Isobutylamine

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:

27 November 2017 to 19 January 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Isobutylamine

EC Number:

201-145-4

EC Name:

Isobutylamine

Cas Number:

78-81-9

Molecular formula:

C4H11N

IUPAC Name:

2-methylpropan-1-amine

Test material form:

liquid

Specific details on test material used for the study:

A volume of 0.25 L of vapour was produced from 1 mL of the test item at 37°C. All concentrations cited in this report are expressed in terms of the CA3324A sample as received.

Method

Target gene:

The S. typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system measures his- → his+ and trp- → trp+ reversions, respectively.

Species / strain

Species / strain / cell type:

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

Remarks:

E.coli WP2 pKM101 and WP2 uvrA pKM101

Details on mammalian cell type (if applicable):

The strains of S.typhimurium and E.coli were obtained from Moltox Inc. Batches of the strains were stored at -90 to -70°C as aliquots of nutrient broth cultures. DMSO was added to the cultures at 8% v/v as a cropreservative. Eachbatch of frozen strain was tested for amino acid requiremetn and , where applicable, for cell membrane permeability (rfa mutation), deficiency in DNA excision repair system (uvrA/uvrB mutation), and the pKM101 plasmid that confers resistantce to antibiotics. The responses of the strains to a series of reference mutagens were also assessed and were within the normal ranges determines in this laboratory.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment 1: Without and with S9 mix: 1.25%, 2.5%, 5%, 10%, 20%, 40%, 70% v/v test substance
Experiment 2: Without S9 mix: 0.08%, 0.16%, 0.31%, 0.63%, 1.25%, 2.5%, 5% v/v test substance
With S9 mix: 0.16%, 0.31%, 0.63%, 1.25%, 2.5%, 5% v/v test substance
Experiment 3: Without and with S9 mix: 0.08%, 0.16%, 0.31%, 0.63%, 1.25%, 2.5%, 5% v/v test substance
Experiment 4: Without and with S9 mix: 0.08%, 0.16%, 0.31%, 0.63%, 1.25%, 2.5%, 5% v/v test substance

Vehicle / solvent:

None

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

sterile air

Controls 2

Untreated negative controls:

yes

Remarks:

incubator controls

Remarks:

Concurrent incubatro controls treated with culture medium in the absence of the test item were placed in an incubator set to 34 to 39°C (i.e. cultures were not placed in the exposure vessels).

Controls 3

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix

Controls 4

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix

Controls 5

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9 mix

Controls 6

Positive controls:

yes

Positive control substance:

other: 4-Nitroquinoline-1-oxide

Remarks:

Without S9 mix

Controls 7

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

without S9 mix

Controls 8

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: The bacteria were exposed to measured volumes of the test item in sealed stainless steel vessels incubated at 34-37°C. Sterile air was introduced inside a class II microbiological safety cabinet to equilibrate to atmospheric pressure.
Four separate independent experiments were conducted in the absence and presence of S9 mix. S9mix (0.5 mL) or 0.1 M pH 7.4 sodium phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10 hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test item, S9 mix and sodium phosphate buffer. The seeded plates were placed in stainless steel vessels. Plates containing S9 mix and buffer were placed in separate vessels. The vessels were sealed and partially evacuated. Appropriate volumes of the test substance were added to the vessels. The vessels were warmed to 34 to 39°C and the contents equilibrated to atmospheric pressure, where necessary, by admitting sterile atmospheric air. The plates were in cubated for ca 48 hours in the vessels at 34 to 39°C and then removed from the vessels under air extraction. The plates were incubated for a further period of ca 24 hours at 37°C to permit the growth of revertant colonies. Further sets of plates were prepared for the liquid positive control compounds. Aliquots of 0.1 mL of the positive control solutions were added to the plates together with the bacteria, buffer or S9 mix and agar overlay. These plates were incubated at 34 to 39°C for 48-72 hours (not in stainless steel vessels). After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter. Concentrations of the test substance up to 70% v/v per plate were tested. This is the maximum
practicable achievable concentration.

NUMBER OF REPLICATIONS: 3

Rationale for test conditions:

The strains were used to detect base changes and frameshift mutations as follows: Base change mutagens: S. typhimurium TA1535 and TA100, and E. coli WP2 uvrA (pKM101). Frameshift mutagens: S. typhimurium TA1537 and TA98. As the test item is known to be in vapour phase at the nominal temperature used within the test system (37°C) a modification of the test procedure was used to ensure consistent exposure.

