Registration Dossier

Administrative data

Endpoint:
extended one-generation reproductive toxicity – with F2 generation and both developmental neuro- and immunotoxicity (Cohorts 1A, 1B with extension, 2A, 2B, and 3)
Type of information:
experimental study planned (based on read-across)
Study period:
Subject to approval following ECHA/MSCA review of the testing proposal; an OECD 422 oral dietary study is planned to start in Q1 2018 and will be used as a range-finder for this study; projected start date for the EOGRTS in Q1 2019
Justification for type of information:
This study forms part of the overall Concawe testing strategy for petroleum substances and the VHGO category (see attached preliminary testing strategy document in Section 13).

TESTING PROPOSAL ON VERTEBRATE ANIMALS
[Please provide information for all of the points below. The information should be specific to the endpoint for which testing is proposed. Note that for testing proposals addressing testing on vertebrate animals under the REACH Regulation this document will be published on the ECHA website along with the third party consultation on the testing proposal(s).]

More detailed information on this testing proposal is found in the document "VHGO EOGRTs study proposal v9" attached below in the "attached justification" field.

NON-CONFIDENTIAL NAME OF SUBSTANCE:
This test is proposed to be conducted on one member of the VHGO category, with the results then read-across to other category members. Brief justification is given below, with additional support for the category given in the category justification document (attached to the category object and to Section 13 of the dossier) and in the document "VHGO EOGRTs study proposal v9" attached below in the