Reaction products of γ-butyrolactone, naphthalene-1,6-diol and phthalic anhydride

Genetic toxicity: in vitro

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:

2016

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(21 Jul 1997)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of γ-butyrolactone, naphthalene-1,6-diol and phthalic anhydride

EC Number:

946-798-2

Molecular formula:

C28H16O5

IUPAC Name:

Reaction products of γ-butyrolactone, naphthalene-1,6-diol and phthalic anhydride

Test material form:

solid

Details on test material:

-Batch: PO 4924335980 - ML 2005975931
- Analytical purity: 100% UVCB
- Physical state: Solid, violet
- Homogeneity: homogeneous by visual inspection
- Storage conditions: Room temperature
- The stability of the test substance under storage conditions is guaranteed until Apr 2019.
-pH of the substance: Ca. 5 (undiluted test substance, moistened with de-ionized water)

Specific details on test material used for the study:

- Batch: PO 4924335980 - ML 2005975931
- Physical state: Solid, violet
- Homogeneity: homogeneous by visual inspection
- Storage conditions: Room temperature
- The stability of the test substance under storage conditions is guaranteed until Apr 2019.
- pH of the substance: Ca. 5 (undiluted test substance, moistened with de-ionized water)

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:

phenobarbital and beta-naphthoflavone induced rat liver S9 microsomal fraction

Test concentrations with justification for top dose:

1st Experiment (Standard plate test with and without S9 mix): 0; 33; 100; 333; 1000; 3925 and 7850 μg/plate
2nd Experiment (Preincubation test with and without S9 mix): 10 μg - 3925 μg/plate

The dose refers to the test material, not to the amount of the pure target substance.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in ultrapure water, ethanol was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD), 9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

Standard plate test:
- Exposure duration: 48 – 72 hours

Preincubation test:
- Preincubation period: 20 min
- Exposure duration: 48 – 72 hours


NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn, reduction in the titer


OTHER:
Titer determination: The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.
The concentrations have been adusted for the low content in the sample.

Positive controls:
With S9 mix: 2-aminoanthracene (2-AA), 2.5 μg/plate, dissolved in DMSO / TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate, dissolved in DMSO / Escherichia coli WP2 uvrA
Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 5 μg/plate, dissolved in DMSO / TA 1535, TA 100; 4-nitro-o-phenylenediamine (NOPD), 10 μg/plate, dissolved in DMSO / TA 98; 9-aminoacridine (AAC), 100 μg/plate, dissolved in DMSO / TA 1537; 4-nitroquinoline-N-oxide (4-NQO) (SIGMA, N-8141), 5 μg/plate, dissolved in DMSO / E. coli WP2 uvrA

Evaluation criteria:

Acceptance criteria:
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10^8 colonies per mL.

Assessment criteria:
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Cytotoxicity criteria:
Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth) was recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables.

In low dose group, single values with a factor ≤ 0.6 were not considered as in indicator of toxicity.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

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:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was found in the standard plate test from about 1000 μg/plate onward with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: A bacteriotoxic effect was observed depending on the strain and test conditions from about 1000 μg/plate onward. For details it is refered to table 1.

