Isobutylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A reverse mutation tests on bacteria (on S. typhimurium and on E. coli) is available on Isobutylamine (Envigo 2018). The study was realised according to the OECD 471 guideline and in compliance with GLP, using the modified plate incorporation method using a vapour phase system in S. typhimurium TA98, TA 1535, TA1537, TA100 strains, at concentration up to 70% v/v/plate. No evidence of mutagenicity was observed with and without metabolic activation.

One in vitro gene mutation test on mammalian cells (CHO/HGPRT) is available (Envigo 2018). Isobutylamine was tested in the CHO/HGPRT Mutation Assay in the absence and presence of metabolic activation, according to the OECD 476 guideline and in compliance with GLP and give homogeneous negative results.

One in vitro mammalian cell micronucleus test was also performed with Isobutylamine (Envigo 2018). The study was conducted according to the OECD 487 guideline and in compliance with GLP. No clastogenic and aneugenic effects were noted at the dose tested and the substance was considered to be non-mutagenic.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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.

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 / 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

Untreated negative controls:

yes

Remarks:

sterile air

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).

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9 mix

Positive controls:

yes

Positive control substance:

other: 4-Nitroquinoline-1-oxide

Remarks:

Without S9 mix

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

without S9 mix

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

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

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

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

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

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.

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.

 

 

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 November 2017 to 30 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

This test system is based on detection and quantitation of forward mutation at the functionally hemizygous hypoxanthine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO-K1) cells.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

mammalian cell line

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: European Collection of Cell Cultures
- Suitability of cells: Spontaneous mutants were eliminated from the cultures by 3-day incubation in the presence of methotrexate (0.3 μg/mL), thymidine (4 μg/mL), hypoxanthine (15 μg/mL) two days prior to storage at -196 to -150°C, in heat-inactivated foetal calf serum (HiFCS) containing 10% dimethyl sulphoxide (DMSO). The cells are screened periodically for the absence of mycoplasma contamination.
- Cell doubling time: ca. 12 hours
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: 20

MEDIA USED
H0. Ham’s Nutrient Mixture F12, supplemented with 1 mM L glutamine and 50 ng/mL amphotericin B / 20 IU/mL penicillin / 20 μg/mL streptomycin. H10. H0 medium supplemented with 10% heat inactivated fetal calf serum H10 medium was used for cell culture unless otherwise specified. The selective medium, in which only HPRT deficient cells will grow, consists of H10 supplemented with 6-TG at a final concentration of 10 mg/mL. All cell cultures were maintained at 34 to 39°C in an atmosphere of 5% CO2 in air.

Metabolic activation:

with and without

Metabolic activation system:

S9 from phenobarbitol/β-naphtoflavone induced rat liver

Test concentrations with justification for top dose:

Preliminary toxicity experiment: 70, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.156% v/v
Additional preliminary toxicity experiment (3 hour -S9 mix): 1.25, 0.75, 0.5, 0.125, 0.0625, 0.031% v/v
Additional preliminary toxicity experiment (3 hour +S9 mix): 1.25, 0.75, 0.5, 0.125, 0.031% v/v
Main experiment (3-hour treatment -S9 mix): 0.7, 0.6, 0.5, 0.4, 0.25, 0.1, 0.01% v/v
Main experiment (3-hour treatment +S9 mix): 0.8, 0.7, 0.6, 0.5, 0.4, 0.25, 0.1, 0.01% v/v

Vehicle / solvent:

None

Untreated negative controls:

yes

Remarks:

sterile air

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

250 μg/mL in DMSO in absence of S9 mix

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

5 μg/mL in DMSO in presence of S9 mix

Untreated negative controls:

other:

Remarks:

incubator controls

Remarks:

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

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Three flasks per culture were seeded with 200 cells each

DURATION
- Preincubation period: 20 hours at between 34 and 39°C, in an atmosphere of 5% CO2 in air, prior to exposure to the test item on Day 1.
- Exposure duration: 7 days

STAIN: methanol:Giemsa solution (4:1 v/v)

