Petroleum resins

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:

23. May 2018 to 30. May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for the Testing of Chemicals Part 471, adopted 21. Jul. 1997 “Bacterial Reverse Mutation Test“

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 440/2008, EU-Method B.13/14 adopted 30. May 2008 “Mutagenicity-Reverse mutation test using bacteria”

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:

Petroleum resins

EC Number:

265-116-8

EC Name:

Petroleum resins

Cas Number:

64742-16-1

Molecular formula:

UVCB substance containing saturated aliphatic hydrocarbon and aromatic hydrocarbons

IUPAC Name:

Petroleum Resins

Test material form:

liquid

Details on test material:

Reference Name: Petroleum Resins (Kendex 0897)
Sponsor Information: CAS Number 64742-16-1
Label Identification: LANXESS Lot # E267112117, 18 Jan 18, Kendex 0897
Quantity & Date Received: 24 Jan 18; 76.1 g (GW)
Physical Description: Brown liquid
Storage: Room temperature
Purity: See Certificate of Analysis
Stability: Not provided to testing facility

Specific details on test material used for the study:

No further details specified in the study report.

Method

Target gene:

histidine

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: 97a

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not specified

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

First Experiment: 5 / 1.5 / 0.5 / 0.15 / 0.05 µL/plate
Second Experiment: 5 / 2.5 / 1.25 / 0.63 / 0.31 / 0.16 µL/plate
The second experiment dose range was based on the findings from experiment one.

Vehicle / solvent:

In a non-GLP pre-test, the solubility of the test item was tested in demineralized water, dimethyl sulfox-ide (DMSO), ethanol, acetone and tetrahydrofurane (THF).
The liquid test item is sufficiently soluble in a concentration of 200 mL/L in tetrahydrofurane, only.
Based on the non-GLP pre-test, THF was chosen as vehicle, because the test item was sufficiently soluble and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO), for the positive controls ni-trophenylendiamine, benzo-a-pyrene and 2-amino-anthracene Demineralised water, for the positive control sodium azide Tetrahydrofurane (THF), for the test item

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene diamine, C6H7N3O2; CAS-No.: 99-56-9; 2-Amino-anthracene, C14H11N; CAS-No.: 613-13-8

Details on test system and experimental conditions:

Culture of Bacteria
Eight hours before the start of each experiment, one vial permanent culture of each strain was taken from the deep freezer and an aliquot was put into a culture flask containing nutrient broth. After incuba-tion for eight hours at 37 ±1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

Conduct of Experiment
Preparations
Different media and solutions were prepared preliminary (exact production dates are documented in the raw data).
On the day of the test, the bacteria cultures were checked for growth visually. The incubation chambers were heated to 37 ±1 °C. The water bath was turned to 43 ±1 °C. The table surface was disinfected.
The S9 mix was freshly prepared and stored at 0 °C.

Experimental Parameters
First Experiment
Concentrations tested: 5 / 1.5 / 0.5 / 0.15 / 0.05 µL/plate
Incubation time: 48 h
Incubation temperature: 37 ±1 °C
Tested strains: TA97a, TA98, TA100, TA102, TA1535
Method: plate incorporation method

Second Experiment
Concentrations tested: 5 / 2.5 / 1.25 / 0.63 / 0.31 / 0.16 µL/plate
Incubation time: 48 h
Incubation temperature: 37 ±1 °C
Tested strains: TA97a, TA98, TA100, TA102, TA1535
Method: pre-incubation method

Description of the Method
General preparation
Per bacteria strain and concentration, three plates with and three plates without metabolic activation (-S9) were used.
The test item solutions were prepared according to chapter 6.1.3.
For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ±1 °C.

Plate incorporation method
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
25 µL test solution at each dose level, resp. 100 µL solvent (negative control) or reference mutagen solution (positive control)
500 µL S9 mix or phosphate buffer (for test without metabolic activation).
100 µL bacteria suspension
2000 µL overlay agar (top agar)

The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.

Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ±1°C for 20 minutes:
25 µL test solution at each dose level, resp. 100 µL solvent (negative control) or reference mutagen solution (positive control)
500 µL S9 mix or phosphate buffer (for test without metabolic activation).
100 µL bacteria suspension

After the pre-incubation for 20 minutes, 2000 µL top agar was added and the tube was gently vortexed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ±1 °C.

References and Validity
Genotype Confirmation
Genotype confirmation is performed for each batch of lyophilized bacteria before stock culture preparation.

Histidine requirement
Each strain was streaked on a biotin and a histidine-biotin-plate, using a sterilized wire loop. The plates were incubated for 24 hours at 37 ±1 °C.

