1,2,3-trichloropropane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Genetic toxicity in vitro: positive, (bacterial reverse mutation assay/Ames test): S. typhimuriurn TA TA 98, TA 100 and TA 1535, study Monsanto (1982) (OECD TG 471)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: - the two highest stated test concentrations are not compatible with the approved solubility of the substance in water - non-GLP

Qualifier:

no guideline followed

Principles of method if other than guideline:

- an in vitro cytochalasin B blocked micronucleus assay was conducted using the human lymphoblastoid cell lines AHH-1 TK +/- (native CYP1A1 activity), h2E1 (native CYP1A1 activity + cDNA expressed human CYP2E1 activity), and MCL-5 (elevated native CYP 1A1 activity + cDNA expressed human CYP1A2, 2A6, 3A4 and 2E1 activity + human microsomal epoxide hydrolase activity)

GLP compliance:

no

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable, as cytogentic effects are analyzed in a micronucleus test

Species / strain / cell type:

other: human lymphoblastoid cell lines: AHH-1 TK +/-, h2E1 and MCL-5

Details on mammalian cell type (if applicable):

human lymphoblastoid cell lines AHH-1 TK +/- (native CYP1A1 activity), h2E1 (native CYP1A1 activity + cDNA expressed human CYP2E1 activity), and MCL-5 (elevated native CYP 1A1 activity + cDNA expressed human CYP1A2, 2A6, 3A4 and 2E1 activity + human microsomal epoxide hydrolase activity)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

0, 0.01, 0.1, 1.0, 2.0, an 5 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none;

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

no

- 1,2,3-Trichloropropane produced 8-fold induction of micronuclei between 0 and 5 mM in the AHH-1 and h2El cell lines. The majority of micronuclei stained negatively with the kinetochore antibody. 1,2,3-Trichloropropane produced a lower but significant induction of micronuclei in the MCL-5 cell line (4-fold). Kinetochore labelling indicated that both K+ve and K-ve micronuclei were produced thereby induction of both aneugenic and clastogenic effects can be assumed.

- 1,2,3 -Trichloropropane induced decreased genotoxic response in the MCL-5 cell line compared with AHH-1 and h2E1.

- These data indicated a direct-acting effect without the requirement for metabolic activation and in the MCL-5 cells a production of a metabolite that was less genotoxic than the parental compounds.

Conclusions:

Interpretation of results: ambiguous

Dose dependent clastogenic and aneugenic effects of ,1,2,3-Trichloropropane were seen in three human lymphoblastoid cell lines. In the cell line with the highest metabolic capacity the least effect was noticed.
The study is partially invalidated by the fact that doses exeeding the water solubility of 1,2,3-Trichloropropane were used. In addition a potential substance loss during preparation of stock solutions due to volatility cannot be precluded as the process description is unprecise.

Executive summary:

In the present study (Doherty, 1996) 3 human lymphoblastoid cell lines with different metabolic capacities were used to test 1,2,3 -trichloropropane in a cytochalasin B blocked micronucleus assay. Human lymphoblastoid cell lines AHH-1 TK +/- (native CYP1A1 activity), h2E1 (native CYP1A1 activity + cDNA expressed human CYP2E1 activity), and MCL-5 (elevated native CYP 1A1 activity + cDNA expressed human CYP1A2, 2A6, 3A4 and 2E1 activity + human microsomal epoxide hydrolase activity) were tested.

Dose dependent clastogenic and aneugenic effects were found in all cell lines. Through kinetochore labeling in addition it was shown that both aneugenic and clastogenic effects contribute to this result. Surprisingly the cell line with the highest metabolic capacity (MCL-5) showed te least cytogenetic effect. The authors conclude that the reason for this result is very likely the production of a metabolite that was less genotoxic than the parental compounds.

In addition they claim that these data indicate a direct-acting effect without the requirement for metabolic activation.

The study is partially invalidated by the fact that doses exceeding the water solubility of 1,2,3-Trichloropropane were used. In addition a potential substance loss during preparation of stock solutions due to volatility cannot be precluded as the process description is unprecise.

Therefore the study cannot be used for a classification and labelling purposes.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

- 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 SIDS report, assessment taken from this report without further analysis of the original data

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

as at 1982

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

freshly prepared S-9 preparations from Aroclor 1254-induced rat liver from male Sprague-Dawley rats.

Test concentrations with justification for top dose:

0.001 - 1.0 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: not precisely described in the SIDS report, probably DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-N-oxide: TA98, TA100 and TA1538; NaNO2: TA1535; 9-aminoacridine: TA1537

Remarks:

without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-acetylaminofluorene: TA98; benzo(a)pyrene: TA100; 2-aminoantrhacene: TA1535 and TA1537; 7-acetylaminofluorene: TA1538

Remarks:

with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
preincubation: modifications to the standard test procedure due to the volatility of the test material:
- Bacteria, test or control solution, and S-9 mix or buffer, mixed in a 1/1/5 (v/v/v) ratio in 5-ml septum vials, sealed with Teflon-lined silicone discs inside an aluminum cap on ice
- Vials incubated at 30 °C for 20 minutes and returned to the ice-bath
- Samples removed with a syringe and added to histidine-biotin top agar, maintained at 44-48 °C, vortexing, spilling of mixture onto the surface of minimal glucose agar medium (Vogel-Bonner, plates gently rotated and tilted to assure uniform distribution of the top agar)
- Inversion of plates after hardening and incubation for 48 hours at 37 °C for two days
, the colonies (revertants to histidine
prototrophy) in both test and control plates were counted.


DURATION
- Preincubation period: 20 min
- Exposure duration: 2 d + 20 min
- Selection time (if incubation with a selection agent): 2 d


SELECTION AGENT (mutation assays): histidine free Vogel Bonner medium


DETERMINATION OF CYTOTOXICITY
- Method: not reported in SIDS report summary

Evaluation criteria:

not reported in SIDS report summary

Statistics:

Performed on assay results after transforming revertant/plate values as log base 10 (revertants/plate). Analysis included Bartlett’s test for homogeneity of variance and comparison of treatments with controls using within-levels pooled variance and a one-sided t-test.
Grubb’s test was performed to determine if outliers were present. Dose response was evaluated with regression analysis for log base 10 transformed doses and revertants/plate. Significant of doseresponse was evaluated by a t-test.

Species / strain:

other: TA 98, TA 100 and TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at 0.02 mg/plate

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at > 1 mg/plate (tested with TA 100 and TA1535)

Vehicle controls validity:

other: not reported in SIDS report summary, but expected to be valid

Untreated negative controls validity:

not examined

Positive controls validity:

other: not reported in SIDS report summary, but expected to be valid

Additional information on results:

Preliminary Cytotoxicity assay:
- Results presented indicate 1,2,3-trichloropropane was toxic at 1 mg/plate in the presence or absence of S-9.
Mutation assay:
- Test compound induced a significant increase in the number of revertant colonies over that shown in the solvent control plates for strains TA100 and TA1535 with activation at 0.02 mg/plate.
- There was an ambiguous result obtained with TA98 in the presence of S-9 that was confirmed as weakly positive in a repeat assay.
- There was no positive effect towards any of the tester strains in the absence of S-9 activation, or in TA1537 and TA1538 with S-9.

Conclusions:

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 1538, TA 98 and TA 100 with and without activation.
Test material was mutagenic in S. typhimurium TA 98, TA100 and TA1535 with activation at levels >= 0.02 mg/plate. It was not mutagenic in any tester strain without S-9 activation, or in TA1537 and TA1538 with S-9.

Executive summary:

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 1538, TA 98 and TA 100 with and without activation.

The preincubation assay was conducted using tightly capped incubation vials to account for the volatility of the substance.

Based on the obtained results the test material is mutagenic in S. typhimurium TA 98, TA100 and TA1535 with activation at levels >= 0.02 mg/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 >= 1 mg/plate.

Reference 3

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

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

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

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

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

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

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

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

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

- 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

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.

Reference 4

Endpoint:

genetic toxicity in vitro, other

Remarks:

Type of genotoxicity: other: summary on gene mutation, chromosome aberration, DNA damage and/or repair and genome mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: summary of summaries on in vitro genetic toxicity testing of two regulatory dossiers

Type of assay:

other: different types of tests

Test concentrations with justification for top dose:

see table 1

Species / strain:

other: different test systems

Metabolic activation:

with and without

Genotoxicity:

other: positive and negative findings, see table 1

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

other: not applicable

Untreated negative controls validity:

other: not applicable

Positive controls validity:

other: not applicable

Additional information on results:

Different studies have been conducted including the bacterial reverse mutation assay (Ames Test), in-vitro cytogenetic assays in mammalian cell lines, a chromosome aberrations assay, UDS assays in mammalian cell lines, sister chromatid exchange assays in mammalian cells and DNA binding studies. The DNA binding studies were reported to be positive. For the other studies both positive and negative results have been found for 1,2,3-trichloropropane only with activation. Instead the two metabolites 1,3-dichloro-2-propanol and 1,3-dichloroacetone were positive in bacterial mutation assays as well as in sister chromatid exchange assays (in V79 cells) with and without activation.

