1,2,3-trichloropropane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: - according to OECD 471 as at 1982 - lack of one of the following strains: E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102. - study only available as summary from the NTP report

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1993

Reference 2

Reference Type:

publication

Title:

Salmonella mutagenicity test results for 250 chemicals.

Author:

Haworth S, Lawlor T, Mortelmans K, Speck W, Zeiger E.

Year:

1983

Bibliographic source:

Environ Mutagen. 1983;5 Suppl 1:1-142. PMID: 6365529

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

as at 1981

Deviations:

no

Principles of method if other than guideline:

.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,2,3-trichloropropane

EC Number:

202-486-1

EC Name:

1,2,3-trichloropropane

Cas Number:

96-18-4

Molecular formula:

C3H5Cl3

IUPAC Name:

1,2,3-trichloropropane

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver)

Test concentrations with justification for top dose:

0, 1, 3, 10, 33, 100, 333 µg/plate SRI, International
0, 10, 33, 100, 333, 666, 1'000 µ g/plate Microbial Associates

Vehicle / solvent:

not reported
- Vehicle(s)/solvent(s) used: DMSO; ethanol; or acetone. Not detailed for 1,2,3-Trichloropropane. Probably DMSO was used
- Justification for choice of solvent/vehicle: none

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: all strains with rat or hamster S9: 2-Aminoanthracene (2-AA); without S9: TA98 - 4-Nitro-o-phenylenediamine (NOPD), TA100 and TA1535 - sodium azide (SA), TA1537 - 9-aminoacridine. (9-AAD) was tested on TA 1537. The

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 20 min preincubation + 48 after plating
- Expression time (cells in growth medium): 20 min
- Selection time (if incubation with a selection agent): 48 h


SELECTION AGENT (mutation assays):
minimal glucose bottom agar (lacking histidine; Vogel HJ, Bonner DM (1956): Acetylornithinase of E coil: Partial purification and some properties. J Biol Chem 218:97-106.)

NUMBER OF REPLICATIONS: each concentration and strain in triplicate, test as a whole in duclicate

DETERMINATION OF CYTOTOXICITY
- Method: test with TA 100 at 10 mg/plate, parameters: viability on complete medium (EGG) and reduced numbers of revertant colonies per plate and/or thinning or absence of the bacterial lawn

Evaluation criteria:

- first assessment: reproducible, dose-related increase, whether it be twofold over background or not.
- second assessment: statistical analysis based on Margolin 1981 (Margolin BH, Kaplan N. Zeiger E (1981): Statistical analysis of the Ames Salmonella/microsome test.)
Proc Natl Acad Sci USA 78:3779-3783.

Statistics:

see above

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: toxicity starting at 333 µg/Plate with activation and at 666 µg without activation

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: SRI: no cytotoxicity up to limit dose 333 µg/plate, which was set based on the sensitivity of TA 100; Microbiological Associates:

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: SRI: no cytotoxicity up to limit dose of 333 µg/plate, which was set based on the sensitivity of TA 100; Microbiological Associates: toxic between 666 and 1000 µg/plate, with and without activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: SRI: no cytotoxicity up to limit dose of 333 µg/plate, which was set based on the sensitivity of TA 100; Microbiological Associates: toxic between 666 and 1000 µg/plate, with and without activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no cytotoxicity up to limit dose of 333 µg/plate, which was set based on the sensitivity of TA 100

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

for details see Tables 1 and 2 below

Any other information on results incl. tables

- Table 1: Mutagenicity of 1,2,3-Trichloropropane in Salmonella typhimurium performed at SRI, International

