Registration Dossier

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:
2019-06-13 to 2019-07-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: “Testing Methods for New chemical Substances”, March 31, 2011 (Yakushokuhatsu No. 0331-7, Heisei 23.03.29 Seikyoku No. 5 and Kampokihatsu No. 110331009)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Specific details on test material used for the study:
Name: cyclic Glucamide C8-C10
Chemical Name: Amides, C8-C10, N-(1-deoxy-D-glucitol-1-yl)-N-methyl, dehydrated (CAS name)
CAS No.: 2290526-77-9
Batch No.: BP 1c (reactor mix)
Molecular Weight: 303.4 g/mol (C8-derivative); 331.45 g/mol (C10-derivative)
Physical State: highly viscous mass
Colour: brown
Purity: 100% (“UVCB”)
Expiry Date: 28 March 2021
Storage Conditions: room temperature
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Method

Target gene:
His, trp
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat S9 liver microsomal fraction
Test concentrations with justification for top dose:
Experiment I:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Experiment II:
1.00, 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
all tester strains (without metabolic activation) except E. coli WP2 uvrA
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
all tester strains (with metabolic activation) except E. coli WP2 uvrA
31.6, 100, 316, 1000, 2500 and 5000 µg/plate
(only E. coli WP2 uvrA)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Remarks:
A. dest., Eurofins Lot No. 190524, 190617, 190606, 190708
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, Applichem Lot No. 0001603375
Positive controls:
yes
Positive control substance:
other:
Remarks:
Positive control without metabolic activation: sodium azide for tester strains TA 100, TA 1535; 4-nitro-o-phenylene-diamine for TA 98, TA 1537; methylmethanesulfonate for E. coli; with metabolic activation: 2-aminoanthracene for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
-For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 µL Overlay agar.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.


- Preincubation period: 60 min; 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
- Exposure duration: at least 48 h; After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.



NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.


DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and E.coli the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
TA 1537, TA98, TA100 and E.coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
experiment I and II
Additional information on results:
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
Toxic effects of the test item were noted in all tester strains evaluated in experiment I and II.
In experiment I toxic effects of the test item were observed in tester strains TA98 and TA100 and at concentrations of 316 µg/plate and higher (without metabolic activation) and at concentrations of 2500 µg/plate (with metabolic activation). In tester strain TA1535 toxic effects of the test item were noted at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation). In tester strain TA1537 toxic effects of the test item were seen at concentrations of 2500 µg/plate and higher (with and without metabolic activation). In tester strain E. coli WP2 uvrA toxic effects of the test item were observed at a concentration of 5000 µg/plate (without metabolic activation).
In experiment II toxic effects of the test item were noted in tester strain TA98 at concentrations of 100 µg/plate and higher (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation). In tester strain TA100 toxic effects of the test item were seen at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation). In tester strain TA1535 toxic effects of the test item were observed at concentrations of 316 µg/plate and higher (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation). In tester strain TA1537 toxic effects of the test item were noted at concentrations of 31.6 µg/plate and higher (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation). In tester strain E. coli WP2 uvrA toxic effects of the test item were observed at concentrations of 2500 µg/plate and higher (without metabolic activation) and at a concentration of 5000 µg/plate (with metabolic activation).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535, TA 1537 of Salmonella typhimurium and

E. coliWP2 uvrA were exposed to cyclic Glucamide C8-C10 at concentrations of

Experiment I:

3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate, in the presence and absence of mammalian metabolic activation

and

Experiment II:

1.00, 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
all tester strains (without metabolic activation) except E. coliWP2 uvrA

3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
all tester strains (with metabolic activation) except E. coliWP2 uvrA

31.6, 100, 316, 1000, 2500 and 5000 µg/plate (onlyE. coliWP2 uvrA)

according to the plate incorporation method (experiment I) and the pre-incubation method (experiment II).

cyclic Glucamide C8-C10 was tested up to the limit concentration of 5000 µg/plate in all tester strains used.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.