Remarks on result:

other: other: a variety of test systems

Remarks:

Migrated from field 'Test system'.

Table 1: Available results ofin vitrogenotoxicity tests for 1,2,3-trichloropropane as published in IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Volume 63, Dry Cleaning, Some Chlorinated Solvents and Other Industrial Chemicals (1995

Test system	Result^a		Dose^b	Reference
	Without exogenous metabolic system	With exogenous metabolic system	(LED/HID)
1,2,3-trichloropropane
SOS chromotest, Escherichia coli PQ37	-	-	0.00	von der Hude et al. (1988)
Salmonella typhimurium TA100, reverse mutation	-	+	5.5 µg/mL	Stolzenberg & Hine (1980)
Salmonella typhimurium TA100, reverse mutation	-	+	4 µg/mL	Haworth et al. (1983)
Salmonella typhimurium TA100, reverse mutation	-	+	2 µg/mL	Ratplan & Plaumann (1988)
Salmonella typhimurium TA100, reverse mutation	-	+	4.0 µg/mL	US National Toxicology Program (1993)
Salmonella typhimurium TA100, reverse mutation	-	(+)	7 µg/mL	Lag et al. (1994)
Salmonella typhimurium TA1535, reverse mutation	-	+	0.4 µg/mL	Haworth et al. (1983)
Salmonella typhimurium TA1535, reverse mutation	-	+	2 µg/mL	Ratplan & Plaumann (1988)
Salmonella typhimurium TA1535, reverse mutation	-	+	4.0 µg/mL	US National Toxicology Program (1993)
Salmonella typhimurium TA1537, reverse mutation	-		128 µg/mL	Haworth et al. (1983)
Salmonella typhimurium TA1537, reverse mutation	-	-	37.0 µg/mL	Ratplan & Plaumann (1988)
Salmonella typhimurium TA1538, reverse mutation	-	-	37.0 µg/mL	Ratplan & Plaumann (1988)
Salmonella typhimurium TA98, reverse mutation	-	+	39 µg/mL	Haworth et al. (1983)
Salmonella typhimurium TA98, reverse mutation	-	-	37.0 µg/mL	Ratplan & Plaumann (1988)
Salmonella typhimurium TA98, reverse mutation	-	+	40.0 µg/mL	US National Toxicology Program (1993)
Salmonella typhimurium TA97, reverse mutation	-	+	4 µg/mL	US National Toxicology Program (1993)
Salmonella. typhimurium TA 1535	-	+	0.02 mg/plate	Monsanto (1982)
Salmonella. typhimurium TA 1537	-	-	0.00	Monsanto (1982)
Salmonella. typhimurium TA 15 38	-	-	0.00	Monsanto (1982)
Salmonella. typhimurium TA 98	-	+	20 µg/plate	Monsanto (1982)
Salmonella. typhimurium TA 100	-	+	20 µg/plate	Monsanto (1982)
Salmonella. typhimurium TA 1535	+	+	20 µg/plate	Dean & Brooks (1979)
Salmonella. typhimurium TA 1537	-	+	20 µg/plate	Dean & Brooks (1979)
Salmonella. typhimurium TA 15 38	-	+	20 µg/plate	Dean & Brooks (1979)
Salmonella. typhimurium TA 98	-	+	20 µg/plate	Dean & Brooks (1979)
Salmonella. typhimurium TA 100	-	+	20 µg/plate	Dean & Brooks (1979)
In vitroMammalian Cell Gene Mutation Test, Mouse lymphoma L5178Y heterozygous TK+/- cells	-	+	2.4 µg/mL	Sawin & Hass (1982)
In vitroChromosome aberration assay, in an epithelial-type cell line (designated as RL 1, derived from rat liver (Carworth Farm E rat))	-	0	1000 µg/plate	Dean & Brooks (1979)
Unscheduled DNA synthesis, Fischer 344 rat primary hepatocytes in vitro	-		10.0	Williams et al. (1989)
Gene mutation, mouse lymphoma L5178Y cells, tk locus in vitro	-	+	14-	US National Toxicology Program (1993)
Sister chromatid exchange, Chinese hamster lung V79 cells in vitro	-	+	44	von der Hude et al. (1987)
Sister chromatid exchange, Chinese hamster ovary (CHO) cells in vitro	-	+	14	US National Toxicology Program11993)
Chromosomal aberrations, Chinese hamster ovary (CHO) cells in vitro	-	+	60	US National Toxicology Program (1993)
Cell transformation, SA7/Syrian hamster embryo cells in vitro	+	0	0.00	Hatch et al. (1983) (Abstract)
human lymphoblastoid cell lines AHH-1 TK +/- (native CYP1A1 activity),	+	0	0.10 mM	Doherty et. al (1996).
h2E1 (native CYP1A1 activity + cDNA expressed human CYP2E1 activity),?	+	0	0.10 mM	Doherty et. al (1996)
MCL-5 (elevated native CYP 1A1 activity + cDNA expressed human CYP1A2, 2A6, 3A4 and 2E1 activity + human microsomal epoxide hydrolase activity)	+	0	1.00 mM	Doherty et. al (1996)
In vitromicronucleus assay, primary human erythrocytes	-	-	8.00 mM	Tafazoli et. al. (1996)
In vitrosingle cell electrophoresis (comet assay), primary human erythrocytes	+	+^c	8.00 mM	Tafazoli et. al. (1996)
Unscheduled DNA synthesis, rat hepatocytes in vivo	-		0.00 po	Mirsalis et al. (1983) (Abstract)
DNA strand breaks, rat hepatocytes in vivo	+		30.0 ip x 1	Weber & Sipes (1990b)
Dominant lethal mutation, rats in vivo	-		80.0 po x 5	Saito-Suzuki et al. (1982)
DNA—DNA and DNA—protein cross-links, rat hepatocytes in vivo	-		300.0 ip x I	Weber & Sipes (1990b)
Covalent DNA binding, rat hepatocytes in vivo	+		30.0 ip x I	Weber & Sipes (1990a)
Metabolites of 1,2,3-trichloropropane
1,3-Dichloro-2-propanol
SOS chromotest, Escherichia coli GC4798	+	0	369.0	Hahn et al. (1991)
Salmonella typhimurium TA100, reverse mutation	+	+	582.0	Hahn et al. (1991)
Salmonella typhimurium TA1535, reverse mutation	+	+	192.0	Hahn et al. (1991)
Sister chromatid exchange, Chinese hamster lung V79 cells in vitro	+	+	147.4	von der Hude et al. (1987)
1,3-Dichloroacetone (1,3-Dichloropropanone)
SOS chromotest, Escherichia coli PQ37	(+)	+	0.7	Le Curieux et al. (1994)
Salmonella typhimurium TA100, reverse mutation	+	0	0.6	Meier et al. (1985)
Salmonella typhimurium TA 100, reverse mutation	+	+	0.8	Merrick et al. (1987)
Salmonella typhimurium TA100, reverse mutation, fluctuation test	+	+	0.03	Le Curieux et al. (1994)
Salmonella typhimurium TA1535, reverse mutation	+	+	0.8	Merrick et al. (1987)
Salmonella typhimurium TA98, reverse mutation	-	0	5.0	Meier et al. (1985)
Pleurodeles waltl, micronucleus induction in vivo	+		0.03, 12 d	Le Curieux et al. (1994)
sister chromatid exchange, Chinese hamster lung V79 cells in vitro	+	+	0.3	von der Hude et al. (1987)

 

In italics:Studies concernin vivogenotoxicity and are all available as study summaries in chapter 7.6.2 in this dossier

a: +, considered to be positive; (+), considered to be weakly positive in an inadequate study; -, considered to be negative; 0, not tested; for in-vivo tests,

no entry of a result under 'with exogenous metabolic system'

b: LED, lowest effective dose; HID, highest ineffective dose; in-vitro tests, µg/mL if not marked otherwise; in-vivo tests, mg/Kg bw;

c: accompanied by high cytotoxicity

0.00, dose not reported; ip, intraperitoneally; po, orally

Original table extracted from:

IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Volume 63, Dry Cleaning, Some Chlorinated Solvents and Other Industrial Chemicals, 1995, IARC Lyon, France, p. 223 -242; http://monographs.iarc.fr/ENG/Monographs/PDFs/index.php

Studies by Dean & Brooks (1979), Sawin & Hass (1982) and Monsanto (1982) were extracted from the “SIDS INITIAL ASSESSMENT REPORT For18 - 1,2,3-Trichloropropane, Published OECD SIDS initial assessments of HPV chemicals: http://www.oecd.org/document/63/0,3343,en_2649_34379_1897983_1_1_1_1,00.html”

Doherty was assessed from the original article but selected based on the citation in the CICADS dossier (review article or handbook; International Program on Chemical Safety (IPCS) Concise International Chemical Assessment Document 56 - 1,2,3-TRICHLOROPROPANE; Marketing and Dissemination, World Health Organization, Marketing and Dissemination, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland; http://www.inchem.org/documents/cicads/cicads/cicad56.htm)

 

Literature:

 

Dean BJ & Brooks TM

Shell Research Limited, London; Sittingbourne Research Centre, Shell Toxicology Laboratory (Tunstall);

Toxicity Studies with Fine Chemicals: In Vitro Mutation Studies with 1,2,3- Trichloropropane (1,2,3-TCP);

Study No. TLGR.79.073;

 Shell Development Company, a Division of Shell Oil Co., Houston, TX; June 1979.