Evaluation criteria:

For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range for the laboratory. The positive control compounds must induce an increase in mean revertant colony numbers of at least twice that of the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 10^9/mL. A minimum of five analysable concentrations should be present with at least four showing no signs of toxic effects, evident as bacterial inhibition and/or a reduction in the number of revertants below the indication factor of 0.5. Mean number +SD of revertant colonies calculated for all groups. Fold-increases relative to vehicle controls calculated and means for treatment groups compared with vehicle control groups. It is considered to exhibit mutagenic activity if a reproducible increase in mean revertant colony numbers of at least twice that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, is seen. If exposure does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system.

If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers.

Statistics:

Not applicable

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:

cytotoxicity

Remarks:

see additional info on results

Vehicle 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:

cytotoxicity

Remarks:

see additinal info on results

Vehicle 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:

cytotoxicity

Remarks:

see additional info on results

Vehicle 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:

cytotoxicity

Remarks:

see additional infor on results

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see additional info on results

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the first experiment, toxicity (observed as thinning of the background lawn of non-revertant colonies, together with a reduction in revertant colony numbers (below an induction factor of 0.5)) was seen in all strains following exposure to the test substance at 5% v/v per plate and above; except in TA98 in the absence of S9 mix and TA1535 in the absence and presence of S9 mix where toxicity was observed at 2.5% v/v per plate and above. Densely coloured plates were seen at 10% v/v and above.

In the second experiment, toxicity (observed as thinning of the background lawn of non-revertant colonies, together with a reduction in revertant colony numbers (below an induction factor of 0.5)) was seen in all strains following exposure to the test substance at 5% v/v/plate. A greater than or equal to 2 fold-increase in revertant colonies was observed following exposure to the test substance at 0.16, 0.31, 0.63 and 1.25 % v/v per plate in the presence of S9 mix in strain TA1535 and 0.31 % v/v/plate in the absence of S9 mix in strain TA1535 and at 0.31 %v/v per plate in the absence of S9 mix in strain TA1537.

In the third experiment, toxicity (observed as thinning of the background lawn of non-revertant colonies, and/or a reduction in revertant colony numbers (below an induction factor of 0.5)) was seen in all strains following exposure to the test substance at 5% v/v per plate: except in strain TA1535 without S9 mix where toxicity was observed at 1.25 and 5% v/v per plate and in strain TA98 without S9 mix where toxicity occurred at 2.5% v/v per plate and above. No evidence of mutagenic activity was seen at any concentration of the test substance. There was also no observed tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

In the fourth experiment, toxicity (observed as thinning of the background lawn of non-revertant colonies, and/or a reduction in revertant colony numbers (below an induction factor of 0.5)) was seen in all strains following exposure to the test substance at 5% v/v per plate: except in strain TA1537 in the absence of S9 mix where toxicity was observed at 2.5% v/v per plate and above. No evidence of mutagenic activity was seen at any concentration of the test substance. There was also no observed tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

As the results observed in the second experiment were not reproduced in the third and fourth independent experiments the overall result is that the test substance is considered not to be mutagenic.