Ampicillin/Tetracycline-Resistance (pKM 101, pAQ1)
Each strain was streaked on an ampicillin agar plate and on an ampicillin-tetracycline agar plate. TA1535 was used as control strain, since it is not ampicillin resistant. The plates were incubated for 24 hours at 37 ±1 °C.

UV-sensitivity (uvrB)
Each strain was streaked on a plate, and one half of the plate covered with aluminium foil so that one half of each streak was protected against light. The plates for the strain TA97a, TA100 and TA102 were irradiat-ed for 8 seconds, the plates for the strain TA 98 were irradiated for 10 seconds and the plates for the strain TA1535 were irradiated for 6 seconds with a germicidal lamp (254 nm, 30W).
Keeping a distance of 33 cm for the strains TA97a, TA102 and TA1535.
Keeping a distance of 66 cm for the following strains: TA98, TA100.
Incubation for 24 hours at 37 ±1 °C followed.

Crystal violet sensitivity (deep rough/rfa)
For each strain, two plates were used. 0.1 mL of bacteria suspension were mixed with 2 mL Top-Agar and poured on nutrient agar. Sterile paper discs (9 mm), each soaked with 10 µL of crystal violet solution (0.1%) were placed into the middle of each plate, followed by incubation for 24 hours at 37 ±1°C.

Spontaneous Revertants
Three replicates, with/without S9, for each solvent which was used in the test, incubation for 48 hours at 37 ±1°C.

Determination of Titre
The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. Incubation for 48 hours at 37 ±1 °C followed. It should give a density of e+9 cells/mL (at the least), two replicates with and without metabolic activation.

Toxicity Control
Performed in experiment 1 only analogously to the titre control with the maximum dose of test item on maximal-soft agar, two replicates with and without metabolic activation, incubation for 48 hours at 37 ±1°C.

Sterility Control
Performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar, incuba-tion for 48 hours at 37 ±1°C, four replicates.

Solubility
Plates were checked for precipitation of test item at the end of the incubation by visual inspection.

Positive Controls
Using diagnostic mutagens, three replicates were prepared. The stock solutions of the substances were diluted to achieve an application volume of 0.1 mL/plate, incubation for 48 hours at 37 ±1°C.

Rationale for test conditions:

In accordance with test guidelines.

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A validated spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Statistics:

Not specified

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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:

not applicable

Positive controls validity:

valid

Test results 3

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:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

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:

not applicable

Positive controls validity:

valid

Additional information on results:

First Experiment
Confirmation of the Criteria and Validity
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the nor-mal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and nearly all (one exception) were within the historical control data ranges.

Solubility and Toxicity
In the first experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.

Mutagenicity
No increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Therefore, the test item is stated as not mutagenic under the conditions of this experiment.
To verify this result, a further experiment was performed.

Second Experiment
Confirmation of the Criteria and Validity
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory.. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.

Solubility and Toxicity
In the second experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.

Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Therefore, the test item is stated as not mutagenic under the conditions of this experiment.

Any other information on results incl. tables

Survey of the Findings– First Experiment

The mean revertant values of the three replicates are presented in the following table.

Mean Revertants First Experiment

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	82	105	36	40	89	103	213	316	14	18
	sd	1.5	6.1	2.3	2.9	7.0	18.6	26.6	68.2	0.6	1.7
DMSO	Mean	101	101	38	42	82	96	289	249	19	20
	sd	6.1	13.3	4.0	2.0	14.6	10.4	22.0	4.6	2.1	4.5
THF	Mean	88	121	39	43	80	81	281	271	18	17
	sd	5.8	37.8	1.2	6.7	4.7	6.4	38.0	58.3	4.0	1.2
Positive Controls*	Mean	536	525	77	119	387	1001	672	652	216	141
	sd	90.9	33.3	4.6	25.0	22.7	0.0	12.0	18.3	50.0	26.0
	f(I)	5.31	5.20	2.03	2.83	4.35	10.43	2.33	2.62	15.43	7.05
5 µL/plate	Mean	118	90	40	39	78	87	295	352	12	13
	sd	17.3	9.0	1.2	9.2	5.6	4.5	36.3	26.2	2.3	3.1
	f(I)	1.34	0.74	1.03	0.91	0.98	1.07	1.05	1.30	0.67	0.76
1.5 µL/plate	Mean	86	127	41	39	79	75	345	340	14	14
	sd	3.5	11.4	2.0	5.1	7.1	5.5	36.3	12.0	0.6	4.4
	f(I)	0.98	1.05	1.05	0.91	0.99	0.93	1.23	1.25	0.78	0.82
0.5 µL/plate	Mean	101	139	40	41	89	83	340	301	17	15
	sd	16.7	8.1	1.2	9.1	11.0	8.6	48.7	15.1	2.3	4.7
	f(I)	1.15	1.15	1.03	0.95	1.11	1.02	1.21	1.11	0.94	0.88
0.15 µL/plate	Mean	95	127	41	41	75	81	360	335	12	13
	sd	14.7	25.5	0.0	0.0	0.6	2.5	31.7	34.5	1.0	2.0
	f(I)	1.08	1.05	1.05	0.95	0.94	1.00	1.28	1.24	0.67	0.76
0.05 µL/plate	Mean	94	100	40	51	82	86	339	313	18	18
	sd	16.9	17.6	1.5	2.0	0.6	21.9	22.0	72.1	3.2	1.5
	f(I)	1.07	0.83	1.03	1.19	1.03	1.06	1.21	1.15	1.00	1.06