NUMBER OF REPLICATIONS: 2

Rationale for test conditions:

This test system is based on detection and quantitation of forward mutation at the functionally hemizygous hypoxanthine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO-K1) cells.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
a) none of the test concentrations exhibits a statistically significant increase in mean mutant frequency compared with the concurrent vehicle control
b) there is no concentration-related increase in mean mutant frequency when evaluated with an appropriate trend test
c) all results (mean mutant frequency) are inside the distribution of the historical vehicle control data (below the 95% control limit).
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
There is no requirement for verification of a clearly positive or negative response.
In cases where the response is neither clearly negative nor clearly positive as descibed above or in order to assist in establishing the biological relevance of a result, the data is evaluated by expert judgement and/or further investigations.

Statistics:

The computer system used to acquire and quantify data was SAS. The statistical significance of the data was analysed by weighted analysis of variance, weighting assuming a Poisson distribution following the methods described by Arlett et al. (1989). Tests were conducted for a linear concentration-response relationship of the test item, for non-linearity and for the comparison of positive control and treated groups to vehicle control.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

mammalian cell line

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

mammalian cell line

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Experiment: The highest final concentration used in the preliminary toxicity in the absence of S9 mix experiment was 70 % v/v. This is the maximum practicable achievable concentration. Overt toxicity was observed at 2.5 % v/v and above, demonstrated by a low cell co
unt at the end of exposure period. The osmolality and pH of medium following 3 hour exposures at 0. 0156-70% test substance were measured by analysing samples of H10 media. For medium exposed to atmospheres of the test substance at 20% v/v and above; fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed, and at 2.5% v/v and above, fluctuations in pH of more than 1.0 unit were observed compared with the vehicle control. Prior to treatment with the test substance, cultures established concurrently to those used in this experiment were assessed to ascertain the cell density. The cell concentration was confirmed to be 1.4 x 10^6 cells/mL (i.e. 14 x 106 cells treated per concentration, 28 x 10^6 cells for the vehicle control). This cell concentration was used in the calculation of the adjusted cloning efficiency. Additional preliminary toxicity test: The test substance was dosed at concentrations up to 1.25% v/v, this was the highest concentration where fluctuation in osmolality was within 50 mOsm/kg of the vehicle control and the pH was within 1 unit of the vehicle control. No precipitate was observed by eye at the end of treatment. Exposure to the test substance for 3 hours at concentrations from 0.031 to 1.25% v/v in both the absence and presence of S9 mix resulted in RS values from 80 to 0% and from 111 to 1% respectively. Concentrations for the main test were based upon these data. The level of cytotoxicity in the preliminary and main tests demonstrates the suitability and efficacy of the vapour phase exposure system for exposing the cells to the test substance. The maximum concentrations evaluated in the main tests were limited by toxicity (RS=10-20%) as required by the guideline.

Main experiment - 3 h treatment -S9 mix: Prior to treatment, cultures established concurrently to those used in this experiment were assessed to ascertain the cell density. The cell concentration was confirmed to be 1.0 x 10^6 cells/mL (i.e. 20 x 10^6 cells treated per concentration, 40 x 10^6 cells for the vehicle control). This cell concentration was used in the calculation of the adjusted cloning efficiency. Cultures were exposed to the test substance at concentrations from 0.01 to 0.7% v/v. No precipitate was seen by eye at the end of treatment. Exposure to the test substance resulted in mean RS values from 95 to 17%. All cultures were plated out for determination of cloning efficiency and mutant frequency. No significant increases in mutant frequency were observed after exposure to the test substance. None of the test substance treated groups induced mutant frequencies above the lab oratory historical control limits. Tests for linear trend and non-linearity were applied across all treatment groups, both were statistically significant (p= 0.008 and p=0.038, respectively). However, this was a negative trend and considered to be not biologically relevant. Therefore, this experiment is concluded to be negative. EMS, the positive control, induced a significant increase in mutant frequency. Main experiment - 3 h treatment +S9 mix: Prior to treatment, cultures established concurrently to those used in this experiment were assessed to ascertain the cell density. The cell concentration was confirmed to be 1.4 x 10^6 cells/mL (i.e. 28 x 10^6 cells treated per concentration, 56 x 10^`6 cells for the vehicle control). This cell concentration was used in the calculation of the adjusted cloning efficiency. Cultures were exposed to the test substance at concentrations from 0.1 to 0.8% v/v. No precipitate was seen by eye at the end of treatment. Five concentrations were evaluated for mutant frequency and the top concentration evaluated (0.6% v/v) resulted in a mean RS of 10%. Exposure to the test substance at 0.1 to 0.6% v/v resulted in mean RS values from 87 to 10%. Cultures treated at 0.8% v/v were not analysed for RS due to toxicity during exposure to the test substance. Cultures treated at 0.7% v/v were not analysed for mutant frequency as mean RS was <10% at this concentration. No significant increases in mutant frequency were observed after exposure to the test substance. None of the test substance treated groups induced mutant frequencies above the laboratory historical control limits and tests for both a linear trend and non-linearity were applied across all treatment groups, neither of which was statistically significant. Therefore, this experiment is concluded to be negative. 3MC, the positive control, induced a significant increase in mutant frequency.