 

Doherty AT, Ellard S, Parry EM, Parry JM.

An investigation into the activation and deactivation of chlorinated hydrocarbons to genotoxins in metabolically competent human cells.

Mutagenesis. 1996 May;11(3):247-74. PMID: 8671747

 

Hahn H, Eder E, Deininger C.

Genotoxicity of 1,3-dichloro-2-propanol in the SOS chromotest and in the Ames test. Elucidation of the genotoxic mechanism.

Chem Biol Interact. 1991;80(1):73-88. PMID: 1913979

 

Hatch, G., Anderson, T., Elmore, E. & Nesnow, S.

In Abstracts of the fifteenth annual meeting of the environmental mutagen society held at San Antonio, Texas March 3-6, 1983:

Status of enhancement of DNA viral trasformation for determination of mutagenic and carcinogenic potential of gaseous and volatile compounds

Environ. Mutag. 1983; 5(3)(Abstract Cd-26): 422

 

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

Salmonella mutagenicity test results for 250 chemicals.

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

 

Låg M, Omichinski JG, Dybing E, Nelson SD, Søderlund EJ.

Mutagenic activity of halogenated propanes and propenes: effect of bromine and chlorine positioning.

Chem Biol Interact. 1994 Oct;93(1):73-84. PMID: 8069951

 

Le Curieux F, Marzin D, Erb F.

Study of the genotoxic activity of five chlorinated propanones using the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test.

Mutat Res. 1994 Nov;341(1):1-15. PMID: 7523939

 

Meier JR, Ringhand HP, Coleman WE, Munch JW, Streicher RP, Kaylor WH, Schenck KM.

Identification of mutagenic compounds formed during chlorination of humic acid.

Mutat Res. 1985 Aug-Sep;157(2-3):111-22. PMID: 3160948

 

Merrick BA, Smallwood CL, Meier JR, McKean DL, Kaylor WH, Condie LW.

Chemical reactivity, cytotoxicity, and mutagenicity of chloropropanones.

Toxicol Appl Pharmacol. 1987 Oct;91(1):46-54. PMID: 3313810

 

Environmental Health Laboratory, Monsanto Company; Ames/Salmonella Mutagenicity Assays of 1,2,3-trichloropropane, 1,2,2,3-Tetrachloropropane, and 1,1,2,2,3- Pentachloropropane;

MSL-2449; Project ML-81-347/810157;

Monsanto Company; August 19, 1982.

Published in: SIDS INITIAL ASSESSMENT REPORT For SIAM 18 - 1,2,3-Trichloropropane; Published OECD SIDS initial assessments of HPV chemicals: http://www.oecd.org/document/63/0,3343,en_2649_34379_1897983_1_1_1_1,00.html

 

Ratpan F, Plaumann H.

Mutagenicity of halogenated propanes and their methylated derivatives.

Environ Mol Mutagen. 1988;12(2):253-9. PMID: 3044786

 

Mutagenicity of 2- and 3-carbon halogenated compounds in the Salmonella/mammalian-microsome test.

Stolzenberg SJ, Hine CH.

Environ Mutagen. 1980;2(1):59-66. PMID: 7035158

 

Sawin VL & Hass BS

Shell Development Company, Westhollow Research Center, Houston, TX;

Assay of 1,2,3- Trichloropropane (1,2,3-TCP) for Gene Mutation in Mouse Lymphoma Cells;

Study No. 85959; Regulatory Information Record No. WRC-RIR-197;

Shell Development Company, a Division of Shell Oil Co., Houston, TX; Feb. 1982.

 

Tafazoli M, Kirsch-Volders M.

In vitro mutagenicity and genotoxicity study of 1,2-dichloroethylene, 1,1,2-trichloroethane, 1,3-dichloropropane, 1,2,3-trichloropropane and 1,1,3-trichloropropene, using the micronucleus test and the alkaline single cell gel electrophoresis technique (comet assay) in human lymphocytes.

Mutat Res. 1996 Dec 20;371(3-4):185-202. PMID: 9008720

 

United States National Toxicology Program (1993) Toxicology and Carcinogenesis Studies of 1,2,3-Trichloropropane (CA No. 96-18-4) in F344/N Rats and B6C3Fl Mice (Gavage Studies)

(NTP Tech. Rep. No. 384; NIH PubL No. 94-2829), Research Triangle Park, NC

 

von der Hude W, Scheutwinkel M, Gramlich U, Fissler B, Basler A.

Genotoxicity of three-carbon compounds evaluated in the SCE test in vitro.

Environ Mutagen. 1987;9(4):401-10. PMID: 3582297

 

von der Hude W, Behm C, Gürtler R, Basler A.

Evaluation of the SOS chromotest.

Mutat Res. 1988 Apr;203(2):81-94. PMID: 3280989

 

Structure-activity relationships in the rat hepatocyte DNA-repair test for 300 chemicals.

Williams GM, Mori H, McQueen CA.

Mutat Res. 1989 Nov;221(3):263-86. PMID: 2682231

Conclusions:

Interpretation of results: positive

In 21 different tests the genetic toxicity of 1,2,3-trichloropropane and its metabolites 1,3-Dichloro-2-propanol and 1,3-Dichloroacetone was tested. For 1,2,3-trichloropropane mainly positive but also negative results depending on test system and conducting laboratory were found. Positive results occurred in most cases only with activation. The two metabolites 1,3-dichloro-2-propanol and 1,3-dichloroacetone were positive in bacterial mutation assays as well as in sister chromatid exchange assays (in V79 cells) with and without activation.
It can therefore be concluded that1,2,3-trichloropropane and ist metabolites 1,3-dichloro-2-propanol and 1,3-dichloroacetone are positive for in vitro genotoxicity.

Executive summary:

In this summary article the results from 21 different tests on the genetic toxicity of 1,2,3-trichloropropane and its metabolites 1,3-Dichloro-2-propanol and 1,3-Dichloroacetone are combined.

For 1,2,3-trichloropropane mainly positive but also negative results depending on test system and conducting laboratory were found. Positive results occurred in most cases only with activation. The two metabolites 1,3-dichloro-2-propanol and 1,3-dichloroacetone were positive in bacterial mutation assays as well as in sister chromatid exchange assays (in V79 cells) with and without activation.

It can therefore be concluded that 1,2,3-trichloropropane and its metabolites 1,3-dichloro-2-propanol and 1,3-dichloroacetone are positive for in vitro genotoxicity.

Results are extracted from:

- IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Volume 63, Dry Cleaning, Some Chlorinated Solvents and Other Industrial Chemicals, 1995, IARC Lyon, France, p. 223 -242; http://monographs.iarc.fr/ENG/Monographs/PDFs/index.php

- SIDS INITIAL ASSESSMENT REPORT For SIAM 18 - 1,2,3-Trichloropropane, Published OECD SIDS initial assessments of HPV chemicals: http://www.oecd.org/document/63/0,3343,en_2649_34379_1897983_1_1_1_1,00.html

- CICADS dossier (review article or handbook; International Program on Chemical Safety (IPCS) Concise International Chemical Assessment Document 56 - 1,2,3-TRICHLOROPROPANE; Marketing and Dissemination, World Health Organization, Marketing and Dissemination, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland; http://www.inchem.org/documents/cicads/cicads/cicad56.htm)

Different studies have been conducted with 1,2,3-trichloropropane including the bacterial reverse mutation assay (Ames Test, positive), in-vitro cytogenetic assays in mammalian cell lines (positive), a chromosome aberrations assay (negative but tested only without activation), a non-standard in vitro micronucleus assay (positive, with inherent activation), UDS assays in mammalian cell lines (negative), sister chromatid exchange assays in mammalian cells (positive, with activation) and DNA binding studies (positive). The DNA binding studies were reported to be positive. For the other studies both positive (mainly only with activation) and negative results have been found for 1,2,3-trichloropropane. The two metabolites 1,3-dichloro-2-propanol and 1,3-dichloroacetone were positive in bacterial mutation assays as well as in sister chromatid exchange assays (in V79 cells) with and without activation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

- Genetic toxicity in vivo: ambiguous, (weight of evidence approach covering all available studies)

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vivo, other

Remarks:

micronucleus, UDS

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: - secondary source - original articles either only short abstracts or hardly available

Principles of method if other than guideline:

The summaries of test results from three in vivo tests are cited from the Concise International Chemical Assessment Document 56 - 1,2,3-TRICHLOROPROPANE. These tests are unavailable and the results (2 x negative, 1 x positive) are only shortly summarized and therefore the reliability is not assignable.

