1,2,3-trichlorobenzene

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:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Salmonella Mutagenicity Test results for 250 Chemicals

Author:

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

Year:

1983

Bibliographic source:

Environmental Mutagenesis Supplement 1:3-142

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1981

Deviations:

yes

Remarks:

Only 4 strain tested instead of 5. Positive control: 2-aminoantracene was only indicator of efficiency of S9-mix. Individual plate reading not reported.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,2,3-trichlorobenzene

EC Number:

201-757-1

EC Name:

1,2,3-trichlorobenzene

Cas Number:

87-61-6

Molecular formula:

C6H3Cl3

IUPAC Name:

1,2,3-trichlorobenzene

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): 1,2,3-Trichlorobenzene
- Analytical purity: 97%
- Lot/batch No.: HC 060787
-Source: Radian Corporation, Austin, TX, USA

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not reported
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: not reported
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: not reported
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: not reported
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): formulated in DMSO
- Final concentration of a dissolved solid, stock liquid or gel: not reported

Method

Target gene:

his

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: male Sprague-Dawley rats and male Syrian hamsters that were injected, ip, with Aroclor 1254 (200 mg/ml in corn oil) at 500 mg/kg. Five days after injection, the animals were sacriftced by decapitation (EGG, SRI) or cervical dislocation (CWR) and the livers were removed aseptically, The animals were fasted for 12-24 hr immediately preceding sacrifice.
- method of preparation of S9 mix: Liver homogenates were prepared aseptically at 0-4°C. Excised livers were rinsed with 0.15 M KCl, then minced and homogenized (3 ml of 0.15 M KCI/g wet tissue) in a Potter-Elvehjem apparatus with a teflon pestle (EGG, SRI) or in a Waring blender (CWR). The homogenate was centrifuged for 10 min at 9,000g at 4°C. The supernatant (S-9) was decanted and distributed into freezing ampules and stored at
-70°C.
The microsornal enzyme reaction mix (S-9 mix) was prepared immediately prior
to each assay. Unused S-9 mix was discarded and not refrozen. One milliliter of S-9 mix has the following composition: S-9, 0.10 ml; 0.04 M MgCl2, 0.02 ml; 1.65 M KCl, 0.02 ml; 0.04 M ß-nicotinamide adenine dinucleotide phosphate (NADP), 0.10 ml; 0.05 M glucose-6-phosphate, 0.10 ml; 1.0 M NaH2P04 (pH 7.4), 0.10 ml; and distilled water, 0.56 ml.
- concentration or volume of S9 mix and S9 in the final culture medium: not reported
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): not reported

Test concentrations with justification for top dose:

0.0, 3.3, 10.0, 33.3, 100.0, 333.3 ug/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: the chemical was not soluble in water
- Justification for percentage of solvent in the final culture medium: not reported

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-aminoanthracene (2-AA) & 4-Nitro-o-phenylenediamine (NOPD)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48h
- Selection time (if incubation with a selection agent): 48h
- Concentration of S9 mix: 10% for both Hamster S9 mix and Rat S9 mix

SELECTION AGENT (mutation assays): L- histidine

NUMBER OF REPLICATIONS: 2 trial per strain and 3 plates per dose

DETERMINATION OF CYTOTOXICITY
- Method: other: viability on complete medium and reduced numbers of revertant colonies per plate and/or thinning or absence of the bacterial lawn

Evaluation criteria:

A positive response was indicated by a reproducible, dose-related increase, whether it be twofold over background or not.
An equivocal response was defined as an increase in revertants which was not dose-related, not reproducible, or was of insufficient magnitude to support a determination of mutagenicity. A negative response was obtained when no increase in revertant colonies is observed following chemical treatment.

Results and discussion

Test results

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no effect reported
- Data on osmolality: no effect reported
- Possibility of evaporation from medium: no effect reported
- Water solubility: no effect reported
- Precipitation and time of the determination: no effect reported

RANGE-FINDING/SCREENING STUDIES (if applicable):
to select the dose range for the mutagenesis assay, the test chemicals were checked for toxicity to TA 100 up to a concentration of 10 mg/plate or the limit of solubility, both in the presence and absence of S-9 mix. If toxicity was not apparent in the preliminary toxicity determination, the highest dose tested was 10 mg/plate; otherwise the upper limit of solubility was used. If toxicity was observed, the doses of test chemical were chosen so that the high dose exhibited some degree of toxicity. Occasionally, in the earlier tests, the high dose was greater than 10 mf/plate.

STUDY RESULTS: see Table 2

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: not reported
- Negative (solvent/vehicle) historical control data: Not reported

Any other information on results incl. tables

Table 2 Mutagenic responses of Salmonella strains TA100, TA 1535, TA 1537, and TA 98 (mean±SEM) to 1,2,3-trochlorobenzene.

TA 100

TA 1535

TA 1537

TA 98

Dose

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

0.0

124±9.9

138±7.0

131±7.3

25±6.0

12±2.9

14±0.9

11±2.7

20±0.3

26±3.5

25±2.4

22±5.0

35±3.4

3.3

122±13.2

122±9.7

114±4.1

17±1.7

10±2.2

16±2.0

8±2.1

15±3.2

18±3.0

20±3.0

26±2.2

32±1.0

10.0

134±14.0

131±7.5

122±7.2

20±4.6

14±0.3

13±0.3

12±1.3

14±3.5

22±3.0

16±4.8

24±1.7

31±3.2

33.3

91±3.8

131±11.6

135±10.6

17±2.3

14±2.9

16±1.5

6±1.3

13±3.8

18±1.5

20±0.7

23±3.7

32±5.1

100.0

11± 6.9s

91±6.4

124±10.2

0±0.0s

8±1.5

14±3.8

0±0.0s

11±1.8

21±3.3

10±2.9s

27±0.6

31±2.0

333.3

t

t

80±2.8s

t

4±2.2s

6±0.7s

t

0±0.0s

6±1.0s

t

6±3.2s

18±6.2s

POS

550±8.1

596±29.5

556±9.5

438±5.3

334±51.3

367±6.2

163±24.0

287±3.6

467±13.0

474±16.3

388±25.1

934±19.9

 

 Abbreviations:NA, not activated; RLI, rat liver S-9, Aroclor1254 induced; HLI, hamster liver S-9, Aroclor1254 induced, t=complete clearing background, s=slight clearing of background lawn

Applicant's summary and conclusion

Conclusions:

negative with metabolic activation;
negative without metabolic activation

Executive summary:

Haworth S, 1983

 

Ames test was performed with theSalmonella typhimurium strains TA1535, TA1537, TA98 and TA100 at 0, 3.3, 10, 33.3, 100, and 333.3µg 1,2,3-TCB/plate with and without S-9 from Aroclor 1254 induced rat and hamster livers using the preincubation procedure. Test doses were chosen following checks for toxicity: in the absence of toxicity a maximum of 10 mg/plate was used. 1,2,3-trichlorobenzene resulted to be not mutagenic.

The test was conducted according to OECD guideline 471 with deviations (Only 4 strains tested instead of 5. Positive control: 2-aminoantracene was only indicator of efficiency of S9-mix.Individual plate reading not reported).