The highest final concentration used in the preliminary toxicity in the absence of S9 mix experiment was 70 % v/v. This is the maximum practicable achievable concentration. Overt toxicity was observed at 2.5 % v/v and above, demonstrated by a low cell count at the end of exposure period.

Table 2: The osmolality and pH of medium following 3 hour exposures at 0.0156-70% CA3324A were measured by analysing samples of H10 media. For medium exposed to atmospheres of CA3324A at 20% v/v and above; fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed, and at 2.5% v/v and above, fluctuations in pH of more than 1.0 unit were observed compared with the vehicle control (see below).

Culture ID	pH	Osmolality (mOsmol/kg)
Vehicle only	8.28	293
CA3324A (70 % v/v)	11.65	682
CA3324A (20 % v/v)	11.10	387
CA3324A (10 % v/v)	10.75	329
CA3324A (5 % v/v)	10.19	308
CA3324A (2.5 % v/v)	9.68	301
CA3324A (1.25 % v/v)	9.21	291
CA3324A (0.625 % v/v)	8.59	288
CA3324A (0.3125 % v/v)	8.61	293
CA3324A (0.156 % v/v)	8.44	286

Table 3: HPRT Summary Table

Test Item	Concentration (% v/v)	3-hour Treatement -S9 mix		3-hour treatement +S9 mix
		Mean RS (%)	Mean MF^a	Mean RS (%)	Mean MF^a
Incubator control	0	-	1.79	-	1.93
Sterile air	0	100	1.97	100	1.94
CA3324A	0.01	95	1.85	NT	NT
CA3324A	0.1	93	1.54	87	1.55
CA3324A	0.25	73	1.53	65	1.23
CA3324A	0.4	66	1.24	42	0.95
CA3324A	0.5	72	1.21	39	1.55
CA3324A	0.6	36	0.63	10	0.90
CA3324A	0.7	17	2.25	3	NA
CA3324A	0.8	NT	NT	NA	NA
Ethyl methanesulphonate	250 µg/mL	71	82.13***	NT	NT
3-methylcholanthrene	5 µg/mL	NT	NT	76	78.68***

a.    Mutant frequencies expressed per 10⁶  viable cells

RS: Relative Survival

MF: Mutant Frequency

NT: Not tested

NA. Not assesse

*** p<0.001; all other cultures p≥0.05

Conclusions:

It was concluded that the test substance did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay, under the experimental conditions described. Hence, the test item is concluded to be negative, i.e. non-mutagenic, in this assay.

Executive summary:

The test substance was tested for mutagenic potential in an in vitro mammalian cell mutation assay using a vapour phase system. This test system is based on detection and quantitation of forward mutation at the functionally hemizygous hypoxanthine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO-K1) cells. The results of two independent tests, one in the absence of exogenous metabolic activation (S9 mix) and one in the presence of S9 mix, are reported.

