the studies were conducted to GLP an in compliance with agreed protocols. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds. As the effects are considered adaptive rather than toxicological, no classification is proposed.
The above results triggered no classification under the CLP Regulation (EC No 1272/2008). No classification for mutagenicity is therefore required.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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