GLP compliance:

not specified

Type of assay:

other: bone marrow micronucleus test, unscheduled DNA synthesis

Species:

other: rat, mouse

Duration of treatment / exposure:

not reported

No. of animals per sex per dose:

not reported

Tissues and cell types examined:

micronucleus test in mouse bone marrow
unscheduled DNA synthesis in rat hepatocytes

Genotoxicity:

ambiguous

Remarks on result:

other: no details reported

- citation from chapter 8.5.2 of the CICADS 56:

" Negative test results for a micronucleus test in mouse bone marrow (Douglas et al., 1985) and unscheduled DNA synthesis in rat hepatocytes in vivo (Mirsalis et al., 1983) mentioned in two abstracts cannot be validated because of lack of documentation (e.g., dose, test conditions).

Exposure of rats to 800 mg 1,2,3-trichloropropane/m³ for 1 week resulted in a disturbance of hepatocyte mitosis. In a comparison of hepatocytes from control and exposed rats, there was a significant shift from binuclear cells to mononuclear polyploid cells. While binuclear diploid and tetraploid cells were significantly reduced in treated animals, mononuclear tetraploid and octaploid cells showed a significant corresponding increase, and cells with a ploidy of 16n appeared (Belyaeva et al., 1974)."

Conclusions:

Interpretation of results: ambiguous

Executive summary:

In the Concise International Chemical Assessment Document 56 - 1,2,3-TRICHLOROPROPANE the results of three studies are summarized that are either only available as short abstracts or difficult to obtained and are therefore only cited here:

" Negative test results for a micronucleus test in mouse bone marrow (Douglas et al., 1985) and unscheduled DNA synthesis in rat hepatocytes in vivo (Mirsalis et al., 1983) mentioned in two abstracts cannot be validated because of lack of documentation (e.g., dose, test conditions).

Exposure of rats to 800 mg 1,2,3-trichloropropane/m³ for 1 week resulted in a disturbance of hepatocyte mitosis. In a comparison of hepatocytes from control and exposed rats, there was a significant shift from binuclear cells to mononuclear polyploid cells. While binuclear diploid and tetraploid cells were significantly reduced in treated animals, mononuclear tetraploid and octaploid cells showed a significant corresponding increase, and cells with a ploidy of 16n appeared (Belyaeva et al., 1974)."

Reference 2

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

abstract

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

DNA was isolated from paraffin-embedded tumor sections (obtained from the forestomach of mice of the NTP bioassay on 1,2,3-trichloropropane, see chapter 7.7), amplified by polymerase chain reaction, and analyzed by direct sequencing for mutations in the ras-genes

GLP compliance:

no

Type of assay:

other: PCR analysis of nature of H-ras and K-ras mutations in tumors

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

see chapter 7.7 "National Toxicology Program (1993) / Mouse"

Route of administration:

oral: gavage

Vehicle:

- corn oil
- see chapter 7.7 "National Toxicology Program (1993) / Mouse"

Details on exposure:

see chapter 7.7 "National Toxicology Program (1993) / Mouse"

Duration of treatment / exposure:

2 years, see chapter 7.7 "National Toxicology Program (1993) / Mouse"

Frequency of treatment:

daily 5 d/wk

Post exposure period:

non

Remarks:

Doses / Concentrations:
0, 6, 20 and 60 mg/kg
Basis:
actual ingested
gavage

No. of animals per sex per dose:

60

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

paraffin-embedded tumor sections from the forestomach

Details of tissue and slide preparation:

DNA was isolated from paraffin-embedded tumor sections (obtained from the forestomach of mice of the NTP bioassay on 1,2,3-trichloropropane, see chapter 7.7), amplified by polymerase chain reaction, and analyzed by direct sequencing for mutations in the ras-genes

Additional information on results:

- 10 of 16 analysed tumors had a highly specific H-ras or K-ras mutation.
- 6 of the 10 had a H-ras mutation at codon 61 with 5 of the 6 showing a AT to TA transversion in base 1.
- 4 of the 10 had a K-ras mutation at codon 13 all of which showed a GC to CG transversion in base 1.
- These transversions aremost probably not caused by S-[1-(hydroxymethyl)-2-(N7- guanypethyl]glutathione which is the major DNA adduct that was found after 1,2,3-trichloropropane treatment.
- Based on preliminary results that are not further specified in the abstract, the authors propose that the found mutations are rather caused through etheno DNA adducts (1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine) which stem from lipid peroxidation.
- Their explanation is that the depletion of glutathion in cells that are exposed to 1,2,3-trichloropropane cause an increase in lipid peroxidation and thereby lead to the detrimental transversion mutations that were found in the ras-genes.

Conclusions:

Interpretation of results: positive
DNA was isolated from paraffin-embedded tumor sections (obtained from the forestomach of mice of the NTP bioassay on 1,2,3-trichloropropane, see chapter 7.7), amplified by polymerase chain reaction, and analyzed by direct sequencing for mutations in the ras-genes.
Based on these results it is confirmed that 1,2,3-trichloropropane induces mutations, but a alternative mode of action is proposed: 1,2,3-trichloropropane exposure leads to glutathion depletion in cells, lipid peroxidation increases and etheno DNA adducts are formed that lead to transversion mutations.

Executive summary:

The present abstract to a poster presentation (Ito, 1996) reports the analysis of mouse forestomach tumor tissues via PCR. DNA was isolated from paraffin-embedded tumor sections (obtained from the forestomach of mice of the NTP bioassay on 1,2,3-trichloropropane, see chapter 7.7), amplified by polymerase chain reaction, and analyzed by direct sequencing for mutations in the ras-genes.

10 of 16 analysed tumors had a highly specific H-ras or K-ras mutation. 6 of the 10 had a H-ras mutation at codon 61 with 5 of the 6 showing a AT to TA transversion in base 1. 4 of the 10 had a K-ras mutation at codon 13 all of which showed a GC to CG transversion in base 1. These transversions are most probably not caused by S-[1-(hydroxymethyl)-2-(N7- guanypethyl]glutathione which is the major DNA adduct that was found after 1,2,3-trichloropropane treatment.

Based on preliminary results that are not further specified in the abstract, the authors propose that the found mutations are rather caused through etheno DNA adducts (1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine) which stem from lipid peroxidation. The proposed explanation is that the depletion of glutathion in cells that are exposed to 1,2,3-trichloropropane causes an increase in lipid peroxidation and thereby lead to the detrimental transversion mutations that were found in the ras-genes.

Reference 3

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: - scientifically sound study - non GLP, no guideline available

Qualifier:

no guideline available

Principles of method if other than guideline:

B6C3F1 mice and F344 rats were treated once with 1,2,3-trichloropropane orally via gavage (mice: 6 and 60 mg/Kg bw, rats 3 and 30 mg/Kg bw). 6 h post treatment animals were sacrificed, several organs excised and DNA extracted from the tissues. DNA was hydrolyzed, separated by cation exchange HPLC and analyzed for DNA adducts by ESI-MS.

GLP compliance:

no

Type of assay:

other: DNA binding study

Species:

other: mouse and rat

Strain:

other: mouse: B6C3F1, rat: F344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, NC, U.S.A.)
- Age at study initiation: not reported
- Weight at study initiation: mice: 22-25 g, rats: 190-210 g,
- no further information given

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: none given, but corn oil is a common vehicle for volatile solvents
- Concentration of test material in vehicle: not reported
- Amount of vehicle (if gavage or dermal): not reported

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- not reported in detail
- Applied amounts of label: labeled TCP was dissolved in unlabeled TCP to give the following specific activities at the respective doses: mice: 2 mCi/Kg bw at the 6 mg/Kg bw dose, 4 mCi/Kg bw at the 60 mg/Kg bw dose; rats: 1 mCi/Kg bw at both the 3 and the 30 mg/Kg bw dose

Duration of treatment / exposure:

once via gavage

Frequency of treatment:

once via gavage

Post exposure period:

animals sacrificed 6 h post treatment

Remarks:

Doses / Concentrations:
mice: 6 and 60 mg/Kg bw, rats: 3 and 30 mg/Kg bw
Basis:
actual ingested

No. of animals per sex per dose:

4

Control animals:

no

Positive control(s):

I ,2-Dibromo-3-chloropropane (DBCP) (97% purity) (provider: Pfaltz and Bauer,Inc. (Waterbury, CT, U.S.A.)