Dose µg/plate	Revertants/plate*
	-S9	+10% hamster S9		+10% rat S9
	Trial 1	Trial 1	Trial 2	Trial 1	Trial 2
Strain TA 100
0	138 ± 11.8	179 ± 9.9	144 ± 4.7	158 ± 6.2	133 ± 4.3
3	145 ± 21.0	267 ± 59.4	210 ± 26.1	141 ± 17.2	130 ± 1.9
10	139 ± 5.6	458 ± 23.9	339 ± 18.6	180 ± 5.3	140 ± 6.5
33	142 ± 14.6	492 ± 75.5	690 ± 24.3	211 ± 16.9	166 ± 9.4
100	135 ± 22.0	816 ± 121.4	1,210 ± 44.4	344 ± 9.8	282 ± 12.8
333	140 ± 7.0	1,005 ± 30.9	1,862 ± 50.8	652 ± 28.6	461 ± 37.9
Trial summary	Negative	Positive	Positive	Positive	Positive
Positive control	352 ± 12.7	2,409 ± 23.4	1,121 ± 67.6	1,079 ± 36.4	688 ± 12.7
Strain TA1535
0	12 ± 4.1	13 ± 0.0	10 ± 2.6	9 ± 2.7	5 ± 1.0
1			41 ± 6.1
3	7 ± 0.9	47 ± 4.4	71 ± 10.0	10 ± 2.6	8 ± 0.9
10	9 ± 1.5	98 ± 18.2	128 ± 20.5	11 ± 3.1	7 ± 1.2
33	7 ± 1.5	209 ± 31.7	266 ± 46.1	31 ± 2.6	21 ± 4.8
100	13 ± 0.6	422 ± 34.6	481 ± 44.6	73 ± 3.5	45 ± 7.9
333	9 ± 0.3	734 ± 109.3		205 ± 7.0	80 ± 7.2
Trial summary	Negative	Positive	Positive	Positive	Positive
Positive control	294 ± 30.5	514 ± 7.3	179 ± 5.7	225 ± 18.5	103 ± 14.3
Strain TA1537
0	5 ± 2.2	6 ± 0.6		6 ± 2.1
3	4 ± 0.9	7 ± 1.3		4 ± 0.9
10	4 ± 0.7	8 ± 0.3		4 ± 0.6
33	5 ± 1.8	8 ± 0.0		5 ± 1.0
100	6 ± 1.3	12 ± 2.4		6 ± 0.9
333	5 ± 1.3	7 ± 3.2		10 ± 2.2
Trial summary	Negative	Negative		Negative
Positive control	330 ± 31.5	657 ± 18.8		269 ± 5.2
Strain TA98
0	19 ± 1.5	26 ± 5.3	54 ± 2.2	26 ± 6.1
1			50 ± 5.8
3	15 ± 3.0	25 ± 0.7	65 ± 2.0	23 ± 2.7
33	18 ± 0.7	58 ± 3.8	70 ± 2.7	22 ± 1.3
100	21 ± 1.7	86 ± 12.4	100 ± 19.8	33 ± 2.6
333	16 ± 1.9	97 ± 19.9		38 ± 0.3
Trial summary	Negative	Positive	Positive	Negative
Positive control	793 ± 43.1	1,884 ± 71.5	395 ± 3.6	697 ± 40.5

*: Revertants are presented as mean ± standard error from three plates.

-Table 2: Mutagenicity of 1,2,3-Trichloropropane in Salmonella typhimurium performed at Microbiological Associate

	Revertants/plate *
Dose µg/plate	-S9		+10% hamster S9			+10% rat S9
	Trial 1	Trial 2	Trial 1	Trial 2	Trial 3	Trial 1	Trial 2
0	78 ± 6.5	106 ± 4.7	241 ± 21.1	81 ± 1.9	89 ± 3.8	93 ± 2.3	219 ± 1.0
10	88 ± 1.2		527 ± 14.5	762 ± 29.7	728 ± 32.7	176 ± 3.0
33	94 ± 2.5	121 ± 2.5	1,008 ± 18.8	1,263 ± 20.0	1,122 ± 29.0	349 ± 10.2	380 ± 7.8
100	86 ± 7.1	106 ± 4.2	1,628 ± 57.7	2,612 ± 269.1	2,728 ± 44.2	748 ± 27.5	700 ± 53.8
333	87 ± 3.8	108 ± 2.7	2,292 ± 136.9	2,879 ± 87.3^$	3,235 ± 210.9	1,518 ± 32.7	1,242 ± 54.3
666	115 ± 4.0		Toxic	Toxic	148 ± 18.6^$	1,924 ± 55.3
667		121 ± 4.5^$					1,786 ± 24.2^$
1,000		Toxic					Toxic
Trial summary	Equivocal	Negative	Positive	Positive	Positive	Positive	Positive
Positive control	446 ± 27.0	410 ± 7.2	524 ± 17.9	355 ± 12.7	2,400 ± 65.0	509 ± 17.4	915 ± 26.9
TA1535
0	19 ± 2.4	21 ± 1.7	4 ± 0.6	8 ± 1.9		22 ± 2.3	47 ± 4.8
10	14 ± 0.9		178 ± 6.7	159 ± 16.5		33 ± 1.9
33	17 ± 1.9	29 ± 5.5	364 ± 12.3	325 ± 5.9		107 ± 2.0	94 ± 4.7
100	19 ± 3.2	24 ± 2.8	786 ± 32.8	720 ± 33.5		203 ± 7.9	203 ± 11.5
333	20 ± 3.8	31 ± 2.3	1,286 ± 22.0^$	1,340 ± 29.7		456 ± 22.6	415 ± 4.2
666	22 ± 2.2		Toxic	Toxic		549 ± 38.7
667		23 ± 1.2^$					544 ± 37.9^$
1,000		12 ± 0.5^$					147 ± 20.4^$
Trial summary	Negative	Negative	Positive	Positive		Positive	Positive
Positive control	280 ± 18.0	330 ± 18.8	59 ± 4.2	256 ± 8.7		239 ± 15.2	254 ± 11.9
TA97
0	74 ± 2.8	142 ± 4.4	108 ± 6.0	137 ± 3.0		111 ± 5.8	183 ± 20.5
10	84 ± 4.2		211 ± 6.4	194 ± 6.5		133 ± 9.1
33	64 ± 8.5	177 ± 2.8	365 ± 5.0	319 ± 20.3		162 ± 8.4	233 ± 5.8
100	78 ± 3.5	131 ± 12.2	779 ± 20.1	691 ± 24.7		219 ± 12.6	270 ± 7.2
333	93 ± 2.5	160 ± 17.0	1,422 ± 50.3	358 ± 54.5^$		408 ± 34.4	391 ± 7.3
666	75 ± 2.6		270 ± 11.3^$	Toxic		489 ± 5.0
667		99 ± 3.8^$					520 ± 15.2
1,000		97 ± 2.0^$					518 ± 16.1
Trial summary	Negative	Negative	Positive	Positive		Positive	Positive
Positive control	105 ± 5.2	345 ± 10.0	521 ± 4.5	532 ± 10.6		1,411 ± 29.8	1,307 ± 28.3
TA98
0	18 ± 4.6	22 ± 2.4	38 ± 1.2	59 ± 4.9		36 ± 6.2	38 ± 1.3
10	19 ± 2.6		35 ± 0.3	59 ± 1.5		28 ± 3.5
33	17 ± 0.6	19 ± 1.9	53 ± 9.6	77 ± 12.5		34 ± 0.9	34 ± 2.3
100	18 ± 3.8	24 ± 2.2	76 ± 5.1	82 ± 9.9		47 ± 5.2	59 ± 3.0
333	13 ± 2.4	18 ± 2.0	193 ± 7.5	191 ± 24.7		67 ± 3.2	68 ± 6.3
666	14 ± 1.8		61 ± 8.7^$			89 ± 10.9
667		22 ± 0.7		181 ± 8.7			91 ± 1.2
1,000		Toxic					43 ± 3.1^$
Trial summary	Negative	Negative	Positive	Positive		Positive	Positive
Positive control	189 ± 10.7	219 ± 11.5	2,226 ± 101.1	151 ± 11.0		263 ± 11.6	229 ± 11.3

