Hexyl chloroformate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP / OECD guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hexyl chloroformate

EC Number:

228-036-4

EC Name:

Hexyl chloroformate

Cas Number:

6092-54-2

Molecular formula:

C7H13ClO2

IUPAC Name:

hexyl carbonochloridate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): N-hexyl chloroformate
- Physical state: liquid, colorless, clear
- Analytical purity: 97.3 area-%
- Stability under test conditions: guaranteed until 16 Oct 2015 as indicated by the sponsor
- Storage condition of test material: RT (protect against humidity, avoid temperatures >40°C)

Method

Target gene:

Salmonella typhimurium: histidine
Escherichia coli WP2 uvrA: tryptophan

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/b-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Standard plate test: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Preincubation test: 10; 33; 100; 333; 1000; and 2500 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test substance insolube in water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: With S9 mix: 2-aminoanthracene (2-AA). W/o S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 4-nitro-o-phenylenediamine (NOPD); 9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO).

Details on test system and experimental conditions:

1. Standard plate test +/- S9 mix:
METHOD OF APPLICATION: in agar (plate incorporation) according to Ames et al. 1975, Maron et al. 1983
NUMBER OF REPLICATIONS: 3 test plates /dose or control

2. Preincubation Test +/- S9 mix (because no mutagenicity was observed in the Standard plate test)
METHOD OF APPLICATION: in medium, according to Yahagi et al. 1977 und Matsushima et al. 1980
NUMBER OF REPLICATIONS: 3 test plates /dose or control

Evaluation criteria:

Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10exp9 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 2500 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No test substance precipitation was found with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
The results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.
In this study with and without S9 mix, the number of revertant colonies in the negative controls was within or marginally below the range of the historical negative control data for each tester strain.
In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test and in the preincubation assay under all test conditions from about 2500 μg/plate onward.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Without S9 Mix

			Standard Plate Test - S9 mix				Preicubation Assay - S9 mix
Strain	Test group	Dose (µg/plate)	Mean revertants per plate	Standard deviation	Factor	Reduced background growth	Mean revertants per plate	Standard deviation	Factor	Reduced background growth
TA 1535	DMSO	-	13,7	3,5	-		12,3	2,1	-
	Test item	10	-	-	-		14,7	3,1	1,2
		33	10,0	2,6	0,7		12,3	4,0	1,0
		100	11,0	1,0	0,8		16,7	8,4	1,4
		333	1,7	5,7	0,9		10,3	2,0	0,8
		1000	11,0	2,0	0,8		11,0	1,7	0,9
		2500	6,0	11,7	0,4	x	7,7	3,8	0,6	x
		5000	0,0	0,0	0,0	x	-	-	-
	MNNG	5,0	5114,7	351,0	374,2		3214,0	84,9	260,6
TA 100	DMSO	-	94,0	1,7	-		81,7	1,5	-
	Test item	10	-	-	-		80,0	9,6	1,0
		33	88,7	1,2	0,9		91,0	10,0	1,1
		100	94,0	12,3	1,0		84,3	8,1	1,0
		333	90,0	10,6	1,0		86,7	406,0	1,1
		1000	87,3	2,1	0,9		80,7	17,4	1,0
		2500	78,3	8,3	0,8	x	29,7	22,5	0,4	x
		5000	0,0	0,0	0,0	x	-	-	-
	MNNG	5,0	4110,0	379,5	43,7		2186,3	90,2	26,8
TA 1537	DMSO	-	6,7	1,2	-		8,0	3,5	-
	Test item	10	-	-	-		8,3	4,5	1,0
		33	6,7	0,6	1,0		9,7	5,5	1,2
		100	6,3	3,1	0,9		8,3	1,2	1,0
		333	8,0	2,0	1,2		10,3	5,0	1,3
		1000	8,7	3,1	1,3		6,0	2,6	0,8
		2500	7,0	4,0	1,0	x	3	1,0	0,4	x
		5000	0,0	0,0	0,0	x	-	-	-
	AAC	100,0	943,0	193,8	141,5		473,3	46,0	59,2
TA 98	DMSO	-	14,3	1,5	-		22	3,6	-
	Test item	10	-	-	-		19,3	3,8	0,9
		33	17,3	0,6	1,2		20	3,6	0,9
		100	19,7	2,5	1,4		19,3	4,5	0,9
		333	14,0	4,4	1,0		24	3,6	1,1
		1000	13,7	1,5	1,0		20	1,0	0,9
		2500	12,7	4,5	0,9	x	5	4,0	0,2	x
		5000	0,0	0,0	0,0	x	-	-	-
	NOPD	10,0	447,0	8,5	31,2		324	35,8	14,7
E. coli	DMSO	-	20,0	2,6	-		21,7	1,2	-
	Test item	10	-	-	-		22,7	7,1	1,0
		33	18,7	6,1	0,9		20,0	1,0	0,9
		100	22,7	2,5	1,1		21,7	6,4	1,0
		333	16,7	7,4	0,8		17,7	4,9	0,8
		1000	19,3	5,9	1,0		17,7	4,2	0,8
		2500	18,0	3,5	0,9	x	12,0	4,6	0,6	x
		5000	0,0	0,0	0,0	x	-	-	-
	4-NQO	5	1150,3	15,0	57,5		433,7	65,7	20,0