Any other information on results incl. tables

Table 1: Experiment 1 without metabolic activation

Strain	Addition	Concentration plate	Mean revertants/plate	SD	Fold Increase (a)
TA98	Incubator Control		19.7	2.3
	Vehicule		17.7	0.6
	Test substance	1.25 % v/v	9.3	0.6	0.5
		2.5 % v/v	6.3	0.6	0.4
		5 % v/v	0.0	0.0	0.0
		10 % v/v	0.0	0.0	0.0
		20 % v/v	0.0	0.0	0.0
TA100	Incubator Control		152.7	31.1
	Vehicule		134.3	1.5
	Test substance	1.25 % v/v	101.0	14.1	0.8
		2.5 % v/v	81.7	11.2	0.6
		5 % v/v	0.0	0.0	0.0
		10 % v/v	0.0	0.0	0.0
		20 % v/v	0.0	0.0	0.0
TA1535	Incubator Control		16.7	8.1
	Vehicule		17.7	2.5
	Test substance	1.25 % v/v	10.3	4.6	0.6
		2.5 % v/v	5.7	6.4	0.3
		5 % v/v	0.3	0.6	0.0
		10 % v/v	0.0	0.0	0.0
		20 % v/v	0.0	0.0	0.0
TA1537	Incubator Control		10.3	3.8
	Vehicule		7.3	2.5
	Test substance	1.25 % v/v	12.0	5.2	1.6
		2.5 % v/v	7.7	2.1	1.0
		5 % v/v	0.0	0.0	0.0
		10 % v/v	0.0	0.0	0.0
		20 % v/v	0.0	0.0	0.0
WP2 uvrA (pKM101)	Incubator Control		108.3	7.0
	Vehicule		114.0	24.9
	Test substance	1.25 % v/v	122.0	137.7	1.1
		2.5 % v/v	103.	6.5	0.9
		5 % v/v	0.3	0.6	0.0
		10 % v/v	0.0	0.0	0.0
		20 % v/v	0.0	0.0	0.0
TA98	2NF	2 µg	265.3	38.4	15.0
TA100	NaN3	2 µg	623.3	46.1	4.6
TA1535	NaN3	2 µg	774.7	25.3	43.8
TA1537	AAC	50 µg	95.0	16.1	13.0
WP2 uvrA(pKM101)	NQO	2 µg	1471.7	92.7	12.9

 

2NF = 2-nitrofluoren; NaN3=sodium azide; AAC = aminoacridine; NQO = 4-Nitroquinoline-1-oxide, D = densely coloured plate; T= thinning of background lawn; (a) = relevant to vehicle. Plates at 40 and 70% v/v/plate were discarded after the 48 hour exposure to test substance.

 

Table 2: Experiment 1 with metabolic activation

Strain	Addition	Concentration plate	Mean revertants/plate	SD	Fold Increase (a)
TA98	Incubator Control		75.0	12.1
	Vehicle		69.3	31.6
	Test substance	1.25 % v/v	53.0	12.1	0.8
		2.5 % v/v	64.3	12.9	0.9
		5 % v/v	0.0	0.0	0.0
		10 % v/v	0.0	0.0	0.0
		20 % v/v	0.0	0.0	0.0
TA100	Incubator Control		193.3	11.7
	Vehicle		196.3	3.8
	Test substance	1.25 % v/v	297.0	6.2	1.5
		2.5 % v/v	174.0	9.8	0.9
		5 % v/v	0.0	0.0	0.0
		10 % v/v	0.0	0.0	0.0
		20 % v/v	0.0	0.0	0.0
TA1535	Incubator Control		15.3	6.7
	Vehicle		30.0	3.5
	Test substance	1.25 % v/v	24.3	2.3	0.8
		2.5 % v/v	12.0	0.0	0.4
		5 % v/v	0.0	0.0	0.0
		10 % v/v	0.0	0.0	0.0
		20 % v/v	0.0	0.0	0.0
TA1537	Incubator Control		14.0	4.6
	Vehicle		17.7	7.6
	Test substance	1.25 % v/v	24.0	2.0	1.4
		2.5 % v/v	30.3	1.2	1.7
		5 % v/v	0.0	0.0	0.0
		10 % v/v	0.0	0.0	0.0
		20 % v/v	0.0	0.0	0.0
WP2 uvrA (pKM101)	Incubator Control		231.0	22.3
	Vehicle		202.3	7.4
	Test substance	1.25 % v/v	219.7	5.5	1.7
		2.5 % v/v	143.0	44.0	0.7
		5 % v/v	0.0	0.0	0.0
		10 % v/v	0.0	0.0	0.0
		20 % v/v	0.0	0.0	0.0
TA98	B[a]P	5 µg	229.0	3.6	3.3
TA100	AAN	5 µg	2814.7	16.3	14.3
TA1535	AAN	5 µg	286.7	28.3	9.6
TA1537	B[a]P	5 µg	61.0	6.0	3.5
WP2 uvrA (pKM101)	AAN	10 µg	1122.3	63.4	5.5

 

B[a]P = benzo[a]pyrene; AAN : 2-aminoanthracene; D = densely coloured plate; T= thinning of background lawn; (a) = relevant to vehicle. Plates at 40 and 70% v/v/plate were discarded after the 48 hour exposure to test substance.

