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Genetic toxicity in vitro

Description of key information

An in vitro mammalian cell gene mutation test (HPRT) was performed to evaluate the mutagenic nature of p-Methylaminophenol sulfate. The study was performed using L5178Y mouse lymphoma cells in the presence and absence of S9 metabolic activation system with a exposure duration of 3 hrs. The doses for the study were 0.5 – 2.0 μg/ml without metabolic activation and 2.5 – 60 μg/ml with metabolic activation. p-Methylaminophenol sulfate did not induce a significant and/or reproducible increase in in the mutant frequencies after the 3-hour treatment in the presence or absence of S9 mix. Hence, p-Methylaminophenol sulfate is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Data is from SCCP
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
An in vitro mammalian cell gene mutation test (HPRT) was performed to evaluate the mutagenic nature of N-Methyl-p-aminophenol sulfate in a mammalian cell line.
GLP compliance:
yes
Type of assay:
other: In vitro mammalian cell gene mutation test (HPRT)
Test material information:
Composition 1
Specific details on test material used for the study:
- Name of test material: N-Methyl-p-aminophenol sulfate- IUPAC name: 4-(Methylamino)phenol Sulfate - Molecular formula: C7H9NO.1/2H2O4S- Molecular weight: 344.386 g/mol- Substance type: Organic- Physical state: No data - Purity: 98.7%- Impurities (identity and concentrations): 1.3%
Target gene:
HPRT locus
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
No data available
Additional strain characteristics:
not specified
Cytokinesis block (if used):
No data available
Metabolic activation:
with and without
Metabolic activation system:
S9 metabolic activation system was isolated from liver of rats induced with aroclor 1254
Test concentrations with justification for top dose:
0.5 – 2.0 μg/ml without metabolic activation2.5 – 60 μg/ml with metabolic activation
Vehicle:
No data available
Negative controls:
yes
Solvent controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Positive control(s) used were in accordance with the OECD guideline
Details on test system and conditions:
METHOD OF APPLICATION: In mediumDURATION- Preincubation period: No data available- Exposure duration: 3 hrs- Expression time (cells in growth medium): No data available- Selection time (if incubation with a selection agent): No data available- Fixation time (start of exposure up to fixation or harvest of cells): No data availableSELECTION AGENT (mutation assays): No data availableSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: 2 independent tests with and without S9 mixNUMBER OF CELLS EVALUATED: No data availableDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No data availableOTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: No data availableOTHER: No data available
Rationale for test conditions:
No data available
Evaluation criteria:
The cell line was observed for a significant and / or reproducible increases in the mutant frequencies.
Statistics:
No data available
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data available- Effects of osmolality: No data available- Evaporation from medium: No data available- Water solubility: No data available- Precipitation: No data available- Other confounding effects: No data availableRANGE-FINDING/SCREENING STUDIES: The doses for the main study were based on the level of toxicity.COMPARISON WITH HISTORICAL CONTROL DATA: No data availableADDITIONAL INFORMATION ON CYTOTOXICITY: No data available
Conclusions:
p-Methylaminophenol sulfate did not induce a significant and/or reproducible increase in mutant frequencies after the 3-hour treatment in the presence or absence of S9 mix, and hence the test chemical is not likely to classify as a gene mutant.
Executive summary:

An in vitro mammalian cell gene mutation test (HPRT) was performed to evaluate the mutagenic nature of p-Methylaminophenol sulfate. The study was performed using L5178Y mouse lymphoma cells in the presence and absence of S9 metabolic activation system with a exposure duration of 3 hrs. The doses for the study were 0.5 – 2.0 μg/ml without metabolic activation and 2.5 – 60 μg/ml with metabolic activation. p-Methylaminophenol sulfate did not induce a significant and/or reproducible increase in in the mutant frequencies after the 3-hour treatment in the presence or absence of S9 mix. Hence, p-Methylaminophenol sulfate is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

