α,α,α,4-tetrachlorotoluene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenic

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro bacterial mutagenicity:

The in vitro mutagenicity of 4 -chloro-benzotrichloride has been tested in an in vitro bacterial system based on the Ames test. The reported GLP study is very similar to the OECD guideline 471.

Hence, 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 theS. 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 theS. typhimurium strain TA 98. Also slightly increased numbers of revertant colonies were obtained in the absence and presence of the metabolic activation system forS. typhimuriumstrains 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.

A second study is available showing no effects on bacteria with and without metabolic activation. Using a conservative approach, data from this study was not taken into account for assessement but was reported for completeness sake.

This study was performed to evaluate parachlorobenzotrichloride for its possible mutagenic activity, by the bacterial reverse mutation test, using five histidine deficient(his-)mutant tester strains ofSalmonella typhimuriumviz., TA1537, TA1535, TA98, TA100 and TA102. The methods followed were as per the OECDN°471(July, 1997). The treatments were performed by the plate incorporation technique both in the absence and presence of metabolic activation (S9 mix). The S9 mix of 5% and 10% v/v consisted of an S9 fraction (Aroclor 1254 induced rat liver homogenate) supplemented with cofactors.

 Before conducting the mutagenicity test parachlorobenzotrichloride was evaluated for its possible cytotoxicity in strain TA100, both in the absence and presence of S9 mix (5% v/v). Cytotoxicity to the tester strain was tested at the concentrations of 0.0098, 0.0195, 0.0391, 0.0781, 0.1563, 0.3125, 0.625,2.5 and 5.0 µL/plateboth in the absence and presence (5% v/vS9 mix) of the metabolic activation system. Cytotoxicity is characterised by inhibition of the background bacterial lawn and reduction in the number of colonies. Inhibition of background bacterial lawn was observed at the test concentrations from 0.3125up to 5.0 µL/plate both in the absence and presence of metabolic activation system. Micro-colonies were observed at the concentration of 0.3125 µL/plateboth in the absence and presence of metabolic activation system.Partial inhibitionof background bacterial lawnand reduction in the number of colonies wereobserved at the concentration of 0.1563 µL/plate both in the absence and presence of metabolic activation system.Normal growth was observed in the other test concentrations and negative control.  Hence, 0.1µL/plateofparachlorobenzotrichloridewas selected as the highest concentration to be tested in the mutagenicity test both in the absence and presence of metabolic activation system, for all the tester strains.

Tria l

Based on the results of the cytotoxicity study,parachlorobenzotrichloridewas evaluated for its possible mutagenic effect in five strains ofSalmonella typhimuriumat thedose levels of 0.0031, 0.0063, 0.0125, 0.025,and 0.1µL/plate both in the absence and presence (5% v/v S9 mix) of metabolic activation system for trial I (factor 2). Triplicate plates were maintained for each test concentration ofparachlorobenzotrichloride,negative and positive controls.

 The results revealed that there was no positive mutagenic effect in strainsTA1537, TA1535, TA98, TA100 and TA102 bothin the absence and presence (5% v/v S9 mix) of metabolic activation system at any of the tested dose levelswhen compared with the concurrent negative control.Statistical analysis did not reveal any significant effects.

Trial II

A second trial was conducted to confirm the negative results obtained in Trial I. In Trial II, the concentration spacing was modified using by factor 2.5 and the concentration of S9 mix was increased to 10% v/v.

The highest concentration being 0.1µL/platefive lower concentrations viz.,0.016, 0.0064, 0.0026 and 0.0010µL/platewere tested both in the absence and presence (10% v/v S9 mix) of metabolic activation system.Triplicateplates were maintained for each test concentration ofparachlorobenzotrichloride,negative and positive controls.

 The results revealed that there was no positive mutagenic effect in strains TA1537, TA1535, TA98, TA100 and TA102 both in the absence and presence (10% v/v S9 mix) of metabolic activation system at any of the tested dose levels when compared with the concurrent negative control. Statistical analysis did not reveal any significant effects.

The values of negative control in all thetester strains during both the trials were within limits.

The positive controls exhibited a clear increase in the number of revertants both in the absence and presence of metabolic activation system (5% and 10% v/v S9 mix) with known mutagens when compared with the respective negative control. This demonstrated the efficiency of the test system and suitability of the procedures employed in the study.

From the results of this study, it is concluded that parachlorobenzotrichloride, up to the concentration of 0.1 µL/plate both in the absence and presence(5% and 10% v/v S9 mix) of metabolic activation system, is non-mutagenic to all the five Salmonella typhimurium tester strains viz., TA1537, TA1535, TA98, TA100 and TA102 when tested under the specified conditions.

Justification for classification or non-classification

So far, based on these results, further testing as required in the annex VIII should be conducted. However, according to the column 2 of the specific rules for adaptation from column 1 of the REACH regulation n° 1907/2006, an in vitro study on cytogenicity in mammalian cells or an in vitro micronucleus study of the annex VIII requirement does not usually need to be conducted if the substance is known to be carcinogenic category 1 or 2 or mutagenic category 1, 2 or 3. In this case, based on the available study, 4-chloro-benzotrichloride is classified in the CLP regulation n° 1272/2008 EC as a carcinogenic category 1B, hence no study on cytogenicity in mammalian cells or an in vitro micronucleus is required.