 

Table 3: Experiment 2 without metabolic activation

Strain	Addition	Concentration plate	Mean revertants/plate	SD	Fold Increase (a)
TA98	Incubator Control		21.3	4.5
	Vehicle		19.0	6.6
	Test substance	0.08 % v/v	18.7	1.2	1.0
		0.16 % v/v	23.7	4.9	1.2
		0.31 % v/v	23.7	9.3	1.8
		0.63 % v/v	21.3	1.5	1.1
		1.25 % v/v	18.7	3.1	1.0
		2.5 % v/v	14.3	3.2	0.8
		5 % v/v	0.7	0.6	0.0
TA100	Incubator Control		148.0	20.2
	Vehicle		117.0	12.1
	Test substance	0.08 % v/v	112.3	15.3	1.0
		0.16 % v/v	118.0	22.3	1.0
		0.31 % v/v	132.0	7.8	1.1
		0.63 % v/v	126.0	13.5	1.1
		1.25 % v/v	107.0	20.9	0.9
		2.5 % v/v	102.7	21.1	0.9
		5 % v/v	0.3	0.6	0.0
TA1535	Incubator Control		17.3	2.5
	Vehicle		12.7	4.0
	Test substance	0.08 % v/v	19.3	7.6	1.5
		0.16 % v/v	17.3	3.2	1.4
		0.31 % v/v	26.3	10.3	2.1
		0.63 % v/v	24.3	17.2	1.9
		1.25 % v/v	17.0	5.6	1.3
		2.5 % v/v	6.7	5.8	0.5
		5 % v/v	1.0	1.0	0.1
TA1537	Incubator Control		12.3	7.4
	Vehicle		6.7	0.6
	Test substance	0.08 % v/v	5.0	1.0	0.7
		0.16 % v/v	8.3	7.5	1.3
		0.31 % v/v	13.3	4.5	2.0
		0.63 % v/v	13.0	9.2	1.9
		1.25 % v/v	4.3	2.5	0.6
		2.5 % v/v	4.3	3.8	0.6
		5 % v/v	1.3	0.6	0.2
WP2 uvrA (pKM101)	Incubator Control		100.0	17.5
	Vehicle		102.3	4.0
	Test substance	0.08 % v/v	121.7	6.5	1.2
		0.16 % v/v	103.3	2.5	1.0
		0.31 % v/v	123.0	4.6	1.2
		0.63 % v/v	117.0	7.2	1.1
		1.25 % v/v	100.3	2.5	1.0
		2.5 % v/v	85.3	25.7	0.8
		5 % v/v	0.3	0.6	0.0
TA98	2NF	2 µg	254.0	79.4	13.4
TA100	NaN3	2 µg	654.3	49.0	5.6
TA1535	NaN3	2 µg	747.7	33.1	59.0
TA1537	AAC	50 µg	113.3	31.4	17.0
WP2 uvrA(pKM101)	NQO	2 µg	2020.0	271.6	19.7

 

2NF = 2-nitrofluoren; NaN3=sodium azide; AAC = aminoacridine; NQO = 4-Nitroquinoline-1-oxide; (a) rel to vehicle

