Reaction mass of N-butylphthalimide and N-sec-butylphthalimide and N-propylphthalimide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 April- 20 June, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21st July, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

May 30, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, 2012

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-propylphthalimide

EC Number:

226-189-1

EC Name:

N-propylphthalimide

Cas Number:

5323-50-2

Molecular formula:

C11H11NO2

IUPAC Name:

2-propyl-1H-isoindole-1,3(2H)-dione

Constituent 2

Reference substance name:

N-sec-butylphthalimide

EC Number:

233-295-1

EC Name:

N-sec-butylphthalimide

Cas Number:

10108-61-9

Molecular formula:

C12H13NO2

IUPAC Name:

2-sec-butyl-1H-isoindole-1,3(2H)-dione

Constituent 3

Reference substance name:

N-butylphthalimide

EC Number:

216-157-5

EC Name:

N-butylphthalimide

Cas Number:

1515-72-6

Molecular formula:

C12H13NO2

IUPAC Name:

2-butyl-1H-isoindole-1,3(2H)-dione

impurity 1

Reference substance name:

Unknown impurities.

Molecular formula:

Not available as unknown impurities.

IUPAC Name:

Unknown impurities.

1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid

Details on test material:

Batch CNAA053604
Color: clear yellowish
Form: liquid
Storage conditions: Room temperature in a closed container, protected from light.

Method

Target gene:

In addition to histidine and tryptophan mutation, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are
defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they
cannot repair DNA damages. rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101
(TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2uvrA) is also
defective in DNA excision repair.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/¿-naphthoflavone-induced rats

Test concentrations with justification for top dose:

3200; 1600; 500; 160; 50 and 16 µg/plate

Vehicle / solvent:

dimethyl sulfoxide (DMSO)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

methylmethanesulfonate

other: 4-Nitro-1,2-phenylenediamine, 2-aminoanthracene

Details on test system and experimental conditions:

The tester strains arrived to the test facility in a form of disc cultures. The origin of the following tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA:
Supplier: Trinova Biochem GmbH; Rathenau Str. 2; D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA.
Frozen stock cultures were prepared from the disc cultures. The identification codes and expiry dates of the actual applied stock cultures are summarized in the Table 3.


Storage of Tester Strains

The strains are stored at -80 ± 10ºC in the Laboratory of TOXI-COOP ZRT. in the form of lyophilized discs and in frozen permanent copies. Frozen permanent cultures of the tester strains are prepared from fresh, overnight cultures to which DMSO (8 % (v/v)) is added as a cryoprotective agent.

Confirmation of Phenotypes of Tester Strains

The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity
(uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames.
Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture and raw data and reports of phenotype confirmation are stored in the Laboratory of TOXI-COOP ZRT.

Each tester strain reverts spontaneously at a frequency that is characteristic for the strain. Spontaneous reversions of the test strains to histidine or tryptophan prototrophs are measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.

Procedure for Bacterial Cultures

The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum was used to inoculate each 50 mL of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for approximately 10-13 hours in a 37oC Benchtop Incubator Shaker.

The viability of each testing culture was determined by plating 0.1 mL of the 10-5, 10-6, 10-7 and 10-8 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by manual colony counting.

The details of the used chemicals (supplier/manufacturer, batch/lot number and expiry/retest date) are summarised in Table 2. This report contains the media composition in the sections 5.4.2 - 5.4.5, 5.5.2 and 5.5.3 referring to 1000 mL.

Ready-to-use minimal glucose agar (MGA) plates were used in the study. The origin of the ready-to use MGA plates:
Supplier: VWR International;
Manufacturer: Merck Life Science GmbH, Germany.
Certificates of Analysis* were obtained from the supplier.
Typical composition (g/1000 mL) of MGA plates:
Glucose 20.0 g
Magnesium sulfate 0.2 g
Citric acid 2.0 g
di-Potassium hydrogenphosphate 10.0 g
Sodium ammonium hydrogenphosphate 3.5 g
Agar agar 13.0 g
* Batch No.: 142584; Expiry date: 22 May 2017; (used in the Informatory Toxicity Test)
143858; Expiry date: 16 August 2017; (used in the Initial and the Confirmatory Mutation Tests)

Nutrient Broth No. 2

Nutrient broth No. 2. 25.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121°C in an autoclave.