1001 colonies per plate means the bacteria growth was too strong for counting.

f(I) = increase factor

* Different positive controls were used

 

Survey of the Findings– Second Experiment

The mean revertant values of the three replicates are presented in the following table.

Mean Revertants Second Experiment

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	87	138	43	42	124	132	305	312	35	32
	sd	7.5	19.3	6.0	10.0	5.3	5.9	18.0	21.2	4.0	3.6
DMSO	Mean	86	104	41	48	110	117	307	328	33	33
	sd	11.7	13.9	9.5	6.0	6.0	8.7	22.0	26.2	3.5	1.7
THF	Mean	92	110	47	48	93	93	284	292	34	40
	sd	19.8	35.6	8.1	3.5	6.4	6.1	61.6	104.8	2.6	0.6
Positive Controls*	Mean	584	696	440	403	675	1001	729	1317	216	163
	sd	77.3	54.1	13.9	101.6	41.1	0.0	18.9	102.9	35.6	9.2
	f(I)	6.79	6.69	10.73	8.40	5.44	8.56	2.37	4.02	6.17	4.94
5 µL/plate	Mean	103	108	47	51	124	102	379	415	33	30
	sd	1.2	6.2	9.1	10.0	4.6	12.8	32.3	32.1	1.2	2.1
	f(I)	1.12	0.98	1.00	1.06	1.33	1.10	1.33	1.42	0.97	0.75
2.5 µL/plate	Mean	93	88	47	43	116	129	349	384	34	33
	sd	15.7	16.5	8.5	7.2	5.3	16.2	11.5	77.1	2.9	2.1
	f(I)	1.01	0.80	1.00	0.90	1.25	1.39	1.23	1.32	1.00	0.83
1.25 µL/plate	Mean	105	88	54	45	104	117	363	377	32	30
	sd	35.5	10.6	5.8	6.8	7.2	7.0	54.0	24.4	1.0	3.5
	f(I)	1.14	0.80	1.15	0.94	1.12	1.26	1.28	1.29	0.94	0.75
0.63 µL/plate	Mean	81	83	50	43	117	103	368	253	30	31
	sd	10.1	9.5	7.8	7.8	4.2	16.8	40.0	31.1	3.1	3.5
	f(I)	0.88	0.75	1.06	0.90	1.26	1.11	1.30	0.87	0.88	0.78
0.31 µL/plate	Mean	103	84	48	48	116	126	305	349	37	39
	sd	19.9	11.8	4.2	8.6	10.4	3.6	20.1	46.7	4.6	7.1
	f(I)	1.12	0.76	1.02	1.00	1.25	1.35	1.07	1.20	1.09	0.98
0.16 µL/plate	Mean	100	71	50	42	122	115	371	340	42	36
	sd	19.6	7.0	4.7	4.0	4.0	14.0	38.0	72.1	1.7	3.5
	f(I)	1.09	0.65	1.06	0.88	1.31	1.24	1.31	1.16	1.24	0.90

1001 colonies per plate means the bacteria growth was too strong for counting.

f(I) = increase factor

* Different positive controls were used

 

First Experiment- Positive Controls

Without metabolic activation:
4-Nitro-1,2-phenylene diamine (NPD) in DMSO, 20 µg/plate
Sodium azide (Na-azide) in demineralized water, 1 µg/plate

 

With metabolic activation:
2-Amino anthracene (2-AA) in DMSO, 1 µg/plate
Benzo-a-pyrene (BaP) in DMSO, 20 µg/plate

 

Diagnostic Mutagens (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Substance	NPD	2-AA	NPD	BaP	Na-azide	2-AA	NPD	2-AA	Na-azide	2-AA
Repl. 1	432	552	74	120	380	1001	672	672	232	140
Repl. 2	576	488	82	94	412	1001	660	636	160	168
Repl. 3	600	536	74	144	368	1001	684	648	256	116
Mean	536	525	77	119	387	1001	672	652	216	141
sd	90.9	33.3	4.6	25.0	22.7	0.0	12.0	18.3	50.0	26.0
f(I)	5.31	5.20	2.03	2.83	4.35	10.43	2.33	2.62	15.43	7.05
Rev. abs.	435	424	39	77	298	905	383	403	202	121