Table 4: Experiment 2 with metabolic activation

Strain	Addition	Concentration plate	Mean revertants/plate	SD	Fold Increase (a)
TA98	Incubator Control		29.0	6.0
	Vehicle		28.3	1.2
	Test substance	0.16 % v/v	39.0	6.9	1.4
		0.31 % v/v	41.3	16.4	1.5
		0.63 % v/v	31.7	6.4	1.1
		1.25 % v/v	22.7	15.0	0.8
		2.5 % v/v	26.0	2.6	0.9
		5 % v/v	0.3	0.6	0.0
TA100	Incubator Control		175.7	12.5
	Vehicle		147.0	15.7
	Test substance	0.16 % v/v	146.7	8.1	1.0
		0.31 % v/v	165.3	10.2	1.1
		0.63 % v/v	141.3	15.2	1.0
		1.25 % v/v	135.3	4.7	0.9
		2.5 % v/v	190.3	10.7	1.3
		5 % v/v	1.3	2.3	0.0
TA1535	Incubator Control		12.0	2.6
	Vehicle		11.3	3.2
	Test substance	0.16 % v/v	23.7	2.5	2.1
		0.31 % v/v	24.7	9.1	2.2
		0.63 % v/v	24.0	7.2	2.1
		1.25 % v/v	28.7	9.9	2.5
		2.5 % v/v	12.7	0.6	1.1
		5 % v/v	2.3	3.2	0.2
TA1537	Incubator Control		14.0	1.7
	Vehicle		14.7	2.9
	Test substance	0.16 % v/v	11.7	9.0	0.8
		0.31 % v/v	15.3	9.2	1.0
		0.63 % v/v	15.7	0.6	1.1
		1.25 % v/v	7.7	1.2	0.5
		2.5 % v/v	10.3	0.6	0.7
		5 % v/v	3.3	2.9	0.2
WP2 uvrA (pKM101)	Incubator Control		148.0	13.1
	Vehicle		148.0	14.8
	Test substance	0.16 % v/v	138.0	6.0	0.9
		0.31 % v/v	168.7	11.8	1.1
		0.63 % v/v	144.7	18.1	1.0
		1.25 % v/v	146.0	18.5	1.0
		2.5 % v/v	151.7	17.0	1.0
		5 % v/v	2.0	2.6	1.0
TA98	B[a]P	5 µg	185.7	24.7	6.6
TA100	AAN	5 µg	1888.0	139.1	12.8
TA1535	AAN	5 µg	270.7	18.5	23.9
TA1537	B[a]P	5 µg	68.7	3.1	4.7
WP2 uvrA(pKM101)	NQO	2 µg	1324.0	82.4	8.9

B[a]P = benzo[a]pyrene; AAN : 2-aminoanthracene;; T= thinning of background lawn24.7; (a) = relevant to vehicle.

Table 5: Experiment 3 without metabolic activation

Strain	Addition	Concentration plate	Mean revertants/plate	SD	Fold Increase (a)
TA98	Incubator Control		23.0	2.6
	Vehicle		28.3	6.4
	Test substance	0.08 % v/v	24.7	3.2	0.9
		0.16 % v/v	22.7	8.5	0.8
		0.31 % v/v	22.7	3.1	0.8
		0.63 % v/v	16.7	6.1	0.6
		1.25 % v/v	30.0	7.5	1.1
		2.5 % v/v	9.7	4.0	0.3
		5 % v/v	0.3	0.6	0.0
TA100	Incubator Control		190.7	11.9
	Vehicle		149.7	7.5
	Test substance	0.08 % v/v	126.7	29.3	0.8
		0.16 % v/v	160.7	5.9	1.1
		0.31 % v/v	145.0	2.0	1.0
		0.63 % v/v	128.7	11.6	0.9
		1.25 % v/v	131.7	11.0	0.9
		2.5 % v/v	153.3	14.2	1.0
		5 % v/v	0.0	0.0	0.0
TA1535	Incubator Control		18.0	3.0
	Vehicle		32.0	3.0
	Test substance	0.08 % v/v	24.7	9.1	0.8
		0.16 % v/v	22.0	11.3	0.7
		0.31 % v/v	21.0	8.7	0.7
		0.63 % v/v	20.7	6.4	0.6
		1.25 % v/v	14.0	3.6	0.4
		2.5 % v/v	18.3	1.5	0.6
		5 % v/v	0.0	0.0	0.0
TA1537	Incubator Control		12.3	5.0
	Vehicle		6.6	0.6
	Test substance	0.08 % v/v	7.0	0.0	1.1
		0.16 % v/v	8.0	1.7	1.3
		0.31 % v/v	12.0	1.7	1.9
		0.63 % v/v	9.0	5.3	1.4
		1.25 % v/v	8.3	1.2	1.3
		2.5 % v/v	7.7	2.9	1.2
		5 % v/v	0.0	0.0	0.0
WP2 uvrA (pKM101)	Incubator Control		184.3	7.8
	Vehicle		148.7	9.5
	Test substance	0.08 % v/v	159.7	21.2	1.1
		0.16 % v/v	180.7	19.6	1.2
		0.31 % v/v	186.3	15.5	1.3
		0.63 % v/v	165.0	10.0	1.1
		1.25 % v/v	190.0	15.4	1.3
		2.5 % v/v	185.0	14.7	1.2
		5 % v/v	2.7	2.1	0.0
TA98	2NF	2 µg	275.0	17.1	9.7
TA100	NaN3	2 µg	602.7	61.0	4.0
TA1535	NaN3	2 µg	819.3	24.4	25.6
TA1537	AAC	50 µg	156.7	51.4	24.7
WP2 uvrA(pKM101)	NQO	2 µg	2111.3	297.3	14.2