Nutrient Agar

Nutrient Agar 20.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121°C in an autoclave.

Top Agar for Salmonella typhimurium Strains

Agar solution:
Agar Bacteriological 4.0 g
NaCl 5.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121°C in an autoclave.

Histidine – Biotin solution (0.5 mM):
D-Biotin 122.2 mg
L-Histidine•HCl H2O 104.8 mg
Ultrapure water ad 1000.0 mL
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete Top Agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM) 100.0 mL
Agar solution 900.0 mL

Top Agar for Escherichia coli Strain

Tryptophan solution (2 mg/mL):
L-Tryptophan 2000.0 mg
Ultrapure water ad 1000.0 mL
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete Top Agar for Escherichia coli strain:
Nutrient Broth by (Section: 5.4.2) 50.0 mL
Tryptophan solution (2 mg/mL) 2.5 mL
Agar solution by (Section: 5.4.4) 947.5 mL

Metabolic Activation System

The test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial fraction (S9).

Rat Liver S9 Fraction

The S9 fraction of Phenobarbital (PB) and ß-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).

The Quality Control & Production Certificate of each lot of S9 was obtained from the supplier1). The original Quality Control & Production Certificates of rat liver S9 are stored in the Laboratory of TOXI-COOP ZRT. The copies of the quality control certificates of the used S9 lots are given in Appendix VI. The following lots of the S9 were applied:
1) Lot Number: 3634; Expiry date: May 12, 2018; Protein content: 40.3 mg/mL
(used in the Informatory Toxicity and Confirmatory Mutation Test);
Lot Number: 3662; Expiry date: July 07, 2018; Protein content: 40.5 mg/mL
(used in the Informatory Toxicity Test);
Lot Number: 3712; Expiry date: November 03, 2018; Protein content: 34.3 mg/mL
(used in the Confirmatory Mutation Test);
Lot Number: 3727; Expiry date: December 01, 2018; Protein content: 33.7 mg/mL
(used in the Initial and Confirmatory Mutation Tests).

The S9 Mix (with Rat Liver S9)

Salt solution for S9 Mix Final concentration in S9 Mix
NADP Na 7.66 g 4 mM
D-glucose-6 phosphate Na 3.53 g 5 mM
MgCl2 1.90 g 8 mM
KCl 6.15 g 33 mM
Ultrapure water ad 1000 mL
Sterilized by filtration through a 0.22 µm membrane filter.

The complete S9 Mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 mL
Rat liver homogenate (S9) 100 mL
Salt solution for S9 Mix 400 mL
The S9 Mix was kept in an ice bath before it was added to the culture medium.

Sodium Phosphate Buffer (0.2 M, pH 7.4)

Solution A:
Na2HPO4 x 12H2O 71.63 g
Ultrapure water ad 1000 mL
Solution B:
NaH2PO4 x H2O 27.6 g
Ultrapure water ad 1000 mL

Solution A 880 mL
Solution B 120 mL*
* The components were mixed in the above ratio; thereafter the pH was checked and corrected. The correction was performed with admixture of the solution A or B.
After the pH setting the sterilization was performed by filtration through a 0.22 µm membrane filter.

Rationale for test conditions:

Justification of concentrations:
Choice of the concentrations was done on the basis of a Solubility Test and a concentration Range Finding Test (Informatory Toxicity Test)

Based on the results of the preliminary Concentration Range Finding Test the following concentrations of the test item were prepared and investigated in
the Initial Mutation Test: 3200; 1600; 500; 160; 50 and 16 µg/plate.

Based on the results of the Initial Mutation Test modification of the originally proposed concentrations levels, ranges was considered as necessary in the Salmonella typhimurium strains for the second main experiment, for Confirmatory Mutation Test. The following concentrations of the test item were prepared and investigated in the Confirmatory Mutation Test in Salmonella typhimurium strains: 1600, 500, 160, 50, 16 and 5 µg/plate; in E. coli WP2 uvrA: 3200, 1600, 500, 160, 50 and 16 µg/plate.
No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the
study.
In the Initial and Confirmatory Mutation Tests, unequivocal inhibitory effect of the test item on bacterial growth was observed. The cytotoxicity was
indicated by absent or decreased revertant colony counts (most of them below the corresponding historical control data ranges) and/or affected
background lawn development: reduced or slightly reduced background lawn. In general, 500 µg/plate was considered as lowest concentration showing
cytotoxicity.
The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO) plates with and without S9 Mix demonstrated the characteristic mean number
of spontaneous revertants that was in line with the corresponding historical control data ranges.