1001 colonies per plate means the bacteria growth was too strong for counting.

f(I) = increase factor

Rev.abs. = absolute revertants

 

Second Experiment- Positive Controls

Without metabolic activation:
4-Nitro-1,2-phenylene diamine (NPD) in DMSO, 20 µg/plate
Sodium azide (Na-azide) in demineralized water, 1 µg/plate

 

With metabolic activation:
2-Amino anthracene (2-AA) in DMSO, 1 µg/plate
Benzo-a-pyrene (BaP) in DMSO, 20 µg/plate

 

Diagnostic Mutagenes (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Substance	NPD	2-AA	NPD	BaP	Na-azide	2-AA	NPD	2-AA	Na-azide	2-AA
Repl. 1	516	640	456	344	720	1001	736	1200	244	152
Repl. 2	668	748	432	520	640	1001	708	1360	176	168
Repl. 3	568	700	432	344	664	1001	744	1392	228	168
Mean	584	696	440	403	675	1001	729	1317	216	163
sd	77.3	54.1	13.9	101.6	41.1	0.0	18.9	102.9	35.6	9.2
f(I)	6.79	6.69	10.73	8.40	5.44	8.56	2.37	4.02	6.17	4.94
Rev. abs.	498	592	399	355	551	884	422	989	181	130

1001 colonies per plate means the bacteria growth was too strong for counting.

f(I) = increase factor

Rev.abs. = absolute revertants

 

 

Historical Data

In the following table, the history of the spontaneous revertants and positive controls of the performed experiments with these strains up to 16. May 2018 (demin. water and DMSO) resp. 20. Mar. 2018 (THF) is stated in comparison with the experiments performed within this study. Only experiments which were performed before the performance of the study were considered.

For the historical data, the plate incorporation method and the pre- incubation method were used.

 

Historical Data of Spontaneous Revertants

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Demin. water	Mean	88	94	22	24	93	97	281	300	18	18
	Min	60	63	6	8	51	64	85	67	6	7
	Max	144	138	52	51	147	141	425	587	36	40
	SD	17	16	12	11	16	15	57	72	6	6
	Exp 1	82	105	36	40	89	103	213	316	14	18
	Exp 2	87	138	43	42	124	132	305	312	35	32
DMSO	Mean	88	97	22	23	90	93	280	293	18	17
	Min	58	67	7	8	44	62	79	80	8	6
	Max	135	144	47	50	138	199	413	459	35	37
	SD	17	16	12	11	16	17	56	60	6	6
	Exp 1	101	101	38	42	82	96	289	249	19	20
	Exp 2	86	104	41	48	110	117	307	328	33	33
THF	Mean	87	101	21	25	97	102	339	332	25	23
	Min	69	78	10	17	61	73	201	269	18	13
	Max	98	121	47	48	187	187	492	433	34	40
	SD	8	15	12	11	33	33	96	57	5	8
	Exp 1	88	121	39	43	80	81	281	271	18	17
	Exp 2	92	110	47	48	93	93	284	292	34	40
Positive Controls*	Mean	534	524	416	127	486	777	1099	1200	264	134
	Min	264	228	100	39	220	273	491	408	55	45
	Max	1165	1181	1001	487	984	1912	2331	6083	515	712
	SD	170	169	168	99	155	284	409	560	85	80
	Exp 1	536	525	77	119	387	1001	672	652	216	141
	Exp 2	584	696	440	403	675	1001	729	1317	216	163

1001 colonies per plate means the bacteria growth was too strong for counting.

* Different positive controls were used

Applicant's summary and conclusion

Conclusions:

The test item Petroleum Resins (Kendex 0897) showed no increase in the number of revertants in all bacteria strains in both experiments.
All negative and strain-specific nearly all positive control values (one exception) were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that Petroleum Resins (Kendex 0897) is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.

Executive summary:

Determination of the mutagenic potential of Petroleum Resins (Kendex 0897) with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14

 

Findings and Results:

Two valid experiments were performed.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test item Petroleum Resins (Kendex 0897) was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535).

The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

 

Experiment 1:

In the first experiment, the test item (dissolved in tetrahydrofurane) was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Experiment 2:

Based on the results of the first experiment, the test item was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in all bacteria strains using the pre-incubation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

 

Based on the results of this study it is concluded that Petroleum Resins (Kendex 0897) is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.