 

2NF = 2-nitrofluoren; NaN3=sodium azide; AAC = aminoacridine; NQO = 4-Nitroquinoline-1-oxide; (a) rel to vehicle

Table 6: Experiment 3 with metabolic activation

Strain	Addition	Concentration plate	Mean revertants/plate	SD	Fold Increase (a)
TA98	Incubator Control		34.0	3.0
	Vehicle		25.7	10.0
	Test substance	0.08 % v/v	28.3	4.0	1.1
		0.16 % v/v	32.7	13.3	1.3
		0.31 % v/v	22.0	6.2	0.9
		0.63 % v/v	29.7	15.2	1.2
		1.25 % v/v	26.0	2.6	1.0
		2.5 % v/v	19.0	3.5	0.7
		5 % v/v	0.7	1.2	0.0
TA100	Incubator Control		161.3	17.4
	Vehicle		126.3	20.2
	Test substance	0.08 % v/v	129.7	5.5	1.0
		0.16 % v/v	113.3	39.4	0.9
		0.31 % v/v	143.3	15.9	1.1
		0.63 % v/v	149.3	5.1	1.2
		1.25 % v/v	147.7	7.5	1.2
		2.5 % v/v	143.3	20.6	1.1
		5 % v/v	0.7	0.6	0.0
TA1535	Incubator Control		13.0	1.7
	Vehicle		26.0	6.1
	Test substance	0.08 % v/v	22.7	1.2	0.9
		0.16 % v/v	15.3	2.9	0.6
		0.31 % v/v	13.7	4.0	0.5
		0.63 % v/v	23.3	4.9	0.9
		1.25 % v/v	26.3	2.5	1.0
		2.5 % v/v	19.3	6.4	0.7
		5 % v/v	0.3	0.6	0.0
TA1537	Incubator Control		12.3	4.6
	Vehicle		13.3	3.8
	Test substance	0.08 % v/v	17.7	9.3	1.3
		0.16 % v/v	15.3	11.0	1.1
		0.31 % v/v	19.7	1.5	1.5
		0.63 % v/v	12.3	5.5	0.9
		1.25 % v/v	10.0	3.6	0.7
		2.5 % v/v	8.3	2.9	0.6
		5 % v/v	0.0	0.0	0.0
WP2 uvrA (pKM101)	Incubator Control		120.7	5.9
	Test substance	0.08 % v/v	131.0	27.9	1.0
		0.16 % v/v	124.7	41.2	0.9
		0.31 % v/v	128.3	11.6	1.0
		0.63 % v/v	141.3	19.1	1.1
		1.25 % v/v	132.3	19.6	1.0
		2.5 % v/v	108.3	7.4	0.8
		5 % v/v	2.3	3.2	0.0
TA98	B[a]P	5 µg	193.3	5.5	7.5
TA100	AAN	5 µg	1629.0	243.6	12.9
TA1535	AAN	5 µg	311.3	20.6	12.0
TA1537	B[a]P	5 µg	75.0	12.8	5.6
WP2 uvrA(pKM101)	NQO	2 µg	760.3	28.4	5.8

B[a]P = benzo[a]pyrene; AAN : 2-aminoanthracene;; T= thinning of background lawn24.7; (a) = relevant to vehicle.

