5-amino-o-cresol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

According to the results, 4-amino-2-hydroxytoluene was not mutagenic in Salmonella typhimurium. However, it induced mutations in mouse lymphoma L5178Y cells in vitro (small colonies indicating clastogenicity), micronuclei  in  human  lymphocytes in  vitro, and  DNA strand breaks in Chinese hamster V79 cells, without metabolic activation. In an in vitro  comet  assay,  the  test  substance was  found positive. On the basis of the available in vitro data, the substance has relevant mutagenic (clastogenic) potential on in vitro test system.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

According the results of the in vivo key studies available, 4-amino-2-hydroxytoluene did not induce micronuclei in mouse bone marrow, or unscheduled DNA synthesis in rat hepatocytes. On the basis of the available data, the substance has no relevant mutagenic potential in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Six in vitro studies were available to assess the potential genotoxicity of the test item 4-amino-2-hydroxytoluene (A027) :

-An in vitro gene mutation test on mammalian cells (L5871Y mouse lymphoma cells) was realised according to OECD method 476 and was GLP compliant. The test targeted the Thymidine Kinase locus. The test item was applied at 50, 100, 250, 500, 600, 700, 800, 900, 1000, 1250 and 1500 µg/mL, with metabolic activation and 2.50, 5.00, 10.0, 25.0, 50.0, 100, 150, 200, 250, 300, 400 and 500 µg/mL  without metabolic activation. The treatment period was about 4 hours. With metabolic activation: A biologically relevant increase of mutant frequencies was observed between a concentration range of 250 µg/mL and 1000 µg/mL. Additionally, a slight decrease in mutants was observed at the highest concentrations evaluated (within or slightly above the historical data). Without metabolic activation: A biologically relevant increase of mutant frequency and a dose-effect relationship was observed at the higher concentrations evaluated in comparison to the negative control. Based upon above, 5-Amino-2-methylphenol was considered mutagenic at the Thymidine Kinase +/- locus in L5178Y mouse lymphoma cells, with or without metabolic activation. (Hamman U, 2005)

- An in vitro micronucleus assay was performed on human lymphocytes. This test was performed accordingly to OECD 476 guideline method and OECD GLP principle. The study was quoted Klimisch 1. The cells were exposed for 3 or 20 hours period , and 28 or 45 hours of recovery for respectively with S9 or without S9 conditions. In experiment 1, before exposure the cells were stimulated 24 hours (experiment 1) and 48 hours (Experiment 2). During experiment 1, without metabolic activation, the frequencies of micronucleated and binucleated cells was significantly elevated. In the second experiment, With and without metabolic activation, Treatment of cells with test substance resulted in increase in frequencies of MNBN cells, which were significantly higher than those observed in concurrent vehicle control cultures for all concentrations analyse with strong dose effect. (Whitwell J, 2005)

- In vitro Comet assay was performed in order to investigate the potential test item genotoxic effect as DNA strand break. The study was quoted as Klimisch 2. Chinese hamster V79 cell were used with and without metabolic activation and were treated at 0, 308, 616 and 1232 µg/mL during 3 hours.  DNA damage were measured % tail DNA appearing as a comet with head and tail. 4-Amino-2-hydroxytoluene induced a clear dose-dependent increase in DNA damage of the cells in the presence and absence of metabolic activation. The percent tail DNA were 4.73, 6.79, 7.28 and 7.68 at 0, 308, 616 and 1232 µg/mL. (Wirnitzer, 2005)

- The bacterial reverse mutation test of 4-Amino-2-Hydroxytoluene (A027) was performed following the OECD guideline 471 and GLP compliance. The study was performed to investigate the potential of the test substance to induce gene mutations according to the plate incorporation test (Experiment 1) and the pre-incubation test (Experiment 2) using the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102. Doses used were 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate. No biological relevant increase in revertant colony numbers was observed in any tester strain following test treatment with and without metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Therefore,4-Amino-2-Hydroxytoluene (A027)was non-mutagenic in Salmonella typhimuriumin a bacterial reverse mutation assay, with and without metabolic activation. (Sokolowski A, 2005)

- The test substance was evaluated for cell transformation assay on Syrian Hamster Cells (SHE) according to OECD Draft and OECD GLP principle. The study was quoted as Klimisch 1. After a preliminary study (dose range finding study), SHE cells were exposed during 24 hours and subsequent 7 days incubation with 20, 40, 60, 80, 100 and 120 µg/mL of the 4-amino-2-hydroxytoluene. The average number of colonies per dish was calculated. Each colony was evaluated and recorded as either normal or morphologically transformed (MT). There was a dose-related trend (p<0.05) in the number of transformed colonies; the results were considered as positive. Two of the test substance concentrations scored (80 and 120 µg/mL) gave a statistically significant increase in the transformation frequency (p<0.05) when compared to the solvent control. (Pant K, 2005)

