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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-06-06 to 2005-06-30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Non-GLP study conducted according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) and EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria) with an acceptable deviation (only three tester strains were used). However, an expert statement is added in attachment and in the field "any other remarks" to justify the fact that no further testing is necessary.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
: Only 3 bacterial strains used and limited details on methodology were provided.
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
: Only 3 bacterial strains used and limited details on methodology were provided.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(p-chlorophenyl)piperidin-4-ol
EC Number:
254-479-8
EC Name:
4-(p-chlorophenyl)piperidin-4-ol
Cas Number:
39512-49-7
Molecular formula:
C11H14ClNO
IUPAC Name:
4-(p-chlorophenyl)piperidin-4-ol
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study reports): JNJ-117676-AAA (T000263)
- Physical state: solid (powder)
- Appearance: White, slight beige amorphous, crystalline, micropowder
Specific details on test material used for the study:
Batch number: Code: 8693
Purity: 100 %
Stability in solvent: not indicated by sponsor
Solubility in water: 0.34 g/L
Solubilityin ethanol: >340 g/L
Storage conditions: Room temperature
Expiry date: 2005-12-31

Method

Target gene:
histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA98, TA100 and TA102
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: See "Any other information on materials and methods"
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
- Pre-experiment/Experiment 1: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
- Experiment 2: 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: On the day of the experiment, the test item was dissolved inethanol and neutralized in 1 N HCl.
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
: untreated
Negative solvent / vehicle controls:
yes
Remarks:
: ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation at 10 µg/plate for TA100
Untreated negative controls:
yes
Remarks:
: untreated
Negative solvent / vehicle controls:
yes
Remarks:
: ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
without metabolic activation at 10 µg/plate for TA98
Untreated negative controls:
yes
Remarks:
: untreated
Negative solvent / vehicle controls:
yes
Remarks:
: ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation at 4.0 µL/plate for TA102
Untreated negative controls:
yes
Remarks:
: untreated
Negative solvent / vehicle controls:
yes
Remarks:
: ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation at 2.5 µg/plate for TA98 and TA100 and 10.0 µg/plate for TA102
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (pre-experiment, experiment I); preincubation (experiment II).
- In Experiment 1 (plate incorporation), the following materials were mixed in a test tube and poured onto the selective agar plates: 100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control), 500 µL S9 mix (for test with S9) or S9 mix substitution buffer (for test without S9), 100 µL bacteria suspension (cf. test system, pre-culture of the strains), and 2000 µL overlay agar.
- In Experiment 2 (pre-incubation), 50 µL test solution, solvent or 100 µL positive control, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45°C) was added to each tube. The mixture was poured on minimal agar plates.
- After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.

DURATION
- Preincubation period: 60 minutes (Experiment 2)
- Exposure duration: at least 48 hours (both experiments)
- Selection time: at least 48 hours (simultaneous with exposure)

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn
Evaluation criteria:
- The test substance was considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control was observed.
- A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent controls such an increase was not considered biologically relevant.
Statistics:
- A statistical analysis of the data was not required.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance in the overlay agar was observed in all strains in Experiment 2 with metabolic activation at 1000 µg/plate and above.

RANGE-FINDING/SCREENING STUDIES:
- The pre-experiment was reported as main Experiment 1.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The positive controls showed a distinct increase in induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Reduced background growth and toxic effects were observed in all tester strans as described in the section "Any other information on results".
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

Reduced background growth was observed at the following concentrations (ug/plate)

Strain

Experiment 1

Experiment 2

 

Without S9

With S9

Without S9

With S9

TA98

5000

5000

2500 -5000

2500 -5000

TA100

5000

5000

1000 -5000

5000

TA102

5000

5000

1000 -5000

2500 -5000

Toxic effects, evident as a reduction in the number of revertants were observed at the following concentrations (µg/plate)

Strain

Experiment 1

Experiment 2

 

Without S9

With S9

Without S9

With S9

TA98

2500-5000

5000

2500 -5000

5000

TA100

5000

5000

2500-5000

5000

TA102

2500-5000

5000

1000 -5000

2500 -5000

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative with and without metabolic activation

The test substance was evaluated for mutagenic potential in the bacterial reverse mutation assay in S. typhimurium strains TA98, TA100 and TA102 in the presence and absence of S9 metabolic activation. Under the conditions of the study, the test substance was determined to be negative for mutagenic potential in all tester strains in the absence and presence of metabolic activation.