Table 7: Experiment 4 without metabolic activation

Strain	Addition	Concentration plate	Mean revertants/plate	SD	Fold Increase (a)
TA98	Incubator Control		19.7	3.5
	Vehicle		22.0	2.6
	Test substance	0.08 % v/v	118.7	3.8	0.8
		0.16 % v/v	21.3	3.1	1.0
		0.31 % v/v	16.7	1.2	0.8
		0.63 % v/v	25.0	5.0	1.1
		1.25 % v/v	19.0	4.4	0.9
		2.5 % v/v	13.0	3.5	0.6
		5 % v/v	0.0	0.0	0.0
TA100	Incubator Control		132.0	4.0
	Vehicle		138.7	3.2
	Test substance	0.08 % v/v	140.7	11.0	1.0
		0.16 % v/v	136.0	7.2	1.0
		0.31 % v/v	135.7	5.7	1.0
		0.63 % v/v	141.7	2.1	0.9
		1.25 % v/v	120.3	4.2	1.0
		2.5 % v/v	132.3	4.5	0.0
		5 % v/v	0.0	0.0
TA1535	Incubator Control		14.7	1.5
	Vehicle		16.3	5.9	0.9
	Test substance	0.08 % v/v	15.3	1.5	1.1
		0.16 % v/v	18.0	1.7	1.1
		0.31 % v/v	17.3	1.5	1.1
		0.63 % v/v	18.3	4.0	1.1
		1.25 % v/v	12.3	4.0	0.8
		2.5 % v/v	15.0	2.6	0.9
		5 % v/v	0.0	0.0	0.0
TA1537	Incubator Control		17.0	2.6
	Vehicle		16.3	4.7
	Test substance	0.08 % v/v	22.0	1.0	1.3
		0.16 % v/v	20.7	505	1.3
		0.31 % v/v	26.0	3.5	1.6
		0.63 % v/v	21.3	4.7	1.3
		1.25 % v/v	16.0	4.4	1.0
		2.5 % v/v	3.0	1.7	0.2
		5 % v/v	0.0	0.0	0.0
WP2 uvrA (pKM101)	Incubator Control		122.0	3.0
	Vehicle		123.3	2.1
	Test substance	0.08 % v/v	109.3	2.5	0.9
		0.16 % v/v	116.0	4.6	0.9
		0.31 % v/v	118.3	2.1	1.0
		0.63 % v/v	115.3	1.5	0.9
		1.25 % v/v	115.0	3.0	0.9
		2.5 % v/v	99.7	3.1	0.8
		5 % v/v	0.7	1.2	0.0
TA98	2NF	2 µg	283.3	48.8	12.9
TA100	NaN3	2 µg	544.7	17.2	3.9
TA1535	NaN3	2 µg	812.7	40.8	49.8
TA1537	AAC	50 µg	193.3	62.0	11.8
WP2 uvrA(pKM101)	NQO	2 µg	1880.3	63.4	15.2

 

2NF = 2-nitrofluoren; NaN3=sodium azide; AAC = aminoacridine; NQO = 4-Nitroquinoline-1-oxide; (a) rel to vehicle

Table 8: Experiment 4 with metabolic activation

Strain	Addition	Concentration plate	Mean revertants/plate	SD	Fold Increase (a)
TA98	Incubator Control		37.7	3.5
	Vehicle		40.3	12.1
	Test substance	0.08 % v/v	37.0	6.0	0.9
		0.16 % v/v	36.0	5.2	0.9
		0.31 % v/v	35.7	8.1	0.9
		0.63 % v/v	40.7	1.2	1.0
		1.25 % v/v	29.0	14.7	0.7
		2.5 % v/v	21.3	4.6	0.5
		5 % v/v	0.0	0.0	0.0
TA100	Incubator Control		175.7	8.1
	Vehicle		157.3	22.8
	Test substance	0.08 % v/v	166.0	9.5	1.1
		0.16 % v/v	174.0	8.5	1.1
		0.31 % v/v	163.3	117.6	1.0
		0.63 % v/v	170.3	9.1	1.1
		1.25 % v/v	153.0	24.8	1.0
		2.5 % v/v	150.7	30.4	1.0
		5 % v/v	3.3	2.1	0.0
TA1535	Incubator Control		15.3	6.0
	Vehicle		11.0	3.5
	Test substance	0.08 % v/v	12.3	0.6	1.1
		0.16 % v/v	9.0	0.0	0.8
		0.31 % v/v	13.3	2.9	1.2
		0.63 % v/v	14.7	1.5	1.3
		1.25 % v/v	11.3	1.5	1.0
		2.5 % v/v	11.7	2.9	1.1
		5 % v/v	0.3	0.6	0.0
TA1537	Incubator Control		16.0	5.3
	Vehicle		13.0	3.0
	Test substance	0.08 % v/v	14.3	2.3	1.1
		0.16 % v/v	15.3	4.9	1.2
		0.31 % v/v	10.7	1.2	0.8
		0.63 % v/v	10.0	2.6	0.8
		1.25 % v/v	7.3	1.5	0.6
		2.5 % v/v	11.0	6.6	0.8
		5 % v/v	0.3	0.6	0.0
WP2 uvrA (pKM101)	Incubator Control		194.0	18.5
	Vehicle		171.0	13.0
	Test substance	0.08 % v/v	210.7	22.6	1.2
		0.16 % v/v	191.7	204.4	1.1
		0.31 % v/v	193.0	5.3	1.1
		0.63 % v/v	195.0	18.2	1.1
		1.25 % v/v	189.0	22.6	1.1
		2.5 % v/v	178.3	3.5	1.0
		5 % v/v	0.0	0.0	0.0
TA98	B[a]P	5 µg	187.7	8.5	4.7
TA100	AAN	5 µg	1729.0	264.2	11.0
TA1535	AAN	5 µg	345.0	5.2	31.4
TA1537	B[a]P	5 µg	70.7	4.6	5.4
WP2 uvrA(pKM101)	NQO	2 µg	730.0	23.6	4.3

B[a]P = benzo[a]pyrene; AAN : 2-aminoanthracene;; T= thinning of background lawn24.7; (a) = relevant to vehicle.

 

 

Applicant's summary and conclusion

Conclusions:

The test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed. Hence, the test item is concluded to be negative, i.e. non-mutagenic, in this assay.

Executive summary:

Histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Eschrichi coli, stain WPU uvrA (pKM101) were wxposed to the test substance vapour diluted in sterile air in a vapour phase system. Sterile air was used as a vehicle control.

Four independent mutation experiments were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The experiments were modified plate incorporation assays using a vapour phase system.

Concentrations of the test item up to 70% v/v/plate were tested. This is the maximum practicable achievable concentration. A series of lower concentration were also used.

In the first experiment, toxicity (observed as thinning of the background lawn of non-revertant colonies, together with a reduction in revertant colony numbers (below an induction factor of 0.5)) was seen in all strains following exposure to the test item at 5% v/v/plate and above; except in TA98 in the absence of S9 mix and TA1535 in the absence and presence of S9 mix where toxicity was observed at 2.5% v/v/plate and above. Densely coloured plates were seen at 10% v/v and above.

In the second experiment, toxicity (observed as thinning of the background lawn of non-revertant colonies, together with a reduction in revertant colony numbers (below an induction factor of 0.5)) was seen in all strains following exposure to the test item at 5% v/v/plate. A greater than or equal to 2 fold-increase in revertant colonies was observed following exposure to the test item at 0.16, 0.31, 0.63 and 1.25 % v/v/plate in the presence of S9 mix in strain TA1535 and 0.31 % v/v/plate in the absence of S9 mix in strain TA1535 and at 0.31 %v/v/plate in the absence of S9 mix in strain TA1537.

In the third experiment, toxicity (observed as thinning of the background lawn of non-revertant colonies, and/or a reduction in revertant colony numbers (below an induction factor of 0.5)) was seen in all strains following exposure to CA3324A at 5% v/v/plate: except in strain TA1535 without S9 mix where toxicity was observed at 1.25 and 5% v/v/plate and in strain TA98 without S9 mix where toxicity occurred at 2.5% v/v/plate and above. No evidence of mutagenic activity was seen at any concentration of CA3324A. There was also no observed tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

In the fourth experiment, toxicity (observed as thinning of the background lawn of non-revertant colonies, and/or a reduction in revertant colony numbers (below an induction factor of 0.5)) was seen in all strains following exposure to the test item at 5% v/v/plate: except in strain TA1537 in the absence of S9 mix where toxicity was observed at 2.5% v/v/plate and above. No evidence of mutagenic activity was seen at any concentration of the test item. There was also no observed tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

As the results observed in the second experiment were not reproduced in the third and fourth independent experiments the overall result is that the test item is considered not to be mutagenic.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in revertant colonies, consistent with the historial positive control range for the laboratory, verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle and incubator controls were within the current historical control range for the laboratory.

It was concluded that the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed. Hence, the test item is concluded to be negative, i.e. non-mutagenic, in this assay.