1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione, zinc salt

Endpoint summary

Description of key information

Background:

Examination of the test material structure indicated that it is a zinc salt with the anion being a strong base (i.e., corresponding protonated form is a weak acid). This anion may associate with cations leading to the formation of salts with limited solubility, resulting in precipitation. In an OECD Guideline 105 study, the water solubility of ZMB2 is 32 mg/L in double distilled water. The maximum solubility of the test material in dechlorinated tap water was reduced by the presence of cations to around 12 mg/L.

 

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, Safepharm (2003) performed a modified version of the standard method for the preparation of aqueous media. An approach endorsed by several important regulatory authorities in the European Union and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test material in cases where the test material is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (150 mg/l) of test material with dechlorinated tap water for 48 hours and then removing the undissolved test material by filtration through a pre-conditioned filter (0.2 µm) to give a saturated solution with a nominal concentration of 12 mg/L.

 

This risk assessment/dossier contains a combined approach for addressing the required endpoints. The required endpoints are addressed using, when available, data on the substance to be registered, 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione, zinc salt (CAS 61617-00-3) (ZMB2). When these data are not available, data from a close structural analog, 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione (MB2) (CAS number 53988-10-6) is used to address the required endpoints.   MB2 is structurally similar to ZMB2, with a smiles code of c1([nH]c2c([nH]1)cccc2C)=S compared to that of c1ccc2[nH]c([nH]c2c1)=S.C*.[Zn] for ZMB2. The zinc salt present in ZMB2 is not expected to cause any significant differences in the ecotoxicological effects compared to MB2. This is based upon the dissociation constant results which indicate that ZMB2 does not remain as a single molecule, with the zinc salt dissociating within the environmental compartment (Harlan Laboratories, 2013). The low impact of zinc on the ecotoxicological impact of ZMB2 can be further seen when comparing the PNECs of zinc and MB2, the strucutural analogue which does not contain the zinc salt. Here, based on the aquatic PNECs derived for zinc (ECHA, 2018), it can be seen that basing the PNECs for ZMB2 on the MB2 effects seen in the read-across Daphnia magna reproduction study, the most sensitive endpoint (Currenta, 2012, OECD 211, 2008), would allow for a conservative and worst-case risk assessment. The PNECs for zinc have been estimated as: PNECfreshwater 20.6 µg/L and PNECmarine 6.1 µg/L. The PNECs for MB2 have been estimated as: PNECfreshwater 3.5 µg/L and PNECmarinewater 0.35

µg/L

. It can be seen that the MB2 PNECs are more than 5 -fold lower than those of zinc. Therefore, when using this read-across approach the precautionary principle applies.

 

The read-across justification is further strengthened by the results seen in the bridging studies for the short-term toxicity endpoints, which used both QSARs and experimental methodologies. These studies indicated that there were good concordance between the results obtained for MB2 and ZMB2, and that in many instances the use of the MB2 results would result in a conservative, worst-case, assessment.

 

MB2 data are used to address the following endpoints: 

 

•  Long-term Toxicity to Aquatic Invertebrates.

 

It is important to note that based on the following information provided in the European Chemical Agency (ECHA) guidance document 'Guidance on information requirements and chemical safety assessment Appendix R.7.13 -2: Environmental risk assessment for metals and metal compounds. July 2008', many quantitative structure–activity relationship ((Q)SAR) models are not suitable or validated to assess/ predict the toxicity of certain metals. Since ZMB2 is an organometallic substance, of which there are no fully accepted organometallic QSAR models by ECHA, the approach has been used with caution.  Furthermore, when the QSARs were used as supporting information for experimental data they showed good corroboration with the experimental results, and were shown to be conservative (i.e. worst-case) predictions in comparison to the QSAR results. They were used to support experimental data of the source and target substance. When available, data published using a reliable scientific method is included within the assessment.

 

Long-term toxicity to fish data were generated using the United States (US) Environmental Protection Agency (EPA) Ecological Structure Activity Relationships (ECOSAR) v1.0 tool. ECOSAR is validated for organosulphates, a chemical class which includes the read-across substance MB2 (CAS Number 53988 -10 -6), and is structurally similar to ZMB2 (CAS Number 61617-00-3).

 

ECOSAR QSAR data are used to address the following endpoints: 

• Long-term toxicity to fish.

In addition, ECOSAR data were generated for endpoints for which testing data are also available, for comparison purposes. ECOSAR results were within 1 degree of magnitude when compared to study data and significantly similar to results from testing data. Due to this consistency, the ECOSAR QSAR model was considered to provide reliable results for the long-term toxicity to fish endpoint.