Tissues and cell types examined:

mice: brain, forestomach, glandular stomach, heart, kidney, liver, lung, spleen, testes
rats: forestornach, glandular stomach, kidney, liver, palate, pancreas, preputial gland, spleen, tongue

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: same as in the NTP 2 year carcinogenicity study on 1,2,3-trichloropropane

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
animals were treated orally via gave with the respective test item concentration in corn oil
6 h post treatment animals were sacrificed, several organs exiced and DNA extracted from the tissues

DETAILS OF METHODS:
- DNA was extracted from tissues by chloroform/phenol extraction and precipitation with ethanol. Where derived amounts were to low, tissues from 4 animals were pooled
- DNA was hydrolysed by neutral thermal or by mild acid treatment and concentrated in Centricon-30 concentrators (Amicon, Beverly, MA, U.S.A. (12.Beranek,D.T., Weis,C.C. and Swenson,D.H. (1980) A comprehensive quantitative analysis of methylated and ethylated DNA using high pressure liquid chromatography. Carcinogenesis, 1, 595-606.).
Hydrolysis products were separated by HPLC cation exchange chromatography, fractioned and analysed for radioactivity by liquid scintillation countingand for guanine content by UV absorbance
- The DBCP-derived DNA adduct, S-[1-(hydroxymethyl)-2-(N 7-guanyl)ethyl]glutathion wasproduced
a) chemically (13.Humphreys,W.G., Kim,D.-H. and Guengerich,F.P. (1991) Isolation and characterization of N7-guanyl adducts derived from 1,2-dibromo-3-chloropropane. Chem. Res. Toxicol., 4, 445-453.)
b) in vivo through ip injection of 300 mg/Kg TCP in rats and isolation of hepatic DNA adducts 8 h post treatment

METHOD OF ANALYSIS:
electron spray ionization mass sprectroscopy (ESI-MS) with samples directly distributed from a HPLC system

Evaluation criteria:

not applicable

Statistics:

not applicable

Sex:

male

Genotoxicity:

positive

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- In both mice and rats one major DNA adduct was formed that was identified by mass spectroscopy spectrum to be identical to S-[1-(hydroxymethyl)-2-(N 7-guanyl)ethyl]glutathion which is also the major DNA adduct arising from treatment with 1,2-dibromo-3-chloropropane (DBCP)
- DNA adducts were found in many tissues reaching the highest values along the portal of entry and the excretion organs. But the levels for the different tissues were not in direct correlation to the tumor incidence as found in the NTP 2-year carcinogenicity study (see Tables 1 and 2)
- A clear dose response in the DNA adduct levels was clearly shown in all tissues of both species.

- Table 1: DNA adduct formation in male Fischer-344 rats exposed to TCP^a

Organ	Dose (mg/kg)	Tumor incidence^c	Adduct level (µmol/mol guanine)^d
Fore stomach^b	3	33/50	3.7
	30	43/52	14.6
Glandular stomach	3	0/50	3.8
	30	0/52	20.4
Kidney^b	3	2/50	6.6 ± 1.4
	30	21/52	38.9 ± 5.0
Liver	3	1/50	5.4 ± 0.7
	30	3/52	47.6 ± 21.0
Palate^b	3	2/50	N.D.
	30	10/52	N.D.
Pancreas^b	3	21/50	5.3 ± 1.0
	30	29/52	37.8 ± 12.8
Preputial gland^b	3	6/47	N.D.
	30	16/50	N.D.
Spleen	3	0/50	0.80 ± 0.06
	30	0/52	7.1 ± 1.8
Tongue^b	3	2/50	4.0
	30	37/52	20.4

a: Rats were administered [14C]TCP by gavage, and organs were excised 6 h later.

b: Organ with statistically significant increase in tumor incidence.

c: Organ specific incidence of tumors in male rats administered TCP by gavage for 2 years (NTP study).

d: Data are expressed as means ± standard deviations of four animals. Results of forestomach, glandular stomach, palate, preputial gland, and tongue are from pooled samples.

N.D., not detected.

- Table 2: DNA adduct formation in male B6C3F1 mice exposed to TCP^a

Organ	Dose (mg/kg)	Tumor incidence^c	Adduct level (µmol/mol guanine)^d
Brain	6	0/50	0.43 ± 0.11
	60	0/56	3.0 ± 0.2
Forestomach^b	6	50/5 I	19.8
	60	55/56	41.0
Glandular stomach	6	0/51	28.1
	60	0/56	208.1
Heart	6	0/5 I	0.38
	60	0/56	2.4
Kidney	6	0/51	4.4 ± 2.9
	60	0/56	32.5 ± 11.3
Liver^b	6	24/51	12.1 ± 4.6
	60	31/56	59.3 ± 21.7
Lung	6	11/51	0.77 ± 0.16
	60	6/56	5.3 ± 0.2
Spleen	6	0/51	0,61
	60	0/56	7.8
Testes	6	0/51	0.32 ± 0.14
	60	0/56	1.2 ± 0.6

a: Rats were administered [14C]TCP by gavage, and organs were excised 6 h later.

b: Organ with statistically significant increase in tumor incidence.

c: Organ specific incidence of tumors in male rats administered TCP by gavage for 2 years (NTP study).

d: Data are expressed as means ± standard deviations of four animals. Results of forestomach, glandular stomach, heart, and spleen are from pooled samples.

N.D., not detected.

Conclusions:

Interpretation of results: positive
Mice and rats were treated once orally via gavage with 3 and 30 and 6 and 60 mg/Kg bw of 1,2,3-trichloropropane respectively to analyze the ability of metabolites of the test item to bind to DNA. The ability of metabolites to bind to DNA of various tissues dose dependently was clearly shown in both species and the main DNA adduct was identified to by S-[1-(hydroxymethyl)-2-(N 7-guanyl)ethyl]glutathion. A clear correlation between DNA adduct levels and tumor incidence in the 2 year NTP study was not found.

Executive summary:

In the present study (La 1995) B6C3F1 mice and F344 rats were treated once orally via gavage with 3 and 30 and 6 and 60 mg/Kg bw of 1,2,3-trichloropropane respectively to analyze the ability of metabolites of the test item to bind to DNA. The ability of metabolites to bind to DNA of various tissues dose dependently was clearly shown in both species and the main DNA adduct was identified to be S-[1-(hydroxymethyl)-2-(N 7-guanyl)ethyl]glutathion. A clear correlation between DNA adduct levels and tumor incidence in the 2 year NTP study was not found.

So 1,2,3 -trichloropropane is positive for the alteration of DNA by its metabolites. Nevertheless this alone is not sufficient to indicate genotoxicity and hence not sufficient for classification purposes.

Mice and rats were treated once with 1,2,3-trichloropropane orally via gavage (mice: 6 and 60 mgmg/Kg bw, rats 3 and 30 mg/Kg bw). 6 h post treatment animals were sacrificed, several organs excised and DNA extracted from the tissues. DNA was hydrolyzed, separated by cation exchange HPLC and analyzed for DNA adducts by ESI-MS.

Reference 4

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: - scientifically sound study - overview article with limited information on animal husbandry and individual data

Qualifier:

no guideline followed

Principles of method if other than guideline:

1,2,3-trichloropropane was injected intraperitoneal once in Male Wistar rats at 1000 to 3000 µmol/Kg bw (147 - 442 mg/Kg bw) in DMSO (0.5 mL/250 g bw) to test for nephrotoxicity via histology and blood parameters 48 h after treatment.
In addition the DNA damage in kidney cells was tested via alkaline elution 60 min after ip administration of 375 - 3000 µmol/LKg bw (55 - 442 mg/Kg bw).