Evaluation criteria:

The colony numbers on the untreated, vehicle control, positive control and the test item treated plates were determined visually by manual counting.
A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times
higher than the reversion rate of the vehicle control.

According to the 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.
Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant
positive response at any of the dose groups, with or without metabolic activation.

Statistics:

The mean values and appropriate standard deviations and mutation rates were calculated by EXCEL software.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

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

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

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:

Validity of the Performed Experiments

The tester strains used in this study demonstrated the specific phenotype characteristics , were in line with the
corresponding historical control data ranges , and showed the adequate strain culture titer.
Each batch of the S9 fraction used in this test had the appropriate biological activity and was active in the applied system
Each of the investigated reference mutagens showed the expected increase (at least a 3-fold increase) in induced revertant colonies over the mean value of the respective vehicle control in both main experimental phases and the number of revertants in most cases fell in the corresponding historical control ranges , thereby meeting the criteria for the positive control in the main experimental phases, in all tester strains .
The spontaneous revertant colony numbers of the dimethyl sulfoxide (DMSO) vehicle control plates showed characteristic mean numbers agreed with the actual historical control data ranges in all strains in both main experimental phases.
Seven concentration levels were investigated in the Informatory Toxicity Test and six in the main mutation experiments (Initial and Confirmatory Mutation
Tests)
In the performed experimental phases there were at least five analyzable concentrations and a minimum of three non-toxic and non-precipitated dose levels at each tester strain.
All criteria for the validity of the performed experiments have therefore been met.

Controls

In the performed Initial and Confirmatory Mutation Test multiple test items were tested with reference values from the common parallel controls.
In the Initial and Confirmatory Mutation Tests the revertant colony numbers of the dimethyl sulfoxide (DMSO) vehicle control plates with and without S9 Mix
were in line with the corresponding historical control data ranges.
The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains. In the Initial Mutation Test, in the case of S. typhimurium TA100 the revertant colony numbers of
Sodium azide (SAZ) were above the corresponding historical control data range; however the higher counts were considered as acceptable without any
effect on the final conclusion of the study.

The revertant colony numbers of the untreated and ultrapure water (ASTM Type I) control plates in different experimental phases were slightly higher or
lower than the dimethyl sulfoxide (DMSO) control plates. The higher or lower revertant counts of these controls remained in the corresponding historical
control data ranges.
In summary, the actual values of untreated, vehicle and positive controls were in line with the criteria for validity of the assay.

Initial and Confirmatory Mutation Tests (Plate Incorporation and Pre-Incubation Tests)

No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with Reaction mass of
N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide at any concentration level, either in the presence or absence of metabolic activation
(S9 Mix) in the performed experiments.
In the performed experiments, sporadically increased revertant colony numbers were observed. These increases did not show a dose-response
relationship, were of minor intensity, and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.
The highest revertant colony number increase was observed in the Confirmatory Mutation Test (Pre-Incubation Test) in S. typhimurium TA1535 strain, at
160 µg/plate, in the presence of metabolic activation (+S9). This value however remained in the range of the corresponding vehicle historical control data
and additional concentration related increase in revertant colony counts was not noticed. The mutation rate was 1.60, which was far below the
genotoxicological threshold for being positive.
In the Initial and Confirmatory Mutation Tests, unequivocal inhibitory effect of the test item on bacterial growth was observed in all examined strains.
The cytotoxicity was indicated by absent or decreased revertant colony counts (most of them below the corresponding historical control data ranges)
and/or affected background lawn development: reduced or slightly reduced background lawn. The table contains the unequivocal cytotoxicity results, only
, where the obtained revertant colony numbers were below the vehicle (in some cases below the corresponding historical control data ranges), and/or
affected background lawn development occurred. All of the further observed lower revertant colony numbers (when compared to the revertant colony
numbers of the corresponding vehicle control) remained in the range of the biological variability of the applied test system.

In general, 500 µg/plate was considered as lowest concentration showing cytotoxicity.

