complex reaction mass of Chinese gum rosin post reacted with acrylic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

This study was conducted according to Safepharm Standard Method 700.03 and was designed to assess the mutagenic potential of the test material using a bacterial/microsome test system. The study was based on the in vitro technique described by Ames and Garner et al.
The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EC and USA, EPA (TSCA) guidelines.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

434-230-1

EC Name:

-

Cas Number:

144413-22-9

Molecular formula:

Not applicable - the substance is an UVCB

IUPAC Name:

1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,4b,5,6,10,10a-decahydrophenanthrene-1-carboxylic acid; prop-2-enoic acid

Test material form:

other: pale yellow blocks

Details on test material:

- Chemical name: Rosin acid reaction products with acrylic acid hydrogenated

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 mix

Test concentrations with justification for top dose:

- Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
- Main test, experiment I and II: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

Dimethyl sulphoxide

Controls

Untreated negative controls:

other: Solvent control served as negative control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

The Salmonella strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 whilst the Escherichia coli strain
WP2uvrA- was obtained from the British Industrial Biological Research Association on 17 August 1987. All of the strains were stored at -196°C in a
Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot numbers 201416 1/03 and 201417 1/03) and incubated at 37°C for approximately 10 hours.

Evaluation criteria:

ACCEPTANCE CRITERIA:
The reverse mutation assay may be considered valid if the following criteria are met:
- All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pkM101 plasmid R-factor etc.
- All tester strain cultures should be in the approximate range of 1 to 9.9 x 10E9 bacteria per mL.
- Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic
sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
- There should be a minimum of four non-toxic test material dose levels. There should be no evidence of excessive contamination.

EVALUATION CRITERIA:
The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria. If a greater than twofold increase in revertant count is observed in two experiments then this is taken as evidence of a positive response.

Statistics:

Dunnett's method of linear regression

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and 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 2

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:

not applicable

Positive controls validity:

valid

Additional information on results:

VEHICLE CONTROL
The results for the negative control (solvent control plates) were considered to be acceptable .

POSITIVE CONTROL
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

TEST MATERIAL
No toxicity was exhibited to any of the strains of bacteria used. A precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation.

Any other information on results incl. tables

PRELIMINARY TOXICICTY STUDY

The test material was non-toxic to the strains of bacteria used (TA 100 and WP2uvrA).

No. of revertant colonies per dose [µg/plate]

S9 -mix	strain	0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
+	TA100	134	127	126	103	102	111	113	105	91	79	96*
-	TA100	116	111	114	95	146	120	93	108	84	84	83*
+	WP2uvrA	27	24	26	24	25	32	37	28	28	28	34*
-	WP2uvrA	29	18	28	25	22	17	35	20	15	20	21*

* Precipitation

MAIN STUDY

EXPERIMENT I - Mean no. of revertant colonies

S9 -mix	test item [µg/plate]	TA100	TA1535	WP2uvrA	TA98	TA1537
-	0	124	20	27	26	12
-	50	109	20	31	30	9
-	150	114	25	22	27	6
-	500	98	10	24	30	9
-	1500	101	17	22	18	12
-	5000	103	17	23	20	6
-	pos. control	440	250	683	140	1018
+	0	114	17	32	32	15
+	50	104	15	26	33	12
+	150	99	15	35	35	15
+	500	99	14	20	33	11
+	1500	83	12	27	31	9
+	5000	76	11	19	22	6
+	pos. control	791	198	706	510	223

EXPERIMENT II - mean no. of revertant colonies

S9 -mix	test item [µg/plate]	TA100	TA1535	WP2uvrA	TA98	TA1537
-	0	135	31	25	34	17
-	50	119	29	23	32	13
-	150	129	22	20	27	6
-	500	118	20	18	25	7
-	1500	99	23	21	31	6
-	5000	106	21	27	36	11
-	pos. control	631	165	486	136	1001
+	0	128	13	26	34	19
+	50	127	18	27	41	14
+	150	123	16	25	34	14
+	500	120	14	30	36	9
+	1500	113	13	26	27	10
+	5000	111	14	27	41	6
+	pos. control	959	252	1184	583	492

Applicant's summary and conclusion

Conclusions:

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EC and USA, EPA (TSCA) guidelines. The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range.

All of the positive controI chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose of 5000 µg/plate. A precipitate was observed at 5000 pg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequeney of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.