With S9 Mix

			Standard Plate Test + S9 mix				Preincubation Assay + S9 mix
Strain	Test group	Dose (µg/plate)	Mean revertants per plate	Standard deviation	Factor	Reduced background growth	Mean revertants per plate	Standard deviation	Factor	Reduced background growth
TA 1535	DMSO	-	10,0	1,0	-		16,0	2,0	-
		10	-	-	-		17,7	2,5	1,1
	Test item	33	11,0	5,0	1,1		11,3	5,1	0,7
		100	12,7	3,2	1,3		13,3	3,5	0,8
		333	9,3	3,1	0,9		10,3	3,5	0,6
		1000	8,7	5,7	0,9		13,0	3,0	0,8
		2500	13,0	5,6	1,3	x	8,3	1,5	0,5	x
		5000	0,0	0,0	0,0	x	-	-	-
	2-AA	2,5	176,3	5,5	27,6		215,7	26,4	13,5
TA 100	DMSO	-	84,0	11,8	-		87,7	2,9	-
		10	-	-	-		84,7	2,5	1,0
	Test item	33	90,3	5,7	1,1		84,7	1,5	1,0
		100	92,0	22,1	1,1		96,7	10,4	1,1
		333	78,0	21,6	0,9		88,7	6,5	1,0
		1000	87,3	4,5	1		82,0	9,5	0,9
		2500	73,3	5,9	0,9	x	14,3	16,2	0,2	x
		5000	0,0	0,0	0,0	x	-	-	-
	2-AA	2,5	1515,7	160,5	18,0		1460,7	75,5	16,7
TA 1537	DMSO	-	11,7	2,5	-		8,0	3,5	-
		10	-	-	-		9,7	1,5	1,2
	Test item	33	11,7	0,6	1,0		7,7	2,5	1,0
		100	11,7	1,5	1,0		9,3	2,1	1,2
		333	16,0	3,5	1,4		10,3	1,5	1,3
		1000	9,0	1,7	0,8		8,0	3,0	1,0
		2500	6,3	3,2	0,5	x	3,3	2,1	0,4	x
		5000	0,0	0,0	0,0	x	-	-	-
	2-AA	2,5	106,7	9,9	9,1		106,7	4,9	13,3
TA 98	DMSO	-	14,3	4,9	-		21,3	2,5	-
		10	-	-	-		19,3	2,5	0,9
	Test item	33	11,0	5,6	0,8		18,7	3,5	0,9
		100	15,7	5,5	1,1		20,7	6,5	1,0
		333	11,7	1,5	0,8		20,0	1,7	0,9
		1000	9,7	1,5	0,7		21,0	1,7	1,0
		2500	9,7	2,5	0,7	x	2,7	1,5	0,1	x
		5000	5,3	1,2	0,4	x	-	-	-
	2-AA	2,5	1192,7	328,9	83,2		1252,7	75,5	57,7
E. coli	DMSO	-	16,7	5,0	-		20,3	6,4	-
		10	-	-	-		19,3	2,9	1,0
	Test item	33	19,0	4,4	1,1		18,7	5,5	0,9
		100	15,7	4,0	0,9		21,6	5,5	1,1
		333	17,0	6,6	1,0		20,7	7,2	1,0
		1000	23,3	3,8	1,4		17,3	6,0	0,9
		2500	18,3	0,6	1,1	x	11,7	2,5	0,6	x
		5000	0,0	0,0	0,0	x	-	-	-
	2-AA	60	91,3	16,2	5,5		132,0	28,5	6,5

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The test substance N-Hexyl chloroformate was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains (S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) in a reverse mutation assay (GLP OECD471 guideline study; BASF SE, 2015).

Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats) were performed with concentration ranges of 33 μg - 5000 μg/plate (SPT) and 10 μg - 2500 μg/plate (PIT).

No precipitation of the test substance was found with and without S9 mix. A bacteriotoxic effect was observed from about 2500 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

Thus, under the experimental conditions of this study, the test substance N-Hexyl chloroformate is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.