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extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
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other justification
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ZDBC – Extended one generation reproductive toxicity study
On November 17th 2017 we received a decision on ZDBC (Decision number: CCH-D-2114375518-38-01/F). ECHA requested to submit the basic extended one-generation reproductive toxicity study with the inclusion of with the inclusion of cohorts 2A and 2B (developmental neurotoxicity). The inclusion of these cohorts was based on a particular concern on (developmental) neurotoxicity based on the results on the structurally analogous substance zinc bis dimethyldithiocarbamate (ziram, ZDMC).
We reviewed ECHAs’ request for the inclusion of cohorts 2A and 2B in light of the newly obtained OECD 408 and 414 study for ZDBC.

OECD 408
In the OECD 408 study, rats (5-6 weeks old) were exposed to doses of ZDBC up to 250 mg/kg bw/day. The results of the weekly detailed clinical observations, neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of ZDBC in rats.
Thyroid hormone levels, TSH and T4, assessed in serum using ELISA, were not affected by the treatment. T4 hormone levels in plasma (inadvertently measured by the Siemens Dimension Clinical Chemistry system) were, however, found to be lower in high-dose females compared to concurrent controls. The lower plasma T4 was not corroborated by treatment-related changes in TSH levels, thyroid weight and pathology, or by effects on plasma total cholesterol levels which are known to be controlled by thyroid hormone action (OECD Guideline 408, 2018). Moreover, this finding was not supported by similar results on T4 obtained by Elisa. Therefore the lower T4 in plasma of high-dose females as obtained by the Siemens Dimension Clinical Chemistry system was considered to be a chance finding. In summary, the weight of evidence from this study does not indicate a perturbation of the thyroid pathway.
Based on decreased total protein and albumin concentrations in males and increased relative weights of the liver (both sexes) and kidneys (males) still present in the mid-dose group, the no-observed-adverse-effect level (NOAEL) was conservatively placed at the lowest level tested (10 mg/kg body weight/day). None of the effects reported in the ZDMC studies, which are regarded as triggers for inclusion of the 2A and 2B cohorts, were observed in animals receiving ZDBC.

OECD 414
In the OECD 414 study, time-mated female Wister Han rats were treated with ZDBC from Day 6 to 20 post-coitum, inclusive by daily oral gavage at dose levels of 25, 75 and 250 mg/kg. Unfortunately, finalisation of this draft report was not possible before the submission deadline of November 26th, consequently this data is not included in the current update of the dossier. Treatment-related effects in the maternal generation were restricted to reduced body weight and food consumption in the highest dose group. This was reflected in a lower fetal body weight, indicating a growth retardation. No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio, external, visceral and skeletal malformations and developmental variations). This included visceral analyzation of the brains for half of the pups. The analysis indicated that the developing brain was not affected by the test substance. The NOAEL for maternal and developmental toxicity of ZDBC was set at 75 mg/kg bw. None of the effects reported in the ZDMC studies, which are regarded as triggers for inclusion of the 2A and 2B cohorts, were observed in animals receiving ZDBC.

Cohort: developmental neurotoxicity
The request by ECHA for the inclusion of the 2A and 2B cohorts is solely based on a particular concern on (developmental) neurotoxicity of a structurally similar substance (ziram, ZDMC). However, in light of newly obtained high quality repeated dose toxicity and pre-natal developmental data for ZDBC, we can conclude that no triggers for the inclusion of the 2A and 2B cohorts were found.
As was reported in the decision, ECHA considers the developmental neurotoxicity effects found in the Nemec (1996) study to be the main trigger for the inclusion of the cohorts. In the US EPA document the validity of the developmental toxicity findings in the Nemec study (1996) was questioned. There are some major deficiencies within the study. The neurobehavioural data (motor activity, startle response and cognitive function) were not analysed statistically. Therefore, it was not indicated that the positive findings were not purely found by chance.
We consider that all observed effects in this study are related to the general state of the pups and were transient (i.e. motor activity and peak startle response), while the developmental neurotoxicological endpoints with a morphological developmental basis did not show any effect. For example, no effect on learning and memory evaluations were found and brain weights and qualitative histopathological evaluation of the nervous system did not reveal any treatment-related findings. Therefore, it appears that the observed effects have a general-toxicity basis instead of a developmental toxicity basis.
Furthermore, we consider that the absence of adverse effects of ZBEC in the recent OECD 408 and 414 study should be the main indicator, overriding the already weak triggers from the study on ziram, to conclude that the inclusion of the 2A and 2B in the OECD 443 is not warranted. In our view, these cohorts should therefore not be included, due to lack of particular concern and also in light of animal welfare reasons. We ask ECHA to revise their decision and agree with a basic OECD 443 study without the developmental neurotoxicity cohort.

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Applicant's summary and conclusion