1-acetyl-4-(3-dodecyl-2,5-dioxo-1-pyrrolidinyl)-2,2,6,6-tetramethylpiperidine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Hostavin 3058 was not mutagenic with or without metabolic activation in two bacterial reverse mutation assays with the strains S. typhimuriumTA 98, TA 100, TA 1535, TA 1537 and TA 1537. Additionally, a negative results in the presence and absence of metabolic activation was obtained with the read-across substance Hostavin 3055 using the strains

S. typhimuriumTA 98, TA 100, TA 1535, TA 1537 and TA102.

The structural analogue, Hostavin 3055, also yielded negative results in an in vitrogene mutation study in mammalian cells with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Species / strain:

S. typhimurium TA 102

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

Key result

Species / strain:

S. typhimurium TA 100

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

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results

Solubility Record
Solvent used	RO (reverse osmosis) Water	Dimethyl sulfoxide
Quantity of test item	50 mg	50 mg
Volume of vehicle added	1000 µL	1000 µL
Final Concentration	50 mg /mL	50 mg /mL
Solubility status	Insoluble	Soluble

As mentioned in the above table, solubility of test item was checked in RO water and found insoluble. So the solubility was checked in Dimethyl sulfoxide (DMSO). The test item was found soluble in DMSO at 50 mg/ml to give final treatment concentration of 5 mg/plate (recommended maximum test concentration for soluble non-cytotoxicity substances). Therefore, DMSO was chosen as solvent for the study.

Precipitation

Precipitation was checked as insolubility to assess precipitation in the final mixture under the actual test conditions and evident to the unaided eye. Test item dissolved in DMSO at 50 mg/mL concentration was checked for precipitation. Details are given in table below:

Precipitation Record
Overlay agar volume	Test item preparation volume	Concentration/Plate	Result
2 mL	100 µL	5 mg	No Precipitation

An amount of test item preparation (50 mg/mL) was added 2 ml of overlay agar (Top agar) in test tubes to give test item concentration of 5 mg/plate and plated on minimal glucose agar (MGA) plates. Precipitation was not observed. Therefore, 5 mg/plate was selected as the highest concentration for pre-experiment.

Range-Finding/Screening studies

The pre-experiment was performed with TA 100 and TA 98 strain ofSalmonella typhimuriumand with eight different concentrations of the test item prepared with half log intervals and three plates were used for each dose level and for the controls. 5 mg/plate were selected as the highest dose in the pre-experiment basedon the solubility and precipitation test. Following doses were selected for the pre-experiment i.e., 0.001, 0.002, 0.005, 0.016, 0.050, 0.501, 1.582 and 5 mg/plate.

The results were evaluated based on the reduction of revertant colony count and bacterial background lawn.

Toxicity was observed in the tested concentrations 5 (T8) and 1.582 (T7) mg/plate. Reduction in colony count and microcolonies were observed in the tested concentrations 0.501 (T6) and 0.050 (T5) mg/plate both in absence and in the presence of metabolic activation. At treated concentrations 0.016 (T4) to 0.001 (T8) mg/plate, no reduction in colony count and background lawn were observed both in absence and in the presence of metabolic activation, when compared to that of the negative control group.

Based on the results of pre-experiment, following doses were selected for the main study trials: 0.0002, 0.001, 0.002, 0.005 and 0.016 mg/plate both in absence (-S9) and in the presence (+S9) of metabolic activation.

Comparision with Historical Control Data

The spontaneous reversion rates in the negative control are in the range of in house historical data.

Conclusions:

It can be concluded that the source substance did not induce gene mutations either by base pair substitution or by frame shifts in the genome of the strains used under the conditions of the assay with and without metabolic activation.

Executive summary:

The study used as source investigated the genotoxicity of Hostavin 3055.The study results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross reference “assessment report”.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Conclusions:

In conclusion it is stated that during the mutagenicity test described and under the experimental conditions reported the source substance did not induce mutations in the HPRT locus in V79 cells of the Chinese hamster in the absence and presence of metabolic activation.

Executive summary:

The study used as source investigated the genotoxicity of Hostavin 3055.The study results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross reference “assessment report”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Hostavin 3058 was assessed for its potential to induce micronuclei in NMRI mice in vivo. The test item did not induce micronuclei when tested at the limit dose of 2000 mg/kg bw.

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:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: 84/449/EWG, B.12 (Mikrokerntest); OECD 474

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: BOR:NMRI (SPF Han.)

Sex:

male/female

Details on test animals or test system and environmental conditions:

41 male, 41 female mice

Route of administration:

oral: gavage

Vehicle:

corn oil

Duration of treatment / exposure:

Limit test

Frequency of treatment:

once

Post exposure period:

24 and 48 h

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 48 hours Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 48 hours

Control animals:

yes

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): commonly accepted
- Route of administration: gavage
- Doses / concentrations: 100 mg/kg bw

Tissues and cell types examined:

bone mark from femurs

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: MTD derived from pretest

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): single application; sample times: 24 and 48 h after treatment

DETAILS OF SLIDE PREPARATION:
Cell suspension was washed by cellulose-chromatography, centrifuged and resuspended in FBS/EDTA. Slide preparation per animal, 24 h drying and staining with May-Grünwald/Giemsa solution.

METHOD OF ANALYSIS:
Analysis by application of "Micronucleus Test V 4.00", Leitz MIAMED)

OTHER:
anaylsis of min. 2000 PCE
- PCE/NCE ratio
- frequency of micro nuclei in polychrmoatic and normochromatic erythrocytes

Statistics:

Application of "Statgraphics, Version 5.0" (Statistical Graphics Corporation)

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

Observations:
No statistically significant increase in number of polychromatic erythrocytes.
No cytotoxicity observed (PCE/NCE).

Conclusions:

Interpretation of results (migrated information): negative
Under the test conditions described above, the test substance showed to be non-mutagenic.

Executive summary:

The mutagenic potential of the test substance was investigated in an in vivo mouse micronucleus tests according to OECD Guideline 474. In this test, the substance showed to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

In the absence of any information on species specific activities or mode of actions the results are regarded as relevant for humans

Additional information

Hostavin 3058 and its structural analogue showed negative results in three studies for the induction of gene mutations (bacterial reverse mutation assay) by frameshift or base-pair substitutions with and without metabolic activation. The study was performed with the test strainsS. typhimuriumTA 98, TA 100, TA 1535, TA 1537 andTA 102. Test concentrations up to the limit concentration of 5000 µg/plate were tested in the experiment. The test compound proved to be not mutagenic to the bacterial strains.

The structural analogue; Hostavin 3055, also yielded negative results in anin vitrogene mutation study in mammalian cells with and without metabolic activation. Additionally, negative results were obtained in an in vivo micronucleus assay in mice at the limit dose of 2000 mg/kg bw.

Justification for classification or non-classification

Hostavin 3058 does not have to be classified as mutagenic according to Regulation (EC) No. 1272/2008 since neither this substance nor it's structural analogue Hostavin 3055 did reveal any mutagenic effect in the available assays.