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

Genetic toxicity in vivo

Description of key information

Based on the prediction done (SSS Nagpur, 2017) using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances, gene mutation was predicted for N-Methyl-p-aminophenol sulfate. The study assumed the use of male and female mice. N-Methyl-p-aminophenol sulfate was predicted to not induce gene mutation in male and female mice and hence, according to the prediction made, it is not likely to classify as a gene mutant in vivo. Based on the predicted result it can be concluded that the substance is considered to not toxic as per the criteria mentioned in CLP regulation.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
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 limited documentation / justification
Justification for type of information:
Data is from OECD QSAR Toolbox version 3.3 and the supporting QMRF report has been attached.
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Prediction is done using OECD QSAR Toolbox version 3.3, 2017
GLP compliance:
not specified
Type of assay:
micronucleus assay
Test material information:
Composition 1
Specific details on test material used for the study:
- Name of test material: N-Methyl-p-aminophenol sulfate- IUPAC name: 4-(Methylamino)phenol Sulfate - Molecular formula: C7H9NO.1/2H2O4S- Molecular weight: 344.386 g/mol- Substance type: Organic- Physical state: No data
Species:
mouse
Strain:
not specified
Details on species / strain selection:
No data available
Sex:
male/female
Details on test animals and environmental conditions:
No data available
Route of administration:
not specified
Vehicle:
No data available
Details on exposure:
No data available
Duration of treatment / exposure:
No data available
Frequency of treatment:
No data available
Post exposure period:
No data available
Remarks:
No data available
No. of animals per sex per dose:
No data available
Control animals:
not specified
Positive control(s):
No data available
Tissues and cell types examined:
No data available
Details of tissue and slide preparation:
No data available
Evaluation criteria:
No data available
Statistics:
No data available
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Additional information on results:
No data available

The prediction was based on dataset comprised from the following descriptors: "Chromosome aberration"
Estimation method: Takes highest mode value from the 7 nearest neighbours
Domain  logical expression:Result: In Domain

(((((((((("a" or "b" or "c" or "d" )  and "e" )  and ("f" and ( not "g") )  )  and ("h" and ( not "i") )  )  and ("j" and ( not "k") )  )  and ("l" and ( not "m") )  )  and "n" )  and ("o" and ( not "p") )  )  and ("q" and ( not "r") )  )  and ("s" and "t" )  )

Domain logical expression index: "a"

Referential boundary: The target chemical should be classified as Phenols (Acute toxicity) by US-EPA New Chemical Categories

Domain logical expression index: "b"

Referential boundary: The target chemical should be classified as Strong binder, NH2 group AND Very strong binder, OH group by Estrogen Receptor Binding

Domain logical expression index: "c"

Referential boundary: The target chemical should be classified as Strong binder, NH2 group OR Very strong binder, OH group by Estrogen Receptor Binding ONLY

Domain logical expression index: "d"

Referential boundary: The target chemical should be classified as Phenols, Poly by Aquatic toxicity classification by ECOSAR

Domain logical expression index: "e"

Referential boundary: The target chemical should be classified as Strong binder, NH2 group AND Very strong binder, OH group by Estrogen Receptor Binding ONLY

Domain logical expression index: "f"

Referential boundary: The target chemical should be classified as Not possible to classify according to these rules (GSH) by Protein binding potency

Domain logical expression index: "g"

Referential boundary: The target chemical should be classified as Moderately reactive (GSH) OR Moderately reactive (GSH) >> Alkyl 2-alkenoates (MA) OR Moderately reactive (GSH) >> Substituted 1-Alken-3-ones (MA) by Protein binding potency

Domain logical expression index: "h"

Referential boundary: The target chemical should be classified as Non-Metals by Groups of elements

Domain logical expression index: "i"

Referential boundary: The target chemical should be classified as Halogens by Groups of elements

Domain logical expression index: "j"

Referential boundary: The target chemical should be classified as o-/ p-Aminophenols (Hemolytic anemia with methemoglobinemia) Rank B AND Oxyphenistain (Hepatotoxicity) Alert AND p-Aminophenols (Renal toxicity) Rank B by Repeated dose (HESS)

Domain logical expression index: "k"

Referential boundary: The target chemical should be classified as Thiocarbamates/Sulfides (Hepatotoxicity) No rank by Repeated dose (HESS)

Domain logical expression index: "l"