Any other information on results incl. tables

Table 2: Standard plate test, without S9 mix

Strain	Test group	Dose (μg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	DMSO	-	10.0	1.7	-	8, 11, 11
	Test item	33	10.0	1.7	1.0	9, 9, 12
		100	14.3	4.2	1.4	13, 19, 11
		333	13.3	2.1	1.3	14, 15, 11
		1000	12.0	1.0	1.2	12 P, 11 P, 13 P
		3925	7.0	4.4	0.7	9 P, 2 P, 10 P
		7850	1.7	1.2	0.2	3 P, 1 P, 1 P
	MNNG	5.0	5616.0	433.2	561.6	5920, 5808, 5120
TA 100	DMSO	-	105.0	5.6	-	106, 99, 110
	Test item	33	112.3	12.5	1.1	100, 112, 125
		100	113.7	8.7	1.1	121, 116, 104
		333	77.3	11.0	0.7	90, 72, 70
		1000	83.0	11.5	0.8	82 P, 95 P, 72 P
		3925	70.0	18.0	0.7	65 P, 55 P, 90 P
		7850	38.3	7.6	0.4	47 P, 35 P, 33 P
	MNNG	5.0	3868.7	245.2	36.8	4071, 3939, 3596
TA 1537	DMSO	-	7.7	0.6	-	8, 7, 8
	Test item	33	9.0	1.0	1.2	10, 9, 8
		100	9.7	2.1	1.3	8, 9, 12
		333	7.7	2.9	1.0	6, 11, 6
		1000	6.0	2.6	0.8	7 P, 8 P, 3 P
		3925	3.7	1.2	0.5	5 P, 3 P, 3 P
		7850	1.0	1.0	0.1	1 P, 2 P, 0 P
	AAC	100	695.7	310.2	90.7	409, 653, 1025
TA 98	DMSO	-	23.3	4.2	-	28, 20, 22
	Test item	33	22.7	4.0	1.0	19, 27, 22
		100	25.0	6.2	1.1	27, 30, 18
		333	19.0	5.3	0.8	17, 15, 25
		1000	18.7	3.1	0.8	18 P, 22 P, 16 P
		3925	13.3	3.8	0.6	9 P, 15 P, 16 P
		7850	8.0	1.0	0.3	9 P, 8 P, 7 P
	NOPD	10	954.7	54.8	40.9	923, 923, 1018
E. coli	DMSO	-	28.3	3.8	-	24, 31, 30
	Test item	33	21.7	3.8	0.8	19, 26, 20
		100	22.7	3.5	0.8	19, 23, 26
		333	21.7	7.5	0.8	13, 26, 26
		1000	23.3	9.5	0.8	14 P, 33 P, 23 P
		3925	9.0	1.0	0.3	9 P, 10 P, 8 P
		7850	11.7	0.6	0.4	12 P, 11 P, 12 P
	4-NQO	5	1454.0	43.5	51.3	1439, 1503, 1420

Table 3, Standard plate test with metabolic activation

Strain	Test group	Dose (μg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	DMSO	-	10.3	0.6	-	10, 11, 10
	Test item	33	11.7	3.5	1.1	12, 8, 15
		100	10.3	2.3	1.0	13, 9, 9
		333	9.3	1.5	0.9	8, 11, 9
		1000	7.3	1.5	0.7	6 P, 9 P, 7 P
		3925	4.3	1.5	0.4	3 P, 6 P, 4 P
		7850	3.7	0.6	0.4	4 P, 3 P, 4 P
	2-AA	2.5	193.0	22.5	18.7	205, 207, 167
TA 100	DMSO	-	114.7	2.5	-	117, 112, 115
	Test item	33	116.3	11.2	1.0	112, 108, 129
		100	114.3	5.1	1.0	110, 113, 120
		333	107.3	3.2	0.9	111, 106, 105
		1000	86.0	8.2	0.8	95 P, 79 P, 84 P
		3925	80.3	19.5	0.7	58 P, 89 P, 94 P
		7850	80.3	8.1	0.7	71 P, 86 P, 84 P
	2-AA	2.5	1732.0	88.1	15.1	1748, 1637, 1811
TA 1537	DMSO	-	8.7	1.2	-	8, 8, 10
	Test item	33	10.3	2.3	1.2	9, 13, 9
		100	8.0	2.6	0.9	11, 7, 6
		333	7.3	1.2	0.8	6, 8, 8
		1000	7.7	2.1	0.9	7 P, 10 P, 6 P
		3925	4.3	1.5	0.5	4 P, 6 P, 3 P
		7850	2.0	1.7	0.2	1 P, 1 P, 4 P
	2-AA	2.5	144.0	19.5	16.6	124, 145, 163
TA 98	DMSO	-	28.0	2.6	-	25, 30, 29
	Test item	33	24.3	6.1	0.9	31, 19, 23
		100	23.3	3.2	0.8	21, 22, 27
		333	31.0	7.2	1.1	29, 25, 39
		1000	23.3	5.7	0.8	25 P, 17 P, 28 P
		3925	15.3	6.0	0.5	21 P, 9 P, 16 P
		7850	7.0	1.0	0.3	8 P, 6 P, 7 P
	2-AA	2.5	1388.7	140.7	49.6	1301, 1551, 1314
E. coli	DMSO	-	24.7	4.5	-	20, 29, 25
	Test item	33	30.7	4.2	1.2	34, 26, 32
		100	26.0	1.7	1.1	25, 25, 28
		333	27.0	7.5	1.1	26, 35, 20
		1000	21.7	6.7	0.9	26 P, 25 P, 14 P
		3925	19.0	3.0	0.8	22 P, 16 P, 19 P
		7850	12.7	3.2	0.5	15 P, 9 P, 14 P
	2-AA	60	133.7	10.1	5.4	139, 122, 140

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in bacteria.