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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 was negative with and without metabolic activation (NTP 1983). In another poorly documented Ames Assay with and without metabolic activation tested in 3 Salmonella Typhimurium strains ( TA1535, TA100, TA98) a weak evidence of mutagenicity was found only in TA 100 with exogenous metabolic activation from hamster-induced liver S9 (NTP 1989).

In an additional Ames test 3-Chloro-p-toluidine hydrochloride (CPT-HCl) was tested in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100. The test and control articles were evaluated in triplicate cultures in all five tester strains; evaluations were performed in the presence and absence of an exogenous metabolic activation system (S9).

Data obtained with this Ames/Salmonella plate incorporation assay indicate that CPT-HCl was not associated with mutagenic effects under the conditions/criteria of the test protocols.

In a chromosomal aberration assay the test substance was tested with and without metabolical activation in Chinese Hamster Ovary Cells. The test concentrations used were: without metabolical activation: 16, 50, 160 µg/ml and with metabolical activation: 50, 160, 500 µg/ml. Without metabolic activation, the rate of chromosomal aberrations was slightly increased; with metabolic activation, no significant increase in the rate of chromosomal aberrations was observed. Due to the test results, the test substance was found to be equivocal for chromosomal aberrations.

In an additional Chromosome aberration assay 3-Chloro-p-toluidine hydrochloride (CPT-HCl) was tested in CHO cells. CPT-HCI was evaluated in duplicate cultures at concentrations of 25, 125, 250, and 350 µg/ml with and without S9. There were no statistically significant or concentration-dependent increases in aberrations/cell or percent aberrant metaphases observed in any of the cultures treated with CPT-HCI in the absence of S9. In contrast, statistically significant, concentration-dependent increases in aberrations/cell and the percent aberrant metaphases were observed in the two highest concentration cultures (250 and 350 µg/ml) treated in the presence of S9.

3-Chloro-p-toluidine hydrochloride (CPT-HCl) was tested in the Chinese hamster ovary/hypoxanthineguanine phosphoribosyltransferase (CHO/HPRT) forward gene mutation assay in CHO cells. Based upon the results of a preliminary cytotoxicity screen, CPT-HCl was evaluated in duplicate cultures in the mutation assay at concentrations of 5, 10, 50, 100, 150, 200, 250, 300, 350, and 400 µg/ml with S9, and 5, 10, 50, 100, 500, 600, 700, 800, 900, and 1000 µg/ml without S9.

There were no statistically significant or concentration-dependent increases in the average mutant frequencies among the cultures treated with CPT-HCl.

In a sister chromatid exchange assay the test substance was tested with and without metabolical activation in Chinese Hamster Ovary Cells. The test concentrations used were: without metabolical activation: 5, 16, 50 µg/ml; with metabolical activation: 50, 160, 500 µg/ml. The rate of sister chromatid exchanges was slightly increased only in the presence of metabolic activation. Therefore the test substance was found to be weak positive in the sister chromatid exchange assay.

In the in vitro rat primary hepatocyte UDS assay from 1.25 µg/ml to 60 µg/ml in five trials a marginal increase in nuclear labeling in all experiments was found and was considered weakly positive.

The UMU test and the microscreen prophage-induction assay were negative.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No OECD guideline or GLP defined. Only 4 Salmonella typhimurium strains tested.
Principles of method if other than guideline:
Bacterial reverse mutation assay (Ames test).
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Ames test
Species / strain / cell type:
other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537
Metabolic activation:
with and without
Test concentrations with justification for top dose:
0.0, 10.0, 33.0, 100.0, 333.0, 1000.0, 3333.0 µg/plate.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
Ames test.
Species / strain:
other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Results:

       TA 100        TA1535        TA 1537        TA 98
 Dose  NA  RLI  KLI  NA RLI   KLI  NA  RLI  KLI  NA  RLI  KLI
0.0  109 +/- 6.2  155 +/- 3.4  158 +/- 12.6  24 +/- 0.9  28 +/- 6.3  24 +/- 3.4  7 +/- 1.2  9 +/- 1.5  16 +/-1.2  23 +/-5.7  22 +/-0.9  37 +/-2.4
10.0        27 +/- 2.7  32 +/- 0.7  32 +/- 2.7        29 +/-2.9  24 +/-1.5  35 +/-6.1
33.0  112 +/- 7.6  150 +/- 10.2  172 +/- 9.1  16 +/- 4.1  39 +/- 2.3  35 +/- 4.0  8 +/- 0.9  12 +/- 0.9 21 +/- 4.7   26 +/-1.5  21 +/-1.5  36 +/-2.2
100.0  122 +/- 10.0  140 +/- 2.6  172 +/- 14.2  25 +/- 3.5  35 +/- 1.3  28 +/- 3.0  5 +/- 0.9 10 +/- 1.2  12 +/- 1.8   23 +/-2.6  25 +/-2.5  31 +/-2.5
333.0  111 +/- 10.0  162 +/- 2.2  150 +/- 3.0  18 +/- 2.6  32 +/- 1.5  21 +/- 11.6  5 +/- 2.0  8 +/- 0.6  14 +/-2.3 18 +/- 4.5   20 +/-0.9  35 +/-3.5
1000.0   81 +/- 9.6  145 +/- 7.0  154 +/- 17.6   9 +/- 5.2  15 +/- 7.7  23 +/- 6.4  3 +/- 1.2  5 +/- 2.6  8 +/- 1.2  30 +/-5.5  8 +/-3.8  21 +/-8.3
 3333.0 POS  120 +/- 5.5  152 +/- 2.0  152 +/- 1.5        0 +/- 0.0  5 +/- 2.3  7 +/-2.0      
 POS  1268 +/- 11.3  2100 +/- 17.9  2458 +/- 49.7  1482 +/- 7.3  186 +/- 23.5  229 +/- 20.2  249 +/- 32.5  170 +/-12.1  100 +/-5.9  304 +/-46.9  1371 +/-78.6  2079 +/-50.4
Executive summary:

In an Ames test the test substance was tested in following strains of Salmonella typhimurium with and without metabolic activation TA 98, TA 100, TA 1535, TA 1537. The test concentrations used were: 0.0, 10.0, 33.0, 100.0, 333.0, 1000.0, 3333.0 µg/plate.

The test substance did not prove to be mutagenic in the Ames test for the strains of Salmonella typhimurium tested.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No OECD guideline or GLP defined, insufficient data documentation, no substance concentrations given.
Principles of method if other than guideline:
Bacterial reverse mutation assay (Ames test).
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Ames Assay
Species / strain / cell type:
other: Salmonella typhimurium TA 98, TA 100, TA 1535
Metabolic activation:
with and without
Test concentrations with justification for top dose:
No substance concentrations given.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
Ames test
Metabolic activation:
with and without
Genotoxicity:
other: weak positive

 Strain: TA1535    
 S9 Activation  S9 Species  Concentration
 5% HLI  Hamster  5%
 30% HLI   Hamster  30%
 10%HLI   Hamster  10%
 Strain: TA100    
S9 Activation   S9 Species  Concentration
 NA  NA  NA
 5% HLI  Hamster  5%
 30% RLI  Rat  30%
 30% HLI  Hamster  30%
 10% HLI  Hamster  10%
 Strain: TA98    
 S9 Activation  S9 Species  Concentration
 NA  NA  NA
 30% RLI  Rat  30%
 30% HLI  Hamster  30%

Concentration denotes the percentage of S9 in the S9 mixture (metabolic activation enzymes and cofactors from Aroclor 1254 -induced male Sprague-Dawley rat or Syrian hamster liver) that was added to cultures. Standard NTP Protocol.

Executive summary:

In a poorly documented Ames Assay with and without metabolic activation tested in 3 Salmonella Typhimurium strains ( TA1535, TA100, TA98) a weak evidence of mutagenicity was found.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

No valid study is available.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

An Ames test in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 was negative with and without metabolic activation (NTP 1983). In another poorly documented Ames Assay in 3 Salmonella Typhimurium strains ( TA1535, TA100, TA98) a weak evidence of mutagenicity was found only in TA 100 with exogenous metabolic activation from hamster-induced liver S9 (NTP 1989).

A further Ames test in in Salmonella typhimurium strains TA1535, TA153 7, TA1538, TA98, and TA100 was negative.

In a chromosomal aberration assay without metabolic activation, the rate of chromosomal aberrations was slightly increased; with metabolic activation, no significant increase in the rate of chromosomal aberrations was observed. Due to the test results, the test substance was found to be equivocal for chromosomal aberrations. A further chromosomal aberration test there were no statistically significant or concentration-dependent increases in aberrations/cell or percent aberrant metaphases observed in any of the cultures treated with CPT-HCI in the absence of S9. In contrast, statistically significant, concentration-dependent increases in aberrations/cell and the percent aberrant metaphases were observed in the two highest concentration cultures (250 and 350 µg/ml) treated in the presence of S9.

The results of the chromosome aberration tests were contradictory.

In a sister chromatid exchange assay the rate of sister chromatid exchanges was slightly increased only in the presence of metabolic activation. Therefore the test substance was found to be weak positive in the sister chromatid exchange assay.

In the in vitro rat primary hepatocyte UDS assay from 1.25 µg/ml to 60 µg/ml in five trials a marginal increase in nuclear labeling in all experiments was found and was considered weakly positive.

The other genotoxicity tests revealed an inconclusive or weak positive result.

Based on the results of the available genotoxicity tests the overall result is inconclusive and a classification according to CLP classification criteria (Regulation (EC) No 1272/2008) is not feasible.