*: Revertants are presented as mean ± standard error from three plates.

$: Slight toxicity

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative without metabolic activation
positive with metabolic activation

1,2,3-trichloropropane was tested in a Bacterial Reverse Mutation Assay (Ames Test) according to OECD Guideline 471 as at 1982, using strains S. typhimurium TA 1535, TA 1537, TA 97, TA 98 and TA 100 in the preincubation assay with and without activation (S9 of hamster and rat) in two different Laboratories.
Test material was mutagenic in S. typhimurium TA 97, TA 98, TA100 and TA1535 with activation at levels >= 1 - 10 µg/plate. It was not mutagenic in any tester strain without S-9 activation, or in TA1537 with S-9. With hamster S9 the sensitivity was usually a bit higher.

Executive summary:

In the present study 1,2,3-trichloropropane at a purity of 99.1 % was tested in a Bacterial Reverse Mutation Assay (Ames Test) according to OECD Guideline 471 as at 1982, using strains S. typhimurium TA 1535, TA 1537, TA 97, TA 98 and TA 100 in the preincubation assay with and without activation (S9 of hamster and rat) in two different Laboratories (Microbial Genetics Department, SRI International 333 Ravenswood Avenue Menlo Park, CA 94025-3493 and Microbiological Associates, 37428, Hills Tech Drive Farmington Hills MI 48331).

Test concentrations were 0, 1, 3, 10, 33, 100, 333 µg/plate (SRI, International) and 0, 10, 33, 100, 333, 666, 1'000 µ g/plate (Microbial Associates). The preincubation assay was conducted using tightly capped incubation vials to account for the volatility of the substance.

The test material was mutagenic in S. typhimurium TA 97, TA 98, TA100 and TA1535 with activation at levels >= 1 - 10 µg/plate. It was not mutagenic in any tester strain without S-9 activation, or in TA1537 with S-9. With hamster S9 the sensitivity was usually a bit higher. Cytotoxicity occured usually at 666 µg/plate.

Based on the obtained results the test material is mutagenic in S. typhimurium TA 98, TA100 and TA1535 with activation at levels >= 1 - 10 µg/plate. It was not mutagenic in any tester strain without S-9 activation, or in TA1537 and TA1538 with S-9. Cytotoxicity to TA 1535 and TA 100 was determined at >= 666 µg/plate.