Referential boundary: The target chemical should be classified as o-/ p-Aminophenols (Hemolytic anemia with methemoglobinemia) Rank B AND Oxyphenistain (Hepatotoxicity) Alert AND p-Aminophenols (Renal toxicity) Rank B by Repeated dose (HESS)

Domain logical expression index: "m"

Referential boundary: The target chemical should be classified as 4,4'-Methylenedianilines/benzidines (Hepatobiliary toxicity) Rank B  by Repeated dose (HESS)

Domain logical expression index: "n"

Similarity boundary:Target: CN{+}(c1ccc(O)cc1).O{-}S(=O)(=O)O{-}.N{+}(C)c1ccc(O)cc1
Threshold=20%,
Dice(Atom centered fragments)
Atom type; Count H attached; Hybridization

Domain logical expression index: "o"

Referential boundary: The target chemical should be classified as Phenols (Acute toxicity) by US-EPA New Chemical Categories

Domain logical expression index: "p"

Referential boundary: The target chemical should be classified as Phenolphthaleins by US-EPA New Chemical Categories

Domain logical expression index: "q"

Referential boundary: The target chemical should be classified as Keratinocyte gene expression Givaudan by Database Affiliation

Domain logical expression index: "r"

Referential boundary: The target chemical should be classified as Estrogen Receptor Binding Affinity OASIS by Database Affiliation

Domain logical expression index: "s"

Parametric boundary:The target chemical should have a value of log Kow which is >= 1.03

Domain logical expression index: "t"

Parametric boundary:The target chemical should have a value of log Kow which is <= 4.74

Conclusions:
N-Methyl-p-aminophenol sulfate was predicted to not induce gene mutation in male and female mice and hence, according to the prediction made, it is not likely to classify as a gene mutant in vivo.
Executive summary:

Based on the prediction done (SSS Nagpur, 2017) using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances, gene mutation was predicted for N-Methyl-p-aminophenol sulfate. The study assumed the use of male and female mice. N-Methyl-p-aminophenol sulfate was predicted to not induce gene mutation in male and female mice and hence, according to the prediction made, it is not likely to classify as a gene mutant in vivo. Based on the predicted result it can be concluded that the substance is considered to not toxic as per the criteria mentioned in CLP regulation.

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

Additional information

Gene mutation in vitro:

An in vitro mammalian cell gene mutation test (HPRT) was performed to evaluate the mutagenic nature of p-Methylaminophenol sulfate. The study was performed using L5178Y mouse lymphoma cells in the presence and absence of S9 metabolic activation system with a exposure duration of 3 hrs. The doses for the study were 0.5 – 2.0 μg/ml without metabolic activation and 2.5 – 60 μg/ml with metabolic activation. p-Methylaminophenol sulfate did not induce a significant and/or reproducible increase in in the mutant frequencies after the 3-hour treatment in the presence or absence of S9 mix. Hence, p-Methylaminophenol sulfate is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

A Salmonella/microsome test was performed in the presence and absence of exogenous metabolic activation by S9-frqactions from the livers of Aroclor-induced male Sprague-Dawley rats or Syrian hamsters to evaluate the mutagenic nature of N-Methyl-p-aminophenol sulfate in Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537. The study was performed as per the preincubation assay with a preincubation time of 20 mins and then an incubation for 48 hrs. The test compound was administered at a dosage level of 0, 0.3, 1.0, 3.3, 10, 33.0, 100, 167, 333, 667, 1000 or 1667 µg/plate and concurrent solvent and positive control chemicals were included in the study. N-Methyl-p-aminophenol sulfate did not induce a reproducible, dose-related increase in his+ revertants over the corresponding solvent in the S. typhimurium tester strains TA1535 and TA98 in the presence and absence of S9 metabolic activation system. It was also non-mutagenic for the strains TA100 (10-1000 µg/plate) in the presence of metabolic activation system from hamster and TA1537 in the absence of S9 metabolic activation system. However, it induced gene mutation in the strains TA100 (combined with S9 factions from rats) and TA1537 in the presence of S9 from 10-1667 µg/plate. Therefore, the results indicate that a mutagenic response can be induced in vitro and thus the results are interpret as ambiguous.