Any other information on results incl. tables

Summary of the Inhibitory Tendencies in the Initial and Confirmatory Mutation Tests

Initial Mutation Test
Concentrations (µg/plate)	Salmonella typhimurium								Escherichia coliWP2uvrA
	TA98		TA100		TA1535		TA1537
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
3200	B0	B0	<< B	<< B	B0	B0	B0	<< B	<< SB	<< SB
1600	<< B	< SB	<< SB	<< SB	< SB	< SB	<< B	<< B	-	-
500	-	-	-	-	-	-	-	-	-	-
160	-	-	-	-	-	-	-	-	-	-
50	-	-	-	-	-	-	-	-	-	-
16	-	-	-	-	-	-	-	-	-	-
Confirmatory Mutation Test
Concentrations (µg/plate)	Salmonella typhimurium								Escherichia coliWP2uvrA
	TA98		TA100		TA1535		TA1537
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
3200									< SB	< SB
1600	<< B	<< SB	<< SB	<< B	B0	< B	<< B	<< B	< SB	-
500	<< SB	<<	<< SB	<< SB	<< SB	SB	B0	< SB	< SB	-
160	-	-	-	-	-	-	-	-	-	-
50	-	-	-	-	-	-	-	-	-	-
16	-	-	-	-	-	-	-	-	-	-
5	-	-	-	-	-	-	-	-

<:         Revertant colony numbers significantly below the vehicle control data range

<<:      Revertant colony numbers below the vehicle and historical control data ranges

B:         Reduced background lawn development

SB:        Slightly reduced background lawn development

B0:        Reduced background lawn development and absent revertant colonies

No precipitation of the test item was observed in the Initial and Confirmatory Mutation Tests on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix).

Historical Control Values for Revertants/Plate (for the Period of 2008-2016)

			Bacterial strains
Historical control data of untreated control	-S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	21.0	105.0	10.5	8.1	25.4
		SD	3.7	25.7	1.4	2.3	5.2
		Minimum	9	66	3	2	11
		Maximum	39	155	23	19	45
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	27.5	117.1	11.8	9.0	33.9
		SD	4.3	18.1	1.4	1.9	5.2
		Minimum	12	75	4	2	17
		Maximum	46	166	23	20	56
			Bacterial strains
Historical control data of DMSO control	-S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	20.4	100.1	10.3	7.9	24.7
		SD	3.6	24.8	1.3	2.4	4.6
		Minimum	10	64	3	2	11
		Maximum	38	147	23	20	45
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	26.5	113.8	11.8	8.8	33.7
		SD	4.1	18.3	1.5	1.9	5.0
		Minimum	15	71	3	3	16
		Maximum	47	162	25	20	57
			Bacterial strains
Historical control data of Water control	-S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	21.9	104.7	10.5	7.6	26.1
		SD	3.7	25.9	1.5	2.2	5.5
		Minimum	12	68	3	2	12
		Maximum	35	154	24	16	48
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	27.4	117.3	11.4	8.7	34.9
		SD	4.0	18.5	1.3	2.2	4.9
		Minimum	15	83	4	3	18
		Maximum	43	167	22	16	57
			Bacterial strains
Historical control data of positive controls	-S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	260.1	977.2	847.3	478.6	724.5
		SD	31.8	150.6	126.3	104.5	65.0
		Minimum	123	521	359	110	320
		Maximum	664	1970	1855	1601	1313
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	1222.7	1436.4	164.1	147.0	257.7
		SD	274.9	318.3	33.1	20.1	72.5
		Minimum	386	583	85	69	140
		Maximum	2676	2988	498	399	477

Abbreviations:   TA98, TA100, TA1535, TA1537: Salmonella typhimuriumTA98, TA100, TA1535,

                               TA1537;E. coli:Escherichia coliWP2uvrA

                               SD: Standard deviation; DMSO: Dimethyl sulfoxide

Summary Table of the Results of the Concentration Range Finding Test

Range Finding Test (Informatory Toxicity Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains
	TA 98				TA 100
	-S9		+S9		-S9		+S9
Mean values of revertants per plate and Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	21.7	0.93	25.7	1.01	103.7	1.05	121.0	1.30
DMSO Control	23.3	1.00	25.3	1.00	99.0	1.00	93.3	1.00
Ultrapure Water Control	–	–	–	–	94.0	1.00	–	–
5000	0.0	0.00	0.0	0.00	6.7	0.07	10.0	0.11
1600	6.0	0.26	16.7	0.66	26.7	0.27	61.0	0.65
500	21.0	0.90	24.3	0.96	81.7	0.82	105.7	1.13
160	18.3	0.79	24.7	0.97	84.3	0.85	103.0	1.10
50	18.0	0.77	26.7	1.05	87.3	0.88	93.0	1.00
16	24.3	1.04	24.0	0.95	83.7	0.85	96.7	1.04
5	24.3	1.04	21.0	0.83	90.0	0.91	109.7	1.18
NPD (4mg)	218.7	9.37	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1944.0	20.68	–	–
2AA (2mg)	–	–	1746.7	68.95	–	–	2360.0	25.29

