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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1986-11-5 to 1986-11-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The authors tested the mutagenicity of 4-chloro-benzotrichloride in the absence and presence of a metabolizing system with a methodology similar to the OECD guideline 471. GLP standards were followed. The study is very well documented, hence the study should be considered a Klimisch 1b: comparable to guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): p-Chlorbenzotrichlorid TTR
- Physical state: colourless liquid
- Analytical purity: >97%
- Impurities (identity and concentrations):
2,4-Dichlorbenzotrichlorid ~1.0%
3,4-Dichlorbenzotrichlorid ~0.4%
2-Chlorbenzotrichlorid ~0.2%
3,4-Chlorbenzalchlorid ~0.2%
2-Chlorbenzalchlorid ~0.4%
Hochsieder ~0.4%
- Storage condition of test material: dark at 22°C

No more data available

Method

Target gene:
Histidine: Salmonella typhimurium strains
One of the genes for tryptophan biosynthesis: Escherichia coli strain

No more data
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: deficient in the complete structure of the lipopolysaccharide layer and in DNA excision repair system
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: deficient in the complete structure of the lipopolysaccharide layer and in DNA excision repair system
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: deficient in the uvrA system of DNA repair
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate (S-9 mix)
Test concentrations with justification for top dose:
Dose range: 0.16 to 500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol

No further data available
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
executed for strain S. typhimurium TA 100 without metabolic activation
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 100, TA 1535; without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537; without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98, TA 1538; without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
Remarks:
WP2uvrA; without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
executed for strain S. typhimurium TA 100 with metabolic activation
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
TA 98, TA 100, TA 1535, TA 1537, TA 1538, WP2uvrA; with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
executed for strain S. typhimurium TA 100 with metabolic activation
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 98, TA 100, TA 1535, TA 1537, TA 1538, WP2uvrA; with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72h

NUMBER OF REPLICATIONS: 3

No further data available
Evaluation criteria:
Amino-acid requiring strains of bacteria are used to detect reverse gene mutations. Based on the number of revertants one can assess the mutagenicity of the test compound.
Statistics:
No data

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
see table 1 and 2 in overall remarks sections
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
thinning of bacterial lawn and reduction in number of colonies at 20 or 100 µg/plate depending on strain
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
see table 1 and 2 in overall remarks sections
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
thinning of bacterial lawn and reduction in number of colonies at 100 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
see table 1 and 2 in overall remarks sections
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
thinning of bacterial lawn and reduction in number of colonies at 100 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: visible precipitation of the test compound on the plates at 2500 and 10000 µg/plate for all strains with and without metabolic activiation


RANGE-FINDING/SCREENING STUDIES:
An experiment was performed with all tester strains (3 replicates per dose) to obtain information on mutagenicity and toxicity for calculation of an appropriate dose range. As indicators for toxicity a reduced rate of spontaneously occuring colonies as well as visible thinning of the bacterial lawn (controlled microscopically) were used.
Based on this test 500 µg/plate was chosen as highest dose for mutagenicity testing.


No further data available
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Result for the mutagenicity experiment with 4 -chloro-benzotrichloride without metabolic activation for all tested strains, including controls. The values presented are the mean value obtained from the three replicates. (ibl: incomplete bacterial lawn; nbl: no bacterial lawn)

S. typhimurium S. typhimurium S. typhimurium S. typhimurium S. typhimurium E. coli
TA-100 TA-1535 TA-1537 TA-1538 TA-98 WP2uvrA
Positive control*
Test compound 0 µg/plate 168 19 8 17 26 46
0.16 µg/plate 7 28
0.8 µg/plate 175 16 10 15 29 50
4 µg/plate 216 22 10 17 37 58
20 µg/plate 318 (ibl) 20 17 16 67 63
60 µg/plate 113 (ibl) 15 (ibl) 22 (ibl) 15 (ibl) 36 (ibl) 67
100 µg/plate 85 (nbl) 2 (ibl) 10 (ibl) 7 (ibl) 20 (ibl) 22 (ibl)
500 µg/plate nbl nbl nbl nbl nbl ibl

* TA-100 Sodium-azide 1 µg/plate 602
TA-1535 Sodium-azide 1 µg/plate 510
TA-1537 9-aminoacridine 50 µg/plate 242
TA-1538 2-nitrofluorene 2.5 µg/plate 603
TA-98 2-nitrofluorene 2.5 µg/plate 366
WP2uvrA MNNG 2.5 µg/plate 330
p-chlorbenzotrichlorid TTR 0.8 µg/plate 0

Table 2: Result for the mutagenicity experiment with 4 -chloro-benzotrichloride with metabolic activation for all tested strains, including controls. The values presented are the mean value obtained from the three replicates. (ibl: incomplete bacterial lawn; nbl: no bacterial lawn)