A Salmonella/microsome test was performed in the presence and absence of exogenous metabolic activation by S9-frqactions from the livers of Aroclor-induced male Sprague-Dawley rats or Syrian hamsters to evaluate the mutagenic nature of N-Methyl-p-aminophenol sulfate in Salmonella typhimurium tester strains TA97, TA98, TA100 and TA1535. The study was performed as per the preincubation assay with a preincubation time of 20 mins and then an incubation for 48 hrs. The test compound was administered at a dosage level of 0, 1.0, 3.3, 10, 33.0, 67, 100, 333, 1000 or 2000 µg/plate in the preincubation assay of 48 hrs. Concurrent solvent and positive control chemicals were included in the study. N-Methyl-p-aminophenol sulfate did not induce a reproducible, dose-related increase in his+ revertants over the corresponding solvent in the S. typhimurium tester strains TA1535 or TA98 in the presence and absence of S9 metabolic activation system. It was also non mutagenic for the strains TA100 and TA97 in the absence of S9 metabolic activation system, however, it induced gene mutation in the strains TA100 (with HLI or RLI) and TA97 (with HLI or RLI). Therefore, the results indicate that a mutagenic response can be induced in vitro and thus the results are interpret as ambiguous.

Gene mutation in vivo:

Based on the prediction done (SSS Nagpur, 2017) using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances, gene mutation was predicted for N-Methyl-p-aminophenol sulfate. The study assumed the use of male and female mice. N-Methyl-p-aminophenol sulfate was predicted to not induce gene mutation in male and female mice and hence, according to the prediction made, it is not likely to classify as a gene mutant in vivo. Based on the predicted result it can be concluded that the substance is considered to not toxic as per the criteria mentioned in CLP regulation.

A rat bone marrow micronucleus assay was performed to determine the mutagenic nature of p-Methylaminophenol sulfate in male and female Sprague-Dawley rats. On the basis of a preliminary toxicity study conducted, the doses for the main study was 0, 100, 200 or 400 mg/kg bw, and concurrent negative and positive controls were included in the study. The highest dosed group animals were sacrificed after 24 and 48 hrs after the treatment. The results showed no indication of bone marrow toxicity in the micronucleus test because the PCE/NCE ratio was not lower in all treated groups than in the negative control group. However, oral bioavailability can be assumed by the systemic clinical signs and the death of one animal treated with 400 mg/kg. The mean MNPCE frequencies were not significantly increased in any of the groups treated with the test substance. In addition, p-Methylaminophenol sulphate did not induce chromosome aberrations or damage to the mitotic apparatus in bone marrow cells of rats after oral treatment. Therefore, since no mutagenic effects could be observed, it is not likely to classify as a gene mutant in vivo.

A rat liver in vivo/in vitro UDS assay was performed to determine the mutagenic nature of p-Methylaminophenol sulfate. The study was performed in male Wistar Han rats at dose level of 0, 50 or 500 mg/kg bw, administered by oral gavage. The animals were sacrificed after 16 hours and for an additional high dose group after 2 hours. p-Methylaminophenol sulphate was investigated for induction of unscheduled DNA synthesis (UDS) in rat hepatocytes in vitro following in vivo dosing. The top dose level was selected on the basis of a preliminary toxicity study, and negative and positive controls were also included in accordance with the OECD guideline. One animal treated with 500 mg/kg died. In none of the groups treated with the test substance there was a significant induction of UDS compared to the control group. There were no differences in the viability of hepatocytes isolated from rats of different dose groups. The results met all the pre-defined criteria for a negative response. Hence, the negative test result indicates that p-Methylaminophenol sulphate does not induce DNA damage that is detectable with the UDS test and thus is not likely to classify as a gene mutant in vivo.

Justification for classification or non-classification

Based on (Q)SAR prediction and the experimental data available for the target chemical, it is indicated that N-Methyl-p-aminophenol sulfate does not exhibit gene mutation in vitro in mammalian cells and in vivo. Since no mutagenic effects were observed in the reviewed in vivo-based studies, these results supersede in vitro studies. Hence, the target chemical it is not likely to classify as a gene mutant in vitro and in vivo as per the criteria mentioned in CLP regulation.