GLP compliance:

not specified

Type of assay:

other: alkaline elution

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: not reported
- Weight at study initiation: 200-250 g
- Diet (e.g. ad libitum): pelleted feed from Special Diet Services, England, ad libitum
- Water: ad libitum

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no
- Concentration of test material in vehicle: 187.5 - 1500 mg/mL
- Amount of vehicle: 2 mL/Kg bw

Details on exposure:

no data

Duration of treatment / exposure:

single application

Frequency of treatment:

once

Post exposure period:

- not applicable
- renal toxicity experiment: sacrifice 48 h post treatment
- DNA damage experiment: sacrifice 60 min post treatment

Remarks:

Doses / Concentrations:
1000, 2000, 3000 µmol/Kg bw (147, 294, 442 mg/Kg bw)
Basis:
other: actual injected in the renal toxicity experiment

Remarks:

Doses / Concentrations:
375, 750, 1500, 3000 µmol/LKg bw (55, 110, 221, 442 mg/Kg bw).
Basis:
other: actual injected in the DNA damage experiment

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

no

Tissues and cell types examined:

- kidneys

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: not reported

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- renal toxicology experiment: ip injection of test item in DMSO, sacrifice 48 h post treatment by exsanguination under anesthesia (combination of fentanyl, fluanisone, and midazolam ip; 0.02, 1.0, and 0.5 mg/100 g, respectively); blood was collected for determination of creatinine and urea levels
- DNA damage experiment: ip injection of test item in DMSO, sacrifice 60 min post treatment by exsanguination under anesthesia (combination of fentanyl, fluanisone, and midazolam ip; 0.02, 1.0, and 0.5 mg/100 g, respectively)

DETAILS OF Methods:
- histology experiment: kidneys were weighted, fixed in buffered formalin, sectioned, stained (hematoxylin, eosin) and analysed blinded by a pathologist (grading of lesion from 0 - 4).
- DNA damage experiment: kidneys excised, buffered in Merchants's solution, minced and nuclei recovered by squeezing the tissue through a stainless steel screen and filtration; filtrated nuclei eluted through polycarbonate filters with 20 mM EDTA, pH = 12.5, DNA content in 2 h fractions determined fluorimetrically with Hoechst 33258 dye(Brunborg, G., Holme, J. A., Soderlund, E. J., Omichinski, J. G., and Dybing, E. (1988) An automated alkaline elution system. DNA damage induced by 1,2-dibromo-3-chloropropane in vivo and in vitro. Anal. Biochem. 174, 522-536.); elution rates expressed as normalized area above curve (NAAC).


METHOD OF ANALYSIS:

OTHER:

Evaluation criteria:

not applicable

Statistics:

not used

Sex:

male

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

- renal toxicity experiment: grade 2 kidney necrosis was only seen in one animal of the 3000 µmol/Kg (442 mg/Kg) group, a dosis at which several of the animals died within 48 h (See Table 1). kidney body weight and creatinine and urea levels were slightly raised in the sole animal that was analysable in this group. In the 1000 and 2000 µmol/Kg (147 and 294 mg/Kg) groups these parameters were marginally raised and no adverse effect was found in the histology (1 fatality in the 2000 µmol/Kg group). Therefore 1,2,3-trichloropropane has very low potency as a nephrotoxicant after single administration.
- DNA damage experiment: 1,2,3-trichloropropane leads to DNA damage but only at doses higher than 375 µmol/Kg bw (= 55 mg/Kg bw) in a dose dependent manner (information presented only graphically).

Table 1: kidney toxicity induced by 1,2,3-trichlorpropane^a

treatment	dose, µmol/kg	kidney/body wt x 10^2	creatinine, µmol/L	urea, mmol/L	kidney necrosis
					0	1+	2+	3+	4+	mean grade^b± SD
control, DMSO		0.74 ± 0.06	61.3 f 14.1	6.5 ± 0.7	5	0	0	0	0	0.01 0.0
1,2,3-trichloropropane (1,2,3-triCP)	1000	0.81 ± 0.06	67.8 ± 4.5	5.4 ± 1.5	5	0	0	0	0	0.0 ± 0.0
	2000	0.92 ± 0.16	63.3 ± 2.6	9.3 ± 4.5	4	0	0	0	0	0.0 + 0.0^c
	3000	1.04	79.5	15.9	1	0	1	0	0	1.0^c

a: Five animals in each treatment group were dosed ip with one of the halogenated propanes. All animals were killed 48 h after administration

of the test compound.

b: Calculated by adding together the necrosis grade for each animal divided by the total number of animals.

c: Deaths were noted in this treatment group.

Conclusions:

Interpretation of results: positive
Wistar rats were ip injected with 1,2,3-trichloropropane and the inflicted DNA damage in kidney cells was assessed 60 min later. In addition 48 h after administration the renal toxicity was assessed by blood creatinine and blood urea levels and histologic analysis of renal tissues. 1,2,3-trichloropropane has very low potency as a nephrotoxicant after single administration of concentrations (294 - 442 mg/Kg bw) that already cause mortality.
In the same dose range (110 -442 mg/Kg bw) it was shown by alkaline elution that 1,2,3-trichloropropane causes DNA damage dose dependently. Based on this results 1,2,3-trichloropropane is genotoxic in vivo at high concentrations and needs to be classified accordingly under CLP.

Executive summary:

In the present study (Lag 1991) Wistar rats were ip injected with 1,2,3-trichloropropane and the inflicted DNA damage in kidney cells was assessed 60 min later. In addition 48 h after administration the renal toxicity was assessed by blood creatinine and blood urea levels and histologic analysis of renal tissues. 1,2,3-trichloropropane has very low potency as a nephrotoxicant after single administration of concentrations (294 - 442 mg/Kg bw) that already cause mortality.

At lower doses (> 55 mg/Kg bw and above) it was shown by alkaline elution that 1,2,3-trichloropropane causes DNA damage dose dependently. Based on this results 1,2,3-trichloropropane is genotoxic in vivo at high concentrations. Therefore 1,2,3 -trichloropropane is deemed to be classified as genotoxic Category 2 (Warning; H341: Suspected of causing genetic defects) according to CLP (REGULATION (EC) No 1272/2008 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL) as implementation of UN-GHS in the EU.

1,2,3-trichloropropane was injected intraperitoneal once in Male Wistar rats at 1000 to 3000 µmol/Kg bw (147 - 442 mg/Kg bw) in DMSO (0.5 mL/250 g bw) to test for nephrotoxicity via histology and blood parameters 48 h after treatment. In addition the DNA damage in in kidney cells was tested via alkaline elution 60 min after ip administration of 375 - 3000 µmol/LKg bw (55 - 442 mg/Kg bw).

In the renal toxicity experiment grade 2 kidney necrosis was only seen in one animal of the 3000 µmol/Kg (442 mg/Kg) group, a dose at which several of the animals died within 48 h. Kidney body weight and creatinine and urea levels were slightly raised in the sole animal that was analysable in this group. In the 1000 and 2000 µmol/Kg (147 and 294 mg/Kg) groups these parameters were marginally raised and no adverse effect was found in the histology (1 fatality in the 2000 µmol/Kg group). It can be concluded that 1,2,3-trichloropropane has very low potency as a nephrotoxicant after single administration.

In the DNA damage experiment 1,2,3 -trichloropropane lead to DNA damage but only at doses higher than 375 µmol/Kg bw (= 55 mg/Kg bw) in a dose dependent manner (information presented only graphically).

Reference 5

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: - scientifically sound study - overview article, only limited information on methods, animal husbandry and individual data

Qualifier:

according to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

GLP compliance:

not specified

Type of assay:

rodent dominant lethal assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Inc.
- Age at study initiation: 10 weeks
- Weight at study initiation: not reported
- Assigned to test groups randomly: not reported
- Fasting period before study: no
- Housing: 1 male per 1 female per cage
- Diet (e.g. ad libitum): not reported
- Water (e.g. ad libitum): not reported
- Acclimation period: >= 7 d

ENVIRONMENTAL CONDITIONS
- not reported

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: not given, but vegetable oils are standard vehicles for volatile organic solvents
- Concentration of test material in vehicle: 66.7 mg/mL
- Amount of vehicle (if gavage or dermal): 1.2 mL/Kg
- Purity: not reported

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- 1,2,3-trichloropropane dissolved in olive oil (66.7 mg/mL)

Duration of treatment / exposure:

5 successive days

Frequency of treatment:

once per day

Post exposure period:

8 weeks

Remarks:

Doses / Concentrations:
80 mg/Kg
Basis:
actual ingested
this dose was regarded as the maximum tolerated dose and was based on the determined acute lethal dose of 500 mg/Kg bw

No. of animals per sex per dose:

15 males paired with in total 8 females per male (= 120 fermales)

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control substance: 1,2-dibromo-3chloropropane (DBCP)
- Justification for choice of positive control(s): shown earlier to induce dominant lethals particularly in the early spermatid stage
- Route of administration: gavage, analogous to the test item
- Doses / concentrations: 50 mg/Kg/d

Tissues and cell types examined:

- females were sacrificed 13 - 14 d after copulation and the female reproductive organs were analysed for corpora lutea, implants, live embryos and early and late embryonic deaths
- it remains unclear whether male reproductive organs were analysed

Details of tissue and slide preparation:

not reported in detail

Evaluation criteria:

The frequency of induced dominant lethal mutations was calculated according to the method of Röhrborn 1970 (Röhrborn, G. (1970) in: F. Vogel and G. Röhrborn (Eds.), Chemical Mutagenesis in Mammals and Man, Springer, New York, pp. 148-155):
(1-live embryos per test female/live embryos per control female) x 100

Statistics:

Fisher's exact method and Mann—Whitney U test

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

- None of the tested parameters showed a significant difference in any of the tested weeks after treatment with 1,2,3-trichloropropane at 80 mg/Kg bw/d as compared to vehicle controls.
- Exposure to the positive test substance DBCP lead to a slight increase in mean dead implants in weeks 1,2 and 8 and to a severe increase in weeks 4 and 5, thereby showing the sensitivity of the test system.