 

During the hazard assessment of the substance priority was given to relevant, reliable test methods on the substance, followed by data on the strucutral analogue and then QSAR data. Furthermore, the precautionairy principle was used where applicable.

 

Short term toxicity to fish:

Key study:

In accordance with OECD Guideline 203 (Fish, Acute Toxicity Test) the 96 -Hour LC50 based on nominal test concentrations of ZMB2 was 5.6 mg/L with 95% confidence limits of 4.5 - 7.1 mg/L. The No Observed Effect Level (NOEL) was 2.1 mg/L (Safepharm Laboratories, 2003).

 

Supporting study:

The acute toxicity of ZMB2 to fish was tested according to "Letale Wirkung beim Zebrabärbling - Brachydanio rerio - 48 - 96 hour LC 0 , LC 50 , LC 100 Berlin, May 1984". A LC0 of 707 mg/L and a LC100 of >1000 mg/L were observed during 96 hours exposure (Bayer AG, 1992).

 

The acute toxicity of the read-across substance, MB2, to fish was tested according to "Letale Wirkung beim Zebrabärbling - Brachydanio rerio - 48 - 96 hour LC 0 , LC 50 , LC 100 Berlin, May 1984". A LC0 of 22 mg/L, LC50 of 37.2 and a LC100 of 63 mg/L were observed during 96 hours exposure (Bayer AG, 1990).

 

Long term toxicity to fish:

Weight of Evidence:

ZMB2 was estimated to have a Chronic Value (ChV) to fish of 9.833 mg/l using the ECOSAR Class (Thioureas) method, and a ChV to fish of 3.517 mg/l using the Neutral Organic SAR (Baseline Toxicity) method (ECOSAR, 2013).

 

The read across substance, MB2, was estimated to have a Chronic Value (ChV) to fish of 42.207 mg/l using the ECOSAR Class (Thioureas) method, and a ChV to fish of 12.086 mg/l using the Neutral Organic SAR (Baseline Toxicity) method

(ECOSAR, 2013).

 

ChV results for both ZMB2 and the read-across substance MB2 both show that fish is expected to be the least sensitive species to aquatic toxicty. This is echoed in the acute testing results for ZMB2, the fish NOEC is 2.1 mg/l and the Daphnia NOEC is 0.47 mg/L (Safepharm Laboratories, 2003), and for MB2 the short-term NOECs of 22 mg/L and 0.38 mg/L for fish and Daphnia, respectively.

 

Short term toxicity to aquatic invertebrates:

In accordance with OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) the acute toxicity of ZMB2 to the freshwater invertebrate Daphnia magna was investigated. The 48 hour EC50 is 1.4 mg/L (mean measured) with 95% confidence limits of 1.1 - 1.6 mg/L. The 48 hour No Observed Effect Concentration (NOEC) is 0.47 mg/L.

 

Supporting Study:

In accordance with OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) the acute toxicity of the read-across substance MB2 to the freshwater invertebrate Daphnia magna was investigated. The 48 hour EC50 is 1.9 mg/L (mean measured) with 95% confidence limits of 1.56 - 2.34 mg/L. The 48 hour No Observed Effect Concentration (NOEC) is 0.38 mg/L.

 

Long term toxicity to aquatic invertebrates:

No chronic aquatic invertebrate data are available on the substance ZMB2. Data from the close structural analog, MB2 (CAS number 53988-10-6) are used to address the endpoint. 

 

ZMB2 will dissociate at environmentally relevant pHs to form MB2 and zinc salts (LANXESS, 2018 - see Section 13.2 of IUCLID). ZMB2, is therefore, not expected to exist in the environment, or if so only very briefly. Thus, there is no need and little relevance in deriving the long-term toxicity for ZMB2. As such it is appropriate to consider the assessment of these two moieties and their environmental hazard seperately. Based on the aquatic PNECs derived for zinc (ECHA, 2018), it can be seen that basing the PNECs for ZMB2 on the MB2 effects seen in the read-across Daphnia magna reproduction study, the most sensitive endpoint (Currenta, 2012, OECD 211, 2008), would allow for a conservative and worst-case risk assessment. The PNECs for zinc have been estimated as: PNECfreshwater 20.6 µg/L and PNECmarine 6.1 µg/L. The PNECs for MB2 have been estimated as: PNECfreshwater 1.38 µg/L and PNECmarinewater 0.138 mg/L. It can be seen that the MB2 PNECs are more than 10 -fold lower than those of zinc. Therefore, when using this read-across approach the precuationary principle applies.

