1,2-dichloro-4-(trichloromethyl)benzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not mutagenic

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

This study was performed to evaluate 3,4-dichloro-benzotrichloride for its possible mutagenic activity, by the bacterial reverse mutation test, using five histidine deficient(his-)mutant tester strains of Salmonella typhimuriumviz., TA1537, TA1535, TA98, TA100 and TA102. The methods followed were as per the OECD N°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 3,4-dichloro-benzotrichloride 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 from concentrations of 0.1563to 5.0 µL/plate both in the absence and presence of metabolic activation system and at the concentration of 0.0781 µL/plate in the absence of metabolic activation system. Partial inhibitionof background bacterial lawnwasobserved at the concentration of 0.0391 µL/plate both in the absence and presence of metabolic activation system and at 0.0781µL/plate in thepresence of metabolic activation system. Reduction in the number of colonies was observed at the concentration of 0.0781µL/plate and micro-colonies were observed at the concentration of 0.1563 µL/plate both in the absence and presence of metabolic activation system. No colonies were observed from 0.3125 to 5.0 µL/plateboth in the absence and presence of metabolic activation system.Normal growth was observed at test concentrations of 0.0195 and 0.0098 µL/plate and negative control.

 Hence,µL/plateof3,4-dichloro-benzotrichloridewas 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.

Trial I

Based on the results of the cytotoxicity study,3,4-dichloro-benzotrichloridewas evaluated for its possible mutagenic effect in five strains ofSalmonella typhimuriumat thedose levels of 0.0025, 0.005,0.02,andµL/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 of3,4-dichloro-benzotrichloride,negative and positive controls.

The results revealed that there was no positive mutagenic effect in strains TA1537, 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 beingµL/platefive lower concentrations viz., 0.032, 0.0128, 0.0051, 0.0020 and 0.0008µL/platewere tested both in the absence and presence (10% v/v S9 mix) of metabolic activation system.Triplicateplates were maintained for each test concentration of3,4-dichloro-benzotrichloride,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 that3,4-dichloro-benzotrichloride, up to the concentration of 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 fiveSalmonella typhimuriumtester strains viz., TA1537, TA1535, TA98, TA100 and TA102 when tested under the specified conditions.

Justification for classification or non-classification

GERM CELL MUTAGENICITY

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

Category 1: Substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans. Substances known to induce heritable mutations in the germ cells of humans.

Categoty 2: Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

Classification for heritable effects in human germ cells is made on the basis of well conducted, sufficiently validated tests as In vitro mutagenicity tests such as these indicated in 3.5.2.3.8:

- in vitro mammalian chromosome aberration test;

- in vitro mammalian cell gene mutation test;

- bacterial reverse mutation tests

The substance to be registeredshows negative results on the strains of Salmonella typhimurium under the performed test, therefore data available are conclusive but not sufficient for the classification, according to the 3.5. of the CLP Regulation n.1272/2008.