The highest final concentration used in the preliminary toxicity experiment was 70% v/v. This is the the maximum practicable achievable concentration. Overt toxicity was observed at 2.5% v/v and above, demonstrated by a low cell count at the end of exposure period.

The osmolality and pH of the test item in medium were measured by analysing samples of H10 media. For medium dosed with the test item at 20% v/v and above; fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed, and at 2.5% v/v and above, fluctuations in pH of more than 1.0 unit were observed compared with the vehicle control of sterile air.

An additional preliminary toxicity experiment was performed where the highest final concentration was 1.25% v/v, this was the highest concentration where fluctuation in osmolality was within 50 mOsm/kg of the vehicle control and the pH was within 1 unit of the vehicle control. No precipitate was observed by eye at the end of treatment. Cytotoxicity was measured as Day 1 relative survival (RS). After exposure to the test item at concentrations from 0.031 to 1.25% v/v the RS values ranged from 80 to 0% and from 111 to 1%, in the absence and presence of S9 mix respectively.

In the main mutation experiment in the absence of S9 mix, cells were exposed to concentrations from 0.01 to 0.7% v/v of the test item. No precipitate was observed by eye at the end of treatment. Mean RS values ranged from 95 to 17% relative to the vehicle control. The test item did not induce a statistically significant increase in mutant frequency. None of the the test item treated groups induced mutant frequencies above the laboratory historical control limits. No biologically relevant dose response trends were noted. Therefore, this experiment is concluded to be negative. The positive control, ethyl methanesulphonate, induced a significant increase in mutant frequency demonstrating the correct functioning of the assay.

In the main mutation experiment in the presence of S9 mix, cells were exposed to concentrations from 0.1 to 0.8% v/v of the test item. No precipitate was observed by eye at the end of treatment. Mean RS values ranged from 87 to10% relative to the vehicle control at concentrations of 0.1 to 0.6% v/v of CA3324A, higher concentrations were not continued. The test item did not induce a statistically significant increase in mutant frequency. None of the test item treated groups induced mutant frequencies above the laboratory historical control limits and tests for both a linear trend and non-linearity were applied across all treatment groups, neither of which was statistically significant. Therefore, this experiment is concluded to be negative. The positive control, 3-methylcholanthrene, induced a significant increase in mutant frequency demonstrating the correct functioning of the assay.

It was concluded that the test item did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay, under the experimental conditions described. Hence, the test item is concluded to be negative, i.e. non-mutagenic, in this assay.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 November 2017 to 19 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes:

Remarks:

Human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Blood taken from healthy adult non-smoking donors
- Suitability of cells: As lymphocytes do not normally undergo cell division, they were stimulated to do so by the addition of phytohaemagglutinin (PHA), a naturally occurring mitogen.
- Cell cycle length, doubling time or proliferation index: Average donor doubling time for this laboratory is approximately 13 hours.
- Sex, age and number of blood donors if applicable: One male and one female 18-35 years for preliminary test (all exposure conditions), secondary additional preliminary test, additional main test (presence of S9 mix), second additional main test (absence of S9 mix for 3 and 20-hour exposures) and the third additional main test (absence of S9 mix for 3 and 20-hour exposures). Two females (age not reported) for additional preliminary test, main test (all exposure conditions), third additional main test (presence of S9 mix) and fourth additional main test (absence of S9 mix for 3 and 20-hour exposures).
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood
- Methods for maintenance in cell culture if applicable: Cultures were established from the prepared (pooled blood) sample and dispensed as 5 mL aliquots (in sterile universal containers) so that each culture contained blood (0.4 mL), HML media (4.5 mL) and PHA solution (0.1 mL). Cultures used in the main tests (except for 3-hour treatment in the absence of S9 mix, main test where 5 mL culture was used) were increased to 10 mL aliquots to ensure sufficient lymphocytes were present for analysis, each culture therefore contained blood (0.8 mL), HML media (9 mL) and PHA solution (0.2 mL). All cultures were then incubated at 34 to 39°C, and the cells were re-suspended (twice daily) by gentle inversion.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640, supplemented with 10% fetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 µg/mL streptomycin and 2.0 mM L glutamine