 

Supporting data were also generated using the United States (US) Environmental Protection Agency (EPA) Ecological Structure Activity Relationships (ECOSAR) v1.0 tool. ECOSAR is validated for organosulphates, a chemical class which includes the read-across substance MB2 (CAS Number 27231-36-3), and is structurally similar to ZMB2 (CAS Number 61617-00-3). The chronic toxicity values (ChV) derived showed a high level of concordance, emphasising the appropriateness of MB2 as a structural analogue for read-across purposes to fulfill this endpoint.

 

Key Study (Read across):

MB2 was found to have a 21 day NOEC (reproduction) value of 0.0346 mg/L (measured) to Daphnia magna (Currenta, 2012).

 

Supporting studies:

ZMB2 was estimated to have a Chronic Value (ChV) to Daphnia of 0.079 mg/l using the ECOSAR Class (Thioureas) method (ECOSAR, 2013).

 

The read across substance, MB2, was estimated to have a Chronic Value (ChV) to Daphnia of 0.09 mg/l using the ECOSAR Class (Thioureas) method (ECOSAR, 2013).

 

Toxicity to aquatic algae:

Key Study:

In accordance with OECD Guideline 201 (Alga, Growth Inhibition Test) the effect of ZMB2 on the growth of Scendesmus subspicatus has been investigated over a 72 hour period. The EbC50 (72 h) (measured) result is 6.6 mg/L; 95% confidence limits 5.8 - 7.5 mg/L and an Er50 (72 h) (measured) result is 10 mg/L. It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits. The 72 hour No Observed Effect Concentration (NOEC) is 0.69 mg/L (Safepharm, 2003).

 

Supporting data were generated using the United States (US) Environmental Protection Agency (EPA) Ecological Structure Activity Relationships (ECOSAR) v1.0 tool. ECOSAR is validated for organosulphates, a chemical class which includes the read-across substance MB2 (CAS Number 53988 -10 -6), and is structurally similar to ZMB2 (CAS Number 61617-00-3). Furthermore, supporting data is also available for the structural analogue MB2. The chronic toxicity values (ChV) derived showed a high level of concordance, emphasising the appropriateness of MB2 as a structural analogue for read-across purposes to fulfill chronic endpoints.

 

Supporting studies:

In accordance with OECD Guideline 201 (Alga, Growth Inhibition Test) the effect of MB2 on the growth of Scendesmus subspicatus has been investigated over a 72 hour period. The EyC50 (72 h) (nominal) result is 41.2 mg/L; 95% confidence limits 39.9 - 42.3 mg/L and the Er50 (72 h) (nominal) result is 62.2 mg/L (95 % CL = 61.5 -63). The 72 hour No Observed Effect Concentration (NOEC) for growth rate inhibition was 25 mg/L (Currenta, 2011).

 

ZMB2 was estimated to have a Chronic Value (ChV) to algae of 0.155 mg/l using the ECOSAR Class (Thioureas) method (ECOSAR, 2013).

 

The read across substance, MB2, was estimated to have a Chronic Value (ChV) to algae of 0.212 mg/l using the ECOSAR Class (Thioureas) method (ECOSAR, 2013).

 

Toxicity to microorganisms:

In accordance with ISO 8192 (Test for Inhibition of Oxygen Consumption by Activated Sludge) ZMB2 was determined to have an EC50 of 6620 mg/L and showed 46 % respiration inhibition on activated sludge within 3 hours (Bayer AG, 1992).

Additional information

According to ECHA Decision on Substance Evaluation (SEV) for 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione, zinc salt (CAS 61617-00-3), requesting an Amphibian Metamorphosis Assay (AMA - OECD TG 231) with 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione (CAS 53988-10-6), this study was performed and summarised in the section 6.1.8 Endocrine disrupter testing in aquatic vertebrates - in vivo.

This AMA study was performed to identify whether the substance may interfere with the normal function of the hypothalamic-pituitary-thyroid (HPT) axis, according to OECD Test Guideline 231 with GLP statement. According to a 14-day preliminary toxicity test, Xenopus laevis tadpoles were exposed to the test item at analytically confirmed nominal concentrations of 0.0048, 0.024, 0.12, 0.60, 3.0 and 15 mg/L and to a control, over a period of 21 days under flow-through conditions. The study met the acceptability criteria and was considered valid. According to the results of this study, neither advanced, nor asynchronous development were observed in the present study. However, remarkable histological effects were noted at concentrations of 0.12 mg/L and higher, including assignment of ‘severe degree’ grading for the core assessed criteria at 3.0 mg/L and 15 mg/L. Consequently, according to the read-across approach and following the decision logic presented in OECD TG 231, the registered substance is concluded to be thyroid active.