Reaction product of salicylaldehyde and formaldehyde and phenol

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:

07 September 2005 to 29 September 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Yakusyoku No.1121002 of the Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: H15.11.13 seikyoku No.2 of the Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: kanpoki No.031121002 of the Environmental Policy Bureau, Ministry of the Environment

Version / remarks:

November 21, 2003

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Notification of Ministry of the Labour, Japan, No.77, September 1, 1988 and No.67 (revised), June 2, 1997.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of salicylaldehyde and formaldehyde and phenol

EC Number:

632-556-0

Cas Number:

304859-44-7

Molecular formula:

C6H5O(C13H10O2)m(C7H6O)nH values on m and n are variable as this is a UVCB substance

IUPAC Name:

Reaction product of salicylaldehyde and formaldehyde and phenol

Test material form:

solid

Details on test material:

- Appearance at ordinary temperature: Red-brownish solid

Method

Target gene:

S. typhimurium: Histidine locus
E. coli: Tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. T. Matsushima, Japan Bioassay Research Center, Japan Industrial Safety and Association, Hadano-City, Kanagawa.
- Strains were stored at -80 °C
- 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 and incubated at 37 C for 10 hours. Each culture was monitored spectrophotometrically for turbidity with titters determined by viable count analysis on nutrient agar plates.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. T. Matsushima, Japan Bioassay Research Center, Japan Industrial Safety and Association, Hadano-City, Kanagawa.
- Strains were stored at -80 °C
- 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 and incubated at 37 C for 10 hours. Each culture was monitored spectrophotometrically for turbidity with titters determined by viable count analysis on nutrient agar plates.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose-Determination test: 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate
Mutagenicity tests: 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was not dissolved in water at 50 mg/mL, but was dissolved in DMSO at 100 mg/mL in solubility checks performed in-house. DMSO was therefore selected as the vehicle of choice.
- The test material was dissolved in DMSO to make a stock solution of 100 mg/mL and further diluted to obtain desired concentrations.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 µg/ 50 µL/ plate for TA100 and WP2uvrA (-S9 mix), and 0.1 µg/ 50 µL/ plate for TA98 (-S9 mix))

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Measured aliquots (0.05 mL) of the test material formulation, vehicle or positive control were dispensed into sets of test tubes followed by either 0.5 mL of S9 mix or phosphate buffer, 0.1 mL of one of the bacterial cultures. The contents of each test tube were incubated at 37 °C for 20 min. and mixed with 2.0 mL of molten, trace histidine and biotin or tryptophan supplemented, top agar and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in duplicate, for each bacterial strain and for each concentration of test material both with and without S9 mix.
- Exposure duration: 48 hours at 37 °C

EVALUATION
- The frequency of revertant colonies was assessed using a colony analyser CA-11S (System Science Co., Ltd.).

OTHER
- Dose-Determination test: Six concentrations of the test material (4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate) were assayed in duplicate against each tester strain, using the pre-incubation method.

Evaluation criteria:

- The response was regarded positive if the maximum number of revertant colonies in the test material group increased ≥ 2 fold relative to the negative control group and dose-response and reproducibility were confirmed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537 TA 98 & TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- The test material did not induce ≥ 2 fold increase in the number of revertant colonies relative to the negative control value for any strain with or without metabolic activation, and the reproducibility of the results was confirmed. From these results, the test material was judged negative for the potential to induce gene mutation (mutagenicity).
- Cytotoxicity were observed at and above 313 µg/plate with or without metabolic activation on dose-determination test, at and above 156 µg/plate without metabolic activation, at and above 313 µg/plate with metabolic activation on mutagenicity test 1 and 2. No precipitate of the test material was observed at any dose level. In the sterility test, no growth of bacteria was observed under any test conditions.
- 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.

Any other information on results incl. tables

Table 1: Summary of Mutagenicity Test 1

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 9.77 19.5 39.1 78.1 156 313	110 93 102 100 107 81* 65*	10 6 8 11 10 8* 0*	34 28 27 31 28 28* 0*	14 14 17 18 16 8* 0*	9 11 9 10 6 0* 0*
+	Solvent 9.77 19.5 39.1 78.1 156 313	114 128 115 115 118 108 102*	11 9 10 8 12 11 6*	31 32 26 33 29 37 39*	25 24 27 26 30 26 25*	10 11 12 11 14 16 9*
Positive Controls
-	Name	AF-2	SA	AF-2	AF-2	9AA
	Concentration (µg/plate)	0.01	0.5	0.01	0.1	80
	Mean no. colonies/plate	639	261	224	513	359
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	1	2	10	0.5	2
	Mean no. colonies/plate	1293	159	725	537	194

AF-2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SA = Sodium azide

When toxicity was observed, "*" was placed to the right of the number of the revertants.

Table 2: Summary of Mutagenicity Test 2

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 9.77 19.5 39.1 78.1 156 313	122 123 96 109 113 74* 0*	9 7 8 10 6 6* 0*	26 35 32 28 23 31* 0*	12 15 15 12 11 10* 0*	9 7 7 7 8 0* 0*
+	Solvent 9.77 19.5 39.1 78.1 156 313	115 127 136 124 113 129 77*	9 10 8 10 8 8 6*	29 29 26 35 34 38 25*	24 22 28 28 32 24 20*	13 13 13 9 11 13 9*
Positive Controls
-	Name	AF-2	SA	AF-2	AF-2	9AA
	Concentration (µg/plate)	0.01	0.5	0.01	0.1	80
	Mean no. colonies/plate	627	314	229	520	331
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	1	2	10	0.5	2
	Mean no. colonies/plate	1317	210	719	576	211

AF-2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SA = Sodium azide

When toxicity was observed, "*" was placed to the right of the number of the revertants.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material was judged negative for the potential to induce gene mutation (mutagenicity).

Executive summary:

The genetic toxicity of the test material was investigated in accordance with Japanese guidelines under GLP conditions. The mutagenic potential was assessed with a bacterial reverse mutation assay.

Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA were treated with the test material using the pre-incubation method at six dose levels, in duplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the dose-determination test was 4.88 to 5000 µg/plate. The experiment was repeated on a separate day using the dose range, 9.77 to 313 µg/plate, fresh cultures of the bacterial strains and fresh test substance formulations.

Cytotoxicity was observed at and above 313 µg/plate with or without metabolic activation on dose-determination test, at and above 156 µg/plate without metabolic activation, at and above 313 µg/plate with metabolic activation on mutagenicity test 1 and mutagenicity test 2. No precipitate of the test material was observed at any dose level.

No significant increases in the frequency of revertant colonies were recorded for any strain with or without metabolic activation.

Under the conditions of this study, the test material was judged negative for the potential to induce gene mutation (mutagenicity).