- Chromosome aberration test was realised in order to assess the potential clastogenicity of the registered substance. The study was quoted as Klimisch 2 and followed OECD guideline 473 method and OECD GLP principle. The test was performed with human peripheral blood lymphocytes treated with62.14, 95.59, 147.07, 226.26, 348.09, 535.52, 823.88, 1267.5, 1950 and 3000 µg/mL . In the assay, lymphocytes were incubated for 44 hours with mitogen, prior to treatment with test substance. Preincubated cells were exposed to test substance or positive/vehicle control treatment for 3 hours. Cultures were washed and further incubated for 25 hours in fresh culture medium. One hour prior to harvest time,Colchicine(1 μg/mL) was added to all cultures. Without metabolic activation, treatment of cells with test substance resulted in large increases in aberration frequencies which were statistically significant at the top dose (3000 µg/mL) in the absence of S9 including gaps, and at the top two doses (1950 and 3000 µg/mL) when gaps were excluded from the data. With metabolic activation, Increases were statistically significant following treatment in the presence of S9 at all test concentrations. (Marshall RR, 1988)

Three relevant in vivo key studies were performed to assess the potential clastogenic effect of the registered substance on in vivo test sytem :

- In vivo UDS was performed on male Sprague Dawley rats (according to OECD method 486, GLP compliant study, Klimisch 1) which were treated by oral gavage with 0, 500, 1000, 2000 mg/kg bw. The test material was administered to 5 male rats/dose level/exposure time (i.e. either 2 to 4 h or 12 to 16 h) giving a total of 10 animals at each dose. The only exception was 2000 mg/kg bw group where 7 rats/ exposure time were used giving a total of 14 animals). After a treatment period of 2-4 h or 12-16 h, the animals was anesthetized by inhalation of isoflurane and a midventral incision was made to expose the liver. Primary hepatocyte cultures were established and exposed for 4 h to (3)H-thymidine which is incorporated if UDS occurs. Hepatocytes were only harvested from the first three successful perfusions and evaluated for UDS. The exposure period did not induce unscheduled DNA synthesis in primary rat hepatocytes in an autoradiographic in vivo unscheduled DNA synthesis assay. (Pant K & San RHC, 2005)

- A micronucleus test was performed according to OECD GLP principle, the method used was in accordance with OECD 474 guideline method using male and female NMRI mice which were treated with 20, 100 and 200 mg/kg bw for 24 h preparation interval and 200 for 48 hours preparation interval. After treatment, animals were sacrificed and the bone marrow tissues were isolated from both femora. Slides were prepared to be examined under microscope. 2000 erythrocytes were counted per animal. Based on above,4-amino-2-hydroxytoluene was non-mutagenic in the micronucleus test with bone marrow cells of the mouse when administered intraperitoneally at 20, 100 and 200 mg/kg bw. (Hornavar, 2005)

- An in vivo COMET assay was performed on wistar male rat with scientifically robust method and was GLP compliant. Five male Wistar rats per group were treated twice via gavage with 0, 500, 1000 and 2000 mg/kg bw, respectively, in Polyethylenglykol (PEG) 400 with a time difference of 20 hours between administrations. Animals were sacrificed 3 hours after the second administration. Histopathology of the liver, stomach, and urinary bladder was assessed in a parallel experiment with three male rats for each concentration. Due to inconclusive results 10 male Wistar rats each were treated in an additional trial analogously with 0 and 2000 mg/kg bw. DNA migrated out from the nucleus in single cell gel electrophoresis was used as a measure of DNA strand breakage. Tail length, moment and tail intensity (% tail DNA) were used as assessment parameter for genotoxicity. Statistically significant increases in mean tail length, mean tail moment, and mean tail intensity were seen at 500 mg/kg bw and for mean tail moment at 1000 mg/kg bw of test substance. Hepatotoxicity was demonstrated at2000 mg/kgwhich may result in loss of cells with primary or secondary genotoxic damage during single cell isolation. The hepatotoxicity and loss in cells at 2000 mg/kg bw, in theory, explained that no liver cells with increased comets were observed at 2000 mg/kg bw (highest concentration tested). Considering both experiments together, no biologically relevant increase in the comet assay parameters were seen in the stomach and urinary bladder. Therefore, 4-amino-2-hydroxytoluene is considered to be non-genotoxic in the comet assay in vivo to stomach and urinary bladder epithelium cells of male Wistar rats. Primary genotoxicity of test substance to rat liver could not be excluded. (Wirnitzer, 2005)

Justification for classification or non-classification

According to the results of the studies, 4-amino-2-hydroxytoluene was not mutagenic in Salmonella typhimurium. However, it induced mutations in mouse lymphoma L5178Y cells in vitro (small colonies indicating clastogenicity), clastogenic effect in micronuclei  in  human  lymphocytes in  vitro, and  DNA strand breaks in Chinese hamster V79 cells, without metabolic activation. In an in vitro  comet  assay,  the  test  substance was  found positive. On the basis of the available in vitro data, the substance has relevant mutagenic (clastogenic) potential on in vitro test system. However, according to the results of the in vivo key studies available, 4-amino-2-hydroxytoluene did not induce micronuclei in mouse bone marrow, or unscheduled DNA synthesis in rat hepatocytes. On the basis of the available data, the substance has no relevant mutagenic potential in vivo.