Metabolic activation (rat liver) S. typhimurium S. typhimurium S. typhimurium S. typhimurium S. typhimurium E. coli
TA-100 TA-1535 TA-1537 TA-1538 TA-98 WP2uvrA
Positive control**
Test compound 0 µg/plate 182 23 10 20 34 54
0.16 µg/plate 6

43

0.8 µg/plate 216 23 9 18 39 54
4 µg/plate 274 26 13 19 43 55
20 µg/plate 392 (ibl) 23 24 23 75 65
60 µg/plate 173 (ibl) 13 (ibl) 14 (ibl) 18 (ibl) 66 (ibl) 71
100 µg/plate 97 (ibl) 13 (ibl) 9 (ibl) 11 (ibl) 25 (ibl) 34 (ibl)
500 µg/plate nbl nbl nbl nbl 20 (nbl) nbl

** TA-100 2-aminoanthracen 0.5 µg/plate 718
TA-100 Benzo(a)pyrene 10 µg/plate 619
TA-1535 2-aminoanthracen 1 µg/plate 160
TA-1535 Benzo(a)pyrene 10 µg/plate 25
TA-1537 2-aminoanthracen 1 µg/plate 74
TA-1537 Benzo(a)pyrene 10 µg/plate 112
TA-1538 2-aminoanthracen 0.5 µg/plate 470
TA-1538 Benzo(a)pyrene 10 µg/plate 162
TA-98 2-aminoanthracen 0.5 µg/plate 444
TA-98 Benzo(a)pyrene 10 µg/plate 652
WP2uvrA 2-aminoanthracen 10 µg/plate 292
WP2uvrA Benzo(a)pyrene 10 µg/plate 88
S-9 mix 500 µl 0
p-chlorbenzotrichlorid TTR 0.8 µg/plate 0

Table 3: Surviving fraction (i.e. ratio of number of colonies from solvent control plates and from plates with test compound) obtained using the solvent control with the Salmonella strain TA 100

Surviving fraction

Without metabolic activation With metabolic activation
Test compound 0 µg/plate 1.0

1.0

0.8 µg/plate

1.0

1.0

4 µg/plate 1.0 1.0
20 µg/plate 0.9 1.0
60 µg/plate 0.00 0.8
100 µg/plate 0.00 0.7
500 µg/plate 0.00 0.00

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with and without metabolic activation

The authors tested the mutagenic potential of 4-chloro-benzotrichloride in the absence and presence of a metabolizing system with a methodology similar to the OECD guideline 471. Under these test conditions the test substance is mutagenic both with and without an exogenous metabolizing system for the tested bacterial test systems (i.e. Salmonella typhimurium TA 100, TA 1535, TA 1537, TA 1538, TA 98 and Escherichia coli WP2uvrA).
Executive summary:

The authors tested the mutagenicity of 4-chloro-benzotrichloride (CAS n° 5216 -25 -1) in the absence and presence of a metabolizing system with a methodology similar to the OECD guideline 471. The Salmonella typhimurium strains TA 100, TA 1535, TA 1537, TA 1538 and TA 98 and the Escherichia coli strain WP2uvrA were used and the metabolizing system was obtained from rat liver homogenates. The number of revertants per plate was estimated and was a basis for comparison with solvent controls.

Based on the range-finding/screening test, 500 µg/plate was chosen as top dose level for the mutagenicity test and the test compound proved to be toxic to most of the bacterial strains at 20 or 100 µg/plate according to observed diminuation or even complete absence of the bacterial lawn.

In the mutagenicity test, seven different doses ranging from 0.16 to 500 µg/plate were used (3 replicates per dose) and the strains were exposed for 48 to 72 h. Control plates without mutagen showed the described number of spontaneous revertant colonies reported in literature and all the positive control compounds gave the expected increase in the number of revertant colonies.

Furthermore the mutagenicity test showed a dose dependant increase in the number of revertant colonies with the S. typhimurium strain TA 98 in absence of the metabolic activation system (S-9 mix) after exposure to the test compound. In the presence of the metabolic activation system, treatment with the test substance resulted in a relevant increase in the number of revertant colonies in the S. typhimurium strain TA 98. Also slightly increased numbers of revertant colonies were obtained in the absence and presence of the metabolic activation system for S. typhimurium strains TA 100 and TA 1537. Considering the overall data, thus, 4 -chloro-benzotrichloride can be considered mutagenic with and without metabolic activation system for the tested strains.

The study was conducted in compliance with to principles of Good Laboratory Paractice and a certificate was even provided. Furthermore this study is comparable to the OECD guideline 471 and should therefore be considered as a klimisch 1.b study and hence reliable without restictions.