- Table 1: dominant lethal mutation test in sd rats with 1,2-dibromo-3chloropropane (DBCP) and 1,2,3-trichloropropane (TCP)

Time of mating after treatment (weeks)	Chemical	Dose (mg/kg/day)	Number of females with implants	Number of corpora lu tea per female^a	Number of implants per female^a	Number of live embryos per female^a	Mean dead implants^b (%)	Dominant lethal mutation index^c
1	Control		14	17.4±3.4	15.1 ± 2.1	13.9 ± 2.7	7.7
	DBCP	50	15	15.9 ± 0.7	15.6 ± 0.7	12.9± 1.8	17.6 **	7.6
	TCP	80	13	16.6±3.7	14.6 ± 1.1	13.5 ± 1.5	7.5	2.8
2	Control		13	18.5 ± 3.9	16.5 ± 1.1	15.6 ± 1.3	5.2
	DBCP	50	15	16.6 ± 1.3	15.9 ± 1.1	13.8 ± 2.4 *	13.1 *	11.6
	TCP	80	14	15.9 ± 2.1 *	14.9 ± 3.0	13.9 ± 3.0	7.5	11.3
3	Control		15	17.7 ± 2.8	16.4 ± 1.6	15.3 ± 1.9	6.7
	DBCP	50	15	16.7 ± 2.1	13.1 ± 5.6	11.7 ± 5.9	9.2	23.1
	TCP	80	15	16.9 ± 1.3	15.4 ± 3.5	14.7 ± 3.5	5.6	3.9
4	Control		12	17.3 ± 2.1	16.9 ± 1.8	15.7 ± 1.7	7.1
	DBCP	50	11	17.3 ± 2.6	15.0 ± 3.3	7.8 ± 5.0 **	50.8 **	50.1
	TCP	80	15	17.4 ± 3.1	16.1 ± 1.6	15.1 ± 1.8	5.8	3.4
5	Control	-	15	17.4 ± 1.8	15.9 ± 2.9	15.1 ± 2.7	4.8
	DBCP	50	12	16.8 ± 2.0	13.7 ± 3.2 *	6.1 ± 4.9 **	59.5 **	59.6
	TCP	80	15	17.1 ± 1.5	15.7 ± 1.0	14.7 ± 1.1	5.9	2.2
6	Control	-	14	16.3 ± 1.6	15.9 ± 1.5	15.2 ± 1.6	4.1
	DBCP	50	12	16.9 ± 1.6	16.1 ± 1.2	14.9 ± 1.8	7.3	2.0
	TCP	80	13	15.8 ± 1.4	13.8 ± 3.9	12.8 ± 3.8 *	8.5	16.1
7	Control		15	16.4 ± 2.0	15.6 ± 1.5	15.01 ± 1.5	3.8
	DBCP	50	13	15.1 ± 3.6	14.3 ± 4.2	13.2 ± 4.2	14.2	11.8
	TCP	80	15	16.5 ± 2.2	15.0 ± 2.9	14.2 ± 3.2	5.9	5.3
8	Control	-	14	16.5 ± 1.1	15.9 ± 1.0	15.3 ± 2.3	4.4
	DBCP	50	12	16.7 ± 2.2	15.7 ± 2.5	14.2 ± 2.5 *	9.2 *	7.3
	TCP	80	15	16.9 ± 1.5	15.5 ± 3.3	14.9 ± 3.2	3.7	2.7

a: Values represent the mean ± S.D.

b: Values are given as mean per pregnancy.

c: (1-live embryos per test female/live embryos per control female) X 100.

Conclusions:

Interpretation of results: negative
1,2,3-trichloropropane was analysed for its ability to induce dominant lethal mutations in the rat (compliant to OECD TG 478) after 5 successive applications of the maximum tolerated dose of 80 mg/Kg bw to the males. None of the test parameters were significantly different treatment group as compared to the vehicle control group, while the positive control group showed unambiguous effects, thereby validating the test system. Therefore it can be concluded that in this test system 1,2,3-trichloropropene showed no tendency to induce dominant lethal mutations.

Executive summary:

In the present study (Saito-Suzuki) 1,2,3-trichloropropane was analysed for its ability to induce dominant lethal mutations in the rat (compliant to OECD TG 478) after 5 successive applications via gavage in olive oil of the maximum tolerated dose of 80 mg/Kg bw to the males. None of the test parameters were significantly different treatment group as compared to the vehicle control group, while the positive control group showed unambiguous effects, thereby validating the test system. Therefore it can be concluded that in this test system 1,2,3-trichloropropene showed no tendency to induce dominant lethal mutations.

15 male mice were treated with the test item for 5 days and subsequently paired with 1 female over night in the same cage for 1 week or until copulation had occurred. The dose was regarded as the maximum tolerated dose and was based on the determined acute lethal dose of 500 mg/Kg bw.

Females were controlled for vaginal plugs or sperm in vaginal smears in the morning and then separated from males for the day period. Each male was paired with a total of 8 females during 8 successive weeks. Females were sacrificed 13 - 14 d after copulation and the female reproductive organs were analysed for corpora lutea, implants, live embryos and early and late embryonic deaths. It remains unclear whether male reproductive organs were analysed. The frequency of induced dominant lethal mutations was calculated according to the method of Röhrborn 1970 (Röhrborn, G. (1970) in: F. Vogel and G. Röhrborn (Eds.), Chemical Mutagenesis in Mammals and Man, Springer, New York, pp. 148-155): (1-live embryos per test female/live embryos per control female) x 100.

None of the tested parameters showed a significant difference in any of the tested weeks after treatment with 1,2,3-trichloropropane at 80 mg/Kg bw/d as compared to vehicle controls. Exposure to the positive test substance DBCP lead to a slight increase in mean dead implants in weeks 1,2 and 8 and to a severe increase in weeks 4 and 5, thereby showing the sensitivity of the test system.

Reference 6

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: - scientifically sound study - short article with limited information on animal husbandry and without individual data

Qualifier:

no guideline followed

Principles of method if other than guideline:

- 1,2,3-trichloropropane was dissolved in soybean oil and applied via ip injected at 30, 100 and 300 mg/Kg (2.5 mL/Kg vehicle) to male F344 rats. Animals were sacrificed at different time points between 1h and 48 h post administration and blood and liver samples recovered. From Blood samples the ALT activity was determined and from liver samples the DNA damage was determined using alkaline elution.
In addition the effect of the test item on DNA-DNA and DNA-protein crosslinking was tested

GLP compliance:

no

Type of assay:

other: alkaline elution

Specific details on test material used for the study:

- Name of test material (as cited in study report): 1,2,3-trichloropropane
- no further details given

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley Inc. (Indianapolis, IN, U.S.A.)
- Age at study initiation: not reported
- Weight at study initiation: not reported
- Assigned to test groups randomly: not reported
- Fasting period before study: not reported
- Housing: not reported
- Diet (e.g. ad libitum): ad libitum, Wayne Lab-Blox®
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C
- Humidity (%): not reported
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: soybean oil
- Justification for choice of solvent/vehicle: not reported
- Concentration of test material in vehicle: 12, 40 and 120 mg/mL
- Amount of vehicle (if gavage or dermal): 2.5 mL/Kg

Details on exposure:

not applicable

Duration of treatment / exposure:

all animals were singly treated between 8:00 and 10:00 am.

