Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 August 2012 - 20 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: Standards for Mutagenicity Tests Using Microorganisms
Version / remarks:
1988 Notification No. 77 of the Ministry of Labor (partial amendment by 1997 Notification No. 67 of the Ministry of Labor)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Methods for Tests Relating to New Chemicals Substances
Version / remarks:
(Ministry of Health, Labour and Welfare Drug & Food Bureau Ordinance No. 0331-7 dated March 31, 2011, Manufacturing & Industrial Bureau Ordinance No. 5 dated March 29, 2011, and Environmental, Safety & Business Bureau Ordinance No. 110331009).
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
1-Propanol, 2-methyl-, reaction products with 1,5-diisocyanatopentane
Cas Number:
1357171-37-9
Molecular formula:
not applicable (a generic formula cannot be provided for this UVCB substance)
IUPAC Name:
1-Propanol, 2-methyl-, reaction products with 1,5-diisocyanatopentane
Test material form:
liquid: viscous
Details on test material:
- Physical appearance: transparent viscous liquid
- Stable at normal temperature. Gently reacts with moisture in the air.

Method

Target gene:
S. typhimurium: histidine gene
E. coli: tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix obtained from rats injected intraperitoneally with phenobarbital and 5,6-benzoflavone.
Test concentrations with justification for top dose:
Dose finding test (with and without S9): 1.2, 4.9, 20, 78, 313, 1250 and 5000 μg/plate

On the basis of the results of the dose finding test, the definitive test was conducted while the dose at which growth inhibition was observed was set as the maximum dose and a total of 6 doses were set at a common ratio of 2. Exception was made for the strains in which the growth inhibition effect at the minimum dose at which growth inhibition was observed was strong.

Doses used for the definitive test:
TA98 and TA 1537 (without S9): 4.9, 9.8, 20, 39, 78, 156 and 313 μg/plate
TA100, TA1535 and WP2 uvrA (without S9): 9.8, 20, 39, 78, 156, 313 μg/plate
TA100, WP2 uvrA and TA98 (with S9): 39, 78, 156, 313, 625, 1250 μg/plate
TA1535 and TA 1537 (with S9): 20, 39, 78, 156, 313, 625 and 1250 μg/plate
Vehicle / solvent:
- Solvent used: acetone
- Justification for choice of solvent: the test item was dissolved in acetone at a concentration of 10% and was found to be stable.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide; ICR-191; 2-aminoanthracene
Remarks:
For details on positive controls, see table 1 in 'any other information on materials and methods'.
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation and in agar (plate incorporation)

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: all strains were tested in triplicate with and without S9

DETERMINATION OF CYTOTOXICITY
- Method:
1) A visual observation using a stereoscopic microscope (40x) was carried out as to whether or not there was growth inhibition, and a direct visual observation was performed as to whether or not there was a precipitation.
2) Colony counting: using an automatic colony counter or manually when the number of colonies was less than 1500.

- OTHER:
A sterility test was performed: 0.5 mL of S9Mix and 0.05 mL of the test substance solution were each put on 2 minimum glucose agar plate media, and the media were piled up with 2.0 mL of top agar. They were cultured at 37°C for 48 hours, and an visual observation was made as to whether or not there was a growth of saprophytic bacteria.
Evaluation criteria:
On the basis of the results of the dose finding test and the definitive test, the test substance was judged to be positive when the number of the revertant colonies in the test substance groups increased not less than 2 times as much as the number of revertant colonies of the negative control group. If any positive results were obtained, the comparative activity value was calculated.
Statistics:
No statistical analysis was performed in the evaluation of the test results.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At and above 156 μg/plate (without S9) and at and above 625 μg/plate (with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 313 μg/plate (without S9) and at and above 625 μg/plate (with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At and above 156 μg/plate (without S9) and at and above 313 μg/plate (with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At and above 156 μg/plate (without S9) and at 1250 μg/plate (with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 313 μg/plate (without S9) and at and above 625 μg/plate (with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- Cytotoxicity: inhibition of growth was observed in all strains with and without S9.
- Precipitation: in the dose finding test at 78 μg/plate and above (without S9) and at 5000 μg/plate (with S9). In the definitive test at and above 39 μg/plate (without S9). No precipitation was observed in all strains at all dose levels with S9 in the definitive test.
- Mutagenicity: the number of revertant colonies did not increase more than 2 times the increase in the number of revertant colonies in the negative control in neither the dose finding test, nor in the definitive test.

Results of negative controls (no. of revertant colonies/plate):
- Without S9: TA100 80-90; TA1535 8-12; TA98 12-21; TA1537 3-8; WP2 uvrA 29-30
- With S9: TA100 81-102; TA1535 7-15; TA98 26-32; TA1537 13-20; WP2 uvrA 31-42

Acceptability of the test:
In either of the dose finding test and the definitive test, the values of the negative control were within the allowable range of the background data of the test facility (average±2.5 SD). Furthermore, the positive controls induced a number of revertant colonies against the bacterial strains more than 2 times as many as that by the negative control. In the sterility test, no increase in saprophytic bacteria was observed. These results indicate that all of the tests were performed properly. See table 2 in 'any other information on results' for the test facility's historical data.

Any other information on results incl. tables

Table 2 Historical data for controls

Strain

Negative control (mean + SD)

Positive control (mean + SD)

 

Without S9

With S9

Without S9

With S9

TA100

101± 10.7

105± 10.7

531± 103.3

802± 101.5

TA1535

11± 4.3

11± 2.1

483± 60.7

243± 27.3

TA1537

8± 2.3

18± 2.6

2205± 467.4

130± 20.1

TA98

20± 3.1

29± 4

455± 46.3

355± 53.7

WP2 uvrA

31± 6.9

34± 7.1

155± 35

814± 157.5

Historical data were collected between January 2011 and December 2011

Applicant's summary and conclusion

Conclusions:
In a reverse mutation assay performed equivalent to OECD guideline 471, D370-N was not mutagenic in S. typhimurium and E. coli strains.
Executive summary:

A reverse mutation assay was performed equivalent to OECD guideline 471 to assess the mutagenic potential of D370 -N in four strains of S. typhimurium (TA100, TA1535, TA1537 and TA98) and one E. coli strain (WP2 uvrA). The test item was tested at different concentrations up to a top dose of 5000 μg/plate in the dose finding study (with and without S9) and a top dose of 1250 μg/plate in the definitive study (with S9) and 313 μg/plate (without S9) (up to and including cytotoxic levels). No increase in the number of revertant colonies greater than 2 times of the increase in the negative control was observed. Growth inhibition was observed in all strains. The test item precipitated at 39 μg/plate and above (without S9). Based on the results of this study, the test item was considered to be not mutagenic in the reverse mutation assay, with and without metabolic activation.

Categories Display