MR:Mutation Rate

NPD:4-Nitro-1,2-phenylenediamine

SAZ:Sodium azide

2AA:2-aminoanthracene

Remarks:   DMSO was applied as vehicle of the test item and the positive control substances NPD and 2AA. The ultrapure water was applied as vehicle of the positive control substance SAZ. The mutation rate of the test item, the untreated control; furthermore NPD and 2AA refers to the DMSO sample. The mutation rate of SAZ refers to ultrapure water.

Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichiacoli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	25.3	1.36	36.3	1.43	97.0	1.27	101.3	0.96	10.7	0.91	13.7	1.00	7.7	1.10	11.3	1.17	27.0	0.95	37.3	1.13
DMSO Control	18.7	1.00	25.3	1.00	76.3	1.00	105.7	1.00	11.7	1.00	13.7	1.00	7.0	1.00	9.7	1.00	28.3	1.00	33.0	1.00
Ultrapure Water Control	–	–	–	–	82.7	1.00	–	–	13.0	1.00	–	–	–	–	–	–	33.7	1.00	–	–
3200	0.0	0.00	0.0	0.00	2.7	0.03	24.3	0.23	0.0	0.00	0.0	0.00	0.0	0.00	1.0	0.10	8.3	0.29	13.3	0.40
1600	4.0	0.21	14.7	0.58	37.3	0.49	53.7	0.51	6.3	0.54	6.0	0.44	0.7	0.10	1.7	0.17	24.0	0.85	35.0	1.06
500	17.0	0.91	19.7	0.78	82.0	1.07	100.3	0.95	10.7	0.91	7.3	0.54	5.7	0.81	10.7	1.10	26.0	0.92	37.3	1.13
160	18.3	0.98	27.3	1.08	79.3	1.04	93.0	0.88	12.3	1.06	11.0	0.80	6.7	0.95	11.3	1.17	22.3	0.79	32.0	0.97
50	18.3	0.98	19.0	0.75	86.0	1.13	97.0	0.92	11.7	1.00	14.0	1.02	6.0	0.86	8.3	0.86	31.0	1.09	31.3	0.95
16	19.0	1.02	25.3	1.00	88.0	1.15	97.7	0.92	12.7	1.09	11.7	0.85	5.7	0.81	10.0	1.03	27.3	0.96	38.3	1.16
NPD (4mg)	271.3	14.54	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	2189.3	26.48	–	–	862.7	66.36	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	914.0	130.57	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	800.0	23.76	–	–
2AA (2mg)	–	–	2397.3	94.63	–	–	2205.3	20.87	–	–	274.7	20.10	–	–	152.0	15.72	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	189.0	5.73

MR:Mutation Rate;          NPD:4-Nitro-1,2-phenylenediamine;SAZ: Sodium azide;9AA:9-Aminoacridine;MMS:Methyl methanesulfonate;2AA: 2-aminoanthracene

Remarks:           DMSO was applied as vehicle of the test item and positive control substances: NPD, 9AA and 2AA and the ultrapure water was applied as vehicle for the SAZ and MMS. The mutation rate of the test item and the untreated control refers to the DMSO. The mutation rate of the NPD, 9AA and 2AA refers to the DMSO and the mutation rate of the SAZ and MMS positive control refers to the ultrapure water.

Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichia coli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	15.0	0.94	22.7	1.19	113.3	1.11	113.3	1.00	13.0	0.87	14.0	1.20	8.7	1.37	8.7	1.08	30.7	1.05	34.0	1.10
DMSO Control	16.0	1.00	19.0	1.00	102.0	1.00	113.3	1.00	15.0	1.00	11.7	1.00	6.3	1.00	8.0	1.00	29.3	1.00	31.0	1.00
Ultrapure Water Control	–	–	–	–	109.3	1.00	–	–	12.3	1.00	–	–	–	–	–	–	33.7	1.00	–	–
3200	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	13.7	0.47	18.0	0.58
1600	3.0	0.19	9.3	0.49	34.3	0.34	42.3	0.37	0.0	0.00	6.7	0.57	0.7	0.11	0.7	0.08	12.3	0.42	26.7	0.86
500	1.7	0.10	11.7	0.61	21.7	0.21	50.7	0.45	2.7	0.18	10.3	0.89	0.0	0.00	4.7	0.58	13.3	0.45	35.0	1.13
160	15.0	0.94	21.3	1.12	73.3	0.72	92.3	0.81	13.0	0.87	18.7	1.60	5.7	0.89	8.0	1.00	36.0	1.23	43.7	1.41
50	15.7	0.98	27.0	1.42	91.7	0.90	87.7	0.77	12.7	0.84	11.3	0.97	5.0	0.79	7.3	0.92	29.7	1.01	38.3	1.24
16	16.0	1.00	29.0	1.53	98.3	0.96	93.0	0.82	13.3	0.89	16.0	1.37	6.7	1.05	6.7	0.83	27.0	0.92	42.0	1.35
5	16.3	1.02	28.0	1.47	95.7	0.94	82.3	0.73	12.7	0.84	12.3	1.06	8.3	1.32	7.3	0.92	–	–	–	–
NPD (4mg)	256.0	16.00	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1584.0	14.49	–	–	1176.0	95.35	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	679.3	107.26	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	1226.7	36.44	–	–
2AA (2mg)	–	–	992.0	52.21	–	–	1464.0	12.92	–	–	211.0	18.09	–	–	115.0	14.38	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	150.7	4.86

MR:Mutation Rate;          NPD:4-Nitro-1,2-phenylenediamine;SAZ: Sodium azide;9AA:9-Aminoacridine;MMS:Methyl methanesulfonate;2AA: 2-aminoanthracene

Remarks:           DMSO was applied as vehicle of the test item and positive control substances: NPD, 9AA and 2AA and the ultrapure water was applied as vehicle for the SAZ and MMS. The mutation rate of the test item and the untreated control refers to the DMSO. The mutation rate of the NPD, 9AA and 2AA refers to the DMSO and the mutation rate of the SAZ and MMS positive control refers to the ultrapure water.

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test itemReaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimidewas tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains ofSalmonella typhimurium(Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain ofEscherichia coli(Escherichia coliWP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).

Based on the results of the Solubility and the Concentration Range Finding Tests the test item was dissolved in dimethyl sulfoxide (DMSO). This vehicle was compatible with the survival of the bacteria and the S9 activity and appropriate historical control database is available in the testing laboratory.

Based on the results of the preliminary Concentration Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial Mutation Test:3200; 1600; 500; 160; 50 and 16 µg/plate.

The selection of the concentration range was based on the recommendations in OECD 471 guideline for cytotoxic, soluble test compounds; accordingly the test item was investigated up to and including the concentration level of 3200 µg/plate.

Based on the results of the Initial Mutation Testmodification of the originally proposed concentrations levels, ranges was considered as necessary in theSalmonella typhimuriumstrains for the second main experiment, for Confirmatory Mutation Test. The following concentrations of the test item were prepared and investigated in the Confirmatory Mutation TestinSalmonella typhimuriumstrains: 1600, 500, 160, 50, 16 and 5 µg/plate; inE. coliWP2uvrA: 3200, 1600, 500, 160, 50 and 16 µg/plate.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

In the Initial and Confirmatory Mutation Tests, unequivocal inhibitory effect of the test item on bacterial growth was observed. The cytotoxicity was indicated by absent or decreased revertant colony counts (most of them below the corresponding historical control data ranges) and/or affected background lawn development: reduced or slightly reduced background lawn.In general, 500 µg/plate was considered as lowest concentration showing cytotoxicity.

The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO) plates with and without S9 Mix demonstratedthe characteristic mean number of spontaneous revertantsthat was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase)in induced revertant coloniesand nearly all the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive controlin all experimental phases, in all tester strains.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment withReaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimideat any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test itemReaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimidehas no mutagenic activity on the applied bacterium tester strainsunder the test conditions used in this study.