Metabolic activation:

with and without

Metabolic activation system:

S9 from phenobarbital/β-naphtoflavone -induced rat liver

Test concentrations with justification for top dose:

Vapour phase exposure chosen as test item is in vapour phase at the nominal temperature in test system. Maximum practicable achievable concentration was 70% v/v. Osmolality and pH of test substance in medium were measured by analysing samples of HML media exposed to varying concentrations of test substance. For medium dosed with test substance at 10% v/v and above; fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed, and at 2.5% v/v and above, fluctuations in pH of more than 1.0 unit were observed compared with the vehicle control of sterile air. Therefore 1.25% v/v was the highest concentration cells were exposed to. For medium dosed with test substance in tetrahydrafuron at 1.25% v/v and above; fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed, and at 2.5% v/v and above, fluctuations in pH of more than 1.0 unit were observed compared with the vehicle control of sterile air. Therefore 1.25% v/v was the highest concentration the cells were exposed to in THF. Four concentrations of test substance were assessed for determination of induction of micronuclei. Highest concentration selected was that which caused a reduction in the cytokinesis-block proliferative index (CBPI) at end of treatment period. Following 3-hour treatment without S9 mix, reductions in CBPI equivalent to 45.5% cytostasis were obtained with test substance at 0.8% v/v. Concentrations of test substance selected for micronucleus analysis were 0.1, 0.4, 0.6 and 0.8% v/v. Following 3 hour treatment with S9 mix, reductions in CBPI equivalent to 40.0% cytostasis were obtained at 0.9% v/v test substance. Concentrations of test substance selected for micronucleus analysis were 0.05, 0.5. 0.7 and 0.9% v/v. Without S9 mix after 20 h treatment, a reduction in CBPI equivalent to 40.4% cytostasis was obtained with test substance at 0.2% v/v. Concentrations of test substance selected for micronucleus analysis were 0.001, 0.1, 0.15 and 0.2% v/v.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile air and tetrahydrafuron (THF) for the 20 hour exposure in the absence of S9 mix.

Untreated negative controls:

yes

Remarks:

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

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent vehicle controls were performed using sterile air

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent THF controls included in 20-h exposure in the absence of S9 mix, where the test item was diluted in THF. THF was added into the exposure vessels at an equivalent volume to that found in the maximum concentration tested for each test

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.3 µg/mL (3-hour treatment without S9); 0.1 µg/mL (20-hour treatment without S9)

Positive controls:

yes

Positive control substance:

other: Colchicine

Remarks:

0.06 and 0.07 µg/mL (3-hour treatment); 0.015 and 0.02 µg/mL (20-hour treatment)

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

(10 µg/mL with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Approximately 48 hours
- Exposure duration: 3 or 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B (followed by incubation for 17 hours)

STAIN (for cytogenetic assays): Acridine orange solution at 0.0125 mg/mL in purified water

NUMBER OF REPLICATIONS: 2 each treatment level and positive control cultures, 4 for vehicle and incubator controls 2 slides prepared from each culture

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cultures centrifuged at 500 g for 5 min and supernatant removed. A homogeneous cell suspension was prepared. Pre-cleaned microscope slides were prepared for each culture by aliquoting the re-suspended cells onto the slides, and allowing the slides to air-dry. One slide was prepared from each culture. The slides were then stained using an acridine orange solution at 0.0125 mg/mL in purified water. Slides were examined by fluorescence microscopy.

NUMBER OF CELLS EVALUATED: 2000 binucleate cells per concentration (4000 for vehicle controls)

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Cells were included in the analysis provided the cytoplasm remained essentially intact and any micronuclei present were separate in the cytoplasm or only just touching the main nucleus (not connected to the nucleus by a nucleoplasmic bridge). Micronuclei should lie in the same focal plane as the cell, and should possess a generally rounded shape with a clearly defined outline. The main nuclei of the binucleate cells scored for micronuclei should be of approximately equal size. The diameter of the micronucleus should be between 1/16 and 1/3 that of the main nucleus. The color of the micronuclei should be the same or lighter than the main nucleus. There should be no micronucleus-like debris in the surrounding area.