Frequency of treatment:

once

Post exposure period:

1 - 48 h

Remarks:

Doses / Concentrations:
30, 100 and 300 mg/Kg
Basis:
other: actual injected

No. of animals per sex per dose:

at least 3 animals per dose and time point

Control animals:

yes, concurrent vehicle

Positive control(s):

- yes, 1,2-dibromoethane
- Route of administration: ip in soybean oil
- Doses / concentrations: 0.068 mmol/Kg bw(= 12.77 mg/Kg bw)

Tissues and cell types examined:

liver

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
the low dose of 30 mg/Kg bw was the highest dose tested in the 2 year carcinogenicity study (NTP)

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
sampling at 1, 2, 3, 8, 12, 24 and 48 h

DETAILS OF METHODS:
- Alkaline elution:
Rat hepatic nuclei were prepared by a modification of the procedure reported by Cox et al. (Cox, R., Damjanov, I., Abanobi, S.E., and Sarma, D.S.R. (1973)). A method for measuring DNA damage and repair in the liver in vivo. Cancer Res. 33, 2214.). Briefly, 2 grams of liver were minced in 2 volumes PBS and gently squashed through a 60 mesh wire screen. Nuclei were pelleted by centrifugation at 50xg for 4 minutes at 4°C. Alkaline elution was performed by a modification of the method of Eastman and Bresnick (Eastman, A., and Bresnick, E. (1978). A technique for the measurement of breakage
and repair of DNA alkylated in vivo. Chem.- Biol. Interactions 23, 369-377). Nuclei (50-100 pg DNA) were applied to 47 mm diameter polycarbonate 2 pm filters (Nucleopore, Corp., Pleasanton, CA) with 20 ml of PBS. The nuclei were lysed with 20 ml of lysing buffer containing 0.2% sodium lauroylsarcosinate (Sarkosyl), 2M NaC1 and 40mM Na2EDTA adjusted to pH 9.7 with NaOH. The nuclei were allowed to lyse for 1 hr then washed twice with 10 ml of buffer containing 20mM H4EDTA adjusted to pH 10.0 with tetrapropylammonium hydroxide (RSA, Corp., Ardsley,NY). The DNA was eluted from the filters with a buffer containing 20mM H4EDTA adjusted to pH 12.3 with tetrapropylammonium hydroxide at a flow rate of 0.1m1/min. Six 3m1 fractions were collected and the DNA quantified fluorometrically using Hoechst 33258 (Murray, D., VanAnkeren, S.C., and Meyn, R. (1987). Applicability of the alkaline elution procedure as modified for the measurement of DNA damage and its repair in nonradioactively labeled cells. Anal. Biochem. 160, 149-159.). The alkaline elution results were plotted as the log of the fraction of DNA retained on the filter versus elution volume. The elution rate constant (k) was calculated as the slope of the linear portion of the elution curve, k=(-2.303) x slope.
- crosslinking analysis: no details reported

Evaluation criteria:

not applicable

Statistics:

one way ANOVA using Duncan's multiple comparisons test.

Sex:

male

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- 1,2,3-trichlorpropane induced strand breaks at already at 1 h post treatment in a dose dependent manner.
- the rate of DNA damage decreased between 1 h and 12 h post treatment but increases ant 24 and decreases again at 48 h.
- the peak at 24 h might be due to the delayed effect of cytotoxicity of 1,2,3-trichloropropane which as show by increased ALT values at 24 h post treatment
- Neither a raise of DNA-DNA nor of DNA-protein cross-links were detected after treatment with the test item

Conclusions:

Interpretation of results: positive
1,2,3-trichloropropane was tested via alkaline elution for its ability to inflict DNA damage after ip injection in male F344 rats at 30, 100 and 300 mg/Kg bw. In addition the effect on blood ALT levels and on DNA-DNA and DNA-protein crosslinking was assayed. Dose dependent DNA damages were determined between 1 h and 48 h post treatment along with raised ALT levels at 24 h which are indicative of cytotoxic liver damage. On the contrary neither a raise of DNA-DNA nor of DNA-protein cross-links was detected after treatment with the test item.

Executive summary:

In the present study (Weber & Sipes (1990B) 1,2,3-trichloropropane was tested via alkaline elution for its ability to inflict DNA damage after ip injection in male F344 rats at 30, 100 and 300 mg/Kg bw. In addition the effect on blood ALT levels and on DNA-DNA and DNA-protein crosslinking was assayed. Dose dependent DNA damages were determined between 1 h and 48 h post treatment along with raised ALT levels at 24 h which are indicative of cytotoxic liver damage. On the contrary neither a raise of DNA-DNA nor of DNA-protein cross-links was detected after treatment with the test item.

Additional information

- Genetic toxicity in vitro:

The classification of 1,2,3-trichloropropane as positive for genetic toxicity in vitro is based on the summary of results from 21 different studies as summarized in the “Summary entry on all other available in vitro test on genetic toxicity” in chapter 7.6.1. In this summary article the results from 20 different tests on the genetic toxicity of 1,2,3-trichloropropane and its metabolites 1,3-Dichloro-2-propanol and 1,3-Dichloroacetone are combined. For 1,2,3-trichloropropane mainly positive but also negative results depending on test system and conducting laboratory were found. Positive results occurred in most cases only with activation (including the bacterial reverse mutation assay (Ames Test, positive), in-vitro cytogenetic assays in mammalian cell lines (positive), a chromosome aberrations assay (negative but tested only without activation), a non-standardin vitromicronucleus assay (positive, with inherent activation), UDS assays in mammalian cell lines (negative), sister chromatid exchange assays in mammalian cells (positive, with activation) and DNA binding studies (positive)). In the Ames Test, genotoxicity occurred at relatively low levels after activation (0.02 mg/plate, Monsanto 1982).
The two metabolites 1,3-dichloro-2-propanol and 1,3-dichloroacetone were positive in bacterial mutation assays as well as in sister chromatid exchange assays (in V79 cells) with and without activation.
It can therefore be concluded that 1,2,3-trichloropropane and its metabolites 1,3-dichloro-2-propanol and 1,3-dichloroacetone are positive for in vitro genotoxicity.
This assumption is in-line with the assessments of IARC ( IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Volume 63, Dry Cleaning, Some Chlorinated Solvents and Other Industrial Chemicals, 1995, IARC Lyon, France, p. 223 -242; http://monographs.iarc.fr/ENG/Monographs/PDFs/index.php) and SCOEL (Recommendation from the Scientific Committee on Occupational Exposure Limits for 1,2,3-trichloropropane, SCOEL/SUM/170, November 2009, For public consultation).

- Genetic toxicity in vivo:

Diverging results have been obtained with test for in vivo genetic toxicity of 1,2,3-trichloropropane. La 1995 (rat and mouse, single gavage of 3 and 30 and 6 and 60 mg/Kg respectively) as well as Weber & Sipes 1990B (single ip injection in male F344 rats at 30, 100 and 300 mg/Kg bw) describe the formation of DNA adducts, namely S-[1-(hydroxymethyl)-2-(N 7-guanyl)ethyl]glutathion. Nevertheless both describe also cytotoxicity at this doses and it remains unclear whether this is the cause of DNA damage or the result of it. A slightly different scenario is seen in Tafazoli (1996). This is an in vitro test, but as human primary blood were tested it might have a special significance for human in vivo. Cells analysed with single cell electrophoresis showed strong DNA damage without S9 activation and even higher with activation but also in the latter case high cytotoxicity (exposure: 3 h at 2 and 4 mM in culture medium, with and without ). Interestingly at the same culture conditions with S9 and at even longer incubation times without S9 activation (24 h) no effect was seen in a micronucleus assay.
Other in vivo tests like a rodent dominant lethal assay (Saito-Suzuki 1982, rat 5 successive applications (gavage) at 80 mg/Kg bw), mouse bone marrow micronucleus test (Douglas et al., 1985 in IPCS CICADS 56 - 1,2,3-trichloropropane ) and unscheduled DNA synthesis in rat hepatocytes in vivo (Mirsalis et al., 1983 in IPCS CICADS 56 - 1,2,3-trichloropropane) gave negative results, but are either not available or only as abstracts and can therefore not be assessed for their reliability.
This is also true for an abstract to a poster presentation by Ito (1996) where some evidence was presented that induction of lipid peroxidation might have an influence on the genotoxicity of 1,2,3-trichloropropane.
Based on the above stated data an unambiguous conclusion on in vivo genotoxicity 1,2,3-trichloropropene is not possible.

This assumption is in-line with the assessments of IARC ( IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Volume 63, Dry Cleaning, Some Chlorinated Solvents and Other Industrial Chemicals, 1995, IARC Lyon, France, p. 223 -242; http://monographs.iarc.fr/ENG/Monographs/PDFs/index.php) and SCOEL (Recommendation from the Scientific Committee on Occupational Exposure Limits for 1,2,3-trichloropropane, SCOEL/SUM/170, November 2009, For public consultation).

Justification for classification or non-classification

As a conclusion to the above discussed data 1,2,3-trichloropropane is deemed genotoxic in vitro and of unclear genotoxicity in vivo. Therefore it is to be classified as Category 2 (H341: Suspected of causing genetic defects) concerning mutagenicity according to CLP (REGULATION (EC) No 1272/2008 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL) as implementation of UN-GHS in the EU.

This classification is in-line with the assessments of IARC ( IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Volume 63, Dry Cleaning, Some Chlorinated Solvents and Other Industrial Chemicals, 1995, IARC Lyon, France, p. 223 -242; http://monographs.iarc.fr/ENG/Monographs/PDFs/index.php) and SCOEL (Recommendation from the Scientific Committee on Occupational Exposure Limits for 1,2,3-trichloropropane, SCOEL/SUM/170, November 2009, For public consultation).