DETERMINATION OF CYTOTOXICITY
- A reduction in cell replication (cytostasis) or to cell death. Cytokinesis-block proliferative index (CBPI) values significantly less than the concurrent vehicle control values are indicative of cytostasis.

Rationale for test conditions:

The purpose of this study was to assess the potential of the test substance to induce an increase in the induction of micronuclei in cultured human lymphocytes in vitro using a vapour phase system. A vapour phase exposure was chosen 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. In this test system, human lymphocytes are cultured in vitro and are stimulated to divide by adding phytohaemagglutinin (PHA) to the culture, resulting in a high mitotic yield. Some substances do not exert a genotoxic effect until they have been metabolized by enzyme systems that are not found in cultured cells. Therefore, the cultures and test item were incubated in both the absence and presence of a supplemented rat liver fraction (S9 mix).

Evaluation criteria:

The test item is clearly positive if, in any of the experimental conditions examined: (a) At least one of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent vehicle control. (b) The increase in the frequency of micronucleated cells is dose-related when evaluated with an appropriate trend test.
(c) Any of the results are outside the distribution of the historical vehicle control data (above the upper 95% confidence limit). If all of these criteria are met, the test item was considered able to induce chromosome breaks and/or gain or loss in the test system.
Test item is negative if, in all of the experimental conditions examined: (a) None of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent vehicle control. (b) There is no concentration-related increase when evaluated with an appropriate trend test. (c) All results are inside the distribution of the historical vehicle control data (below the upper 95% confidence limit). If all of these criteria are met, the test item was considered unable to induce chromosome breaks and/or gain or loss in the test system.
In rare cases, even after further investigations, the data set will not allow a conclusion of positive or negative response and will therefore be concluded as equivocal.

Statistics:

The analysis assumed that the replicate was the experimental unit. An arcsine transformation was used to transform the data. Test substance treated groups were then compared to control using Williams' tests. Positive controls were compared to control using t tests. Trend tests have also been carried out using linear contrasts by group number. These were repeated, removing the top dose group, until there were only 3 groups. Statistical significance was declared at the 5% level for all tests.

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.8% without S9 (3h); 0.9% with S9 (3h); 0.2% without S9 (20h)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In both the absence and presence of S9 mix, following 3-hour treatment, the test substance did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared to the vehicle controls. There was no evidence of a dose concentration relationship and none of the mean micronuclei frequencies were outside of the laboratory 95% confidence limits.

In the absence of S9 mix, following 20-hour treatment, the test substance did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls. A statistically significant linear trend (p<0.05) was observed at 0.2% v/v. However, the increase in micronuclei was not considered to be biologically relevant as the micronucleus frequency was within the laboratory 95% confidence limits of the historical vehicle control data at all concentrations analysed. This treatment arm was therefore concluded to be negative overall based on expert judgement.

The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

Table 1: Summary of results (number of micronucleated cells determined in a sample of 2000 binucleate cells (4000 for vehicle)

Test item	Conc       (% v/v)	Exposure time (hours)	Metabolic activation	Cytostasis (%)	Mean no. binucleated cells containing micronuclei	p-value	trend test    p-value
Incubator control	-	3	without	-	5.3
Vehicle (sterile air)	-	3	without	0	6.3
test item	0.1	3	without	6.3	6.0	1.000
test item	0.4	3	without	11.0	6.5	1.000	0.966
test item	0.6	3	without	28.1	6.5	1.000	0.992
test item	0.8	3	without	45.5	3.5	0.255	0.269
MMC	0.3µg/mL	3	without	39.3	17.0	0.005**
COL	0.07µg/mL	3	without	13.3	15.5	0.009**
Incubator control	-	3	with	-	3.3
Vehicle (sterile air)	-	3	with	0	4.3
test item	0.05	3	with	10.0	4.0	1.000
test item	0.5	3	with	29.6	3.5	1.000	0.612
test item	0.7	3	with	33.8	4.5	1.000	0.987
test item	0.9	3	with	40.0	4.5	1.000	0.870
CPA	10 µg/mL	3	with	50.8	16.0	<0.001***
Incubator control	-	20	without	-	3.3
THF	-	20	without	-	4.0
Vehicle (sterile air)	-	20	without	0.0	5.3
test item	0.001	20	without	0.0^e	3.5	1.000
test item	0.1	20	without	25.5	4.0	1.000	0.412
test item	0.15	20	without	32.2	6.0	0.647	0.463
substance	0.2	20	without	40.4	8.5	0.062	0.021*
MMC	0.1µg/mL	20	without	22.4	16.0	<0.001***
COL	0.02µg/mL	20	without	24.6	14.5	<0.001***

*p<0.05, **p<0.01, ***p<0.001, CPA: Cyclophosphamide, MMC: Mitomycin C, COL: Colchicine

Conclusions:

It was concluded that the test item did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described. Hence, the test item is concluded to be negative in this in vitro micronucleus assay.

Executive summary:

This study was designed to assess the potential of the test item to cause an increase in the induction of micronuclei in cultured human peripheral blood lymphocytes in vitro using a vapour phase system. The study consisted of preliminary toxicity experiments and main micronucleus experiments. Human lymphocytes in whole blood culture, were exposed to the test item for 3 hours in both the absence and presence of exogenous metabolic activation (S9 mix) and for 20 hours in the absence of S9 mix. One preliminary toxicity experiment for 3 hours in both the absence and presence of S9 mix and three preliminary toxicity experiments were performed in the absence of S9 mix for 20-hour exposure. Three main experiments for the 3-hour exposure in the presence of S9 mix were performed. Four main experiments for the 3-hour exposure in the absence of S9 mix and for the 20-hour exposure in the absence of S9 mix were performed. Vehicle (sterile air), incubator, tetrahydrafuron (THF; where it had been used to dilute the test item) and positive control cultures were included in all appropriate test conditions.

 

The maximum practicable achievable concentration in this modified test system was 70% v/v. The osmolality and pH of the test item in medium were measured by analysing samples of HML media exposed to varying concentrations of test item. For medium dosed with the test item at 10% v/v and above; fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed, and at 2.5% v/v and above, fluctuations in pH of more than 1.0 unit were observed compared with the vehicle control of sterile air. Therefore 1.25% v/v was the highest concentration the cells were exposed to. For medium dosed with the test item in tetrahydrafuron at 1.25% v/v and above; fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed, and at 2.5% v/v and above, fluctuations in pH of more than 1.0 unit were observed compared with the vehicle control of sterile air. Therefore 1.25% v/v was the highest concentration the cells were exposed to in THF.

 

Four test item concentrations were assessed for determination of induction of micronuclei. The highest concentration selected was that which caused a reduction in the cytokinesis-block proliferative index (CBPI) at the end of the treatment period. Following 3-hour treatment in the absence of S9 mix, reductions in CBPI equivalent to 45.5% cytostasis were obtained with the test item at 0.8% v/v. Concentrations of the test item selected for micronucleus analysis were 0.1, 0.4, 0.6 and 0.8% v/v. Following 3‑hour treatment in the presence of S9 mix, reductions in CBPI equivalent to 40.0% cytostasis were obtained with the test item at 0.9% v/v. Concentrations of the test item selected for micronucleus analysis were 0.05, 0.5. 0.7 and 0.9% v/v. In the absence of S9 mix following 20 hour treatment, a reduction in CBPI equivalent to 40.4% cytostasis was obtained with the test item at 0.2% v/v. Concentrations of the test item selected for micronucleus analysis were 0.001, 0.1, 0.15 and 0.2% v/v.

 

In both the absence and presence of S9 mix, following 3-hour treatment, the test item did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared to the vehicle controls. There was no evidence of a dose concentration relationship and none of the mean micronuclei frequencies were outside of the laboratory 95% confidence limits.

 

In the absence of S9 mix, following 20-hour treatment, the test item did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls. A statistically significant linear trend (p<0.05) was observed at 0.2% v/v. However, the increase in micronuclei was not considered to be biologically relevant as the micronucleus frequency was within the laboratory 95% confidence limits of the historical vehicle control data at all concentrations analysed. This treatment arm was therefore concluded to be negative overall based on expert judgement.

 

The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

It was concluded that the test item did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described.  Hence, the test item is concluded to be negative in this in vitro micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test (bacterial reverse mutation in vitro): Negative

A single study conducted under GLP according to a method equivalent to guideline OECD 471 is available (Woods 2018). The study is considered to be relevant, reliable and adequate for the purposes of risk assessment, classification and labelling. No increase in the incidence of revertant colonies was elicited in strains S. typhimurium TA1535, TA1537, TA1538, TA 98, TA 100 and E. Coli strain WP2 uvrA pKM 101 with and without metabolic activation. Four independent mutation experiments were performed in the presence and absence of liver preparation (S9 mix) from rats treated with phenobarbital and 5,6 -benzoflavone. The experiments were modified to plate incorporation assays using vapour phase system. 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.

The study reported by Florin et al. (Toxicology 18, 1980) is considered to be relevant, reliable with restrictions and adequate for the purposes of risk assessment, classification and labelling. No increase in the incidence of revertants colonies was elicited in strains S. typhirmurium TA1535, TA1537, TA98 and TA100 with and without metabolic activation. The test item was reported to be non-mutagenic.

The study reported by Mortelmans et al. (Environmental Mutagenesis Volume 8, 1986) considered to be relevant, reliable with restrictions and adequate for the purposes of risk assessment, classification and labelling. No increase in the incidence of revertants colonies was elicited in strains S. Typhirmurium TA1535, TA1537, TA100, TA98 and TA97 with and without metabolic activation. The test item was reported to be non-mutagenic.

The study reported by the National Toxicology Program (1981) considered to be relevant, reliable with restrictions and adquate for the purposes of risk assessment, classification and labelling. No increase in the incidence of revertants colonies was elicited in strains S. Typhirmurium TA1535, TA1537, TA100 and TA 98 with and without metabolic activation. The test item was reported to be non-mutagenic.

In vitro HPRT Mutation assay using CHO cells: Negative

A single study (in vitro HPRT Mutation Test Using Chinese Hamster Ovary Cells) conducted according to guideline OECD 476 and under GLP is available (Woods 2018). The study is considered to be relevant, reliable and adequate for the purposes of risk assessment, classification and labelling. The test substance was tested for mutagenic potential in an in vitro mammalian cell mutation assay using a vapour phase system. The results of two independent tests, one in the absence of exogenous metabolic activation (S9 mix) and one in the presence of S9 mix demonstrated no mutagenic potential in this in vitro HPRT cell mutation assay, under the experimental conditions described. Hence, the test item is concluded to be negative, i.e. non-mutagenic, in this assay.

In vitro cytogenicity / micronucleus study in human lymphocytes Negative

A single study (in vitro micronucleus Test Using human lymphocytes) conducted according to guideline OECD 487 and under GLP is available (Woods 2018). The study is considered to be relevant, reliable and adequate for the purposes of risk assessment, classification and labelling. The test substance was tested for clastogenicity in an in vitro micronucleus assay using a vapour phase system. The results in the absence of exogenous metabolic activation (S9 mix) and in the presence of S9 mix demonstrated no increase in the induction of micronuclei in this in vitro micronucelus assay, under the experimental conditions described. Hence, the test item is concluded to be negative in this assay.

Justification for selection of genetic toxicity endpoint
No study was selected since the results in all three studies in vitro were negative.

Justification for classification or non-classification

Based on the results of the available studies, the substance does not meet the criteria for classification under Directive 2001/59/EC, Annex VI, 4.2.2.3 and under Regulation (EC) No. 1272/2008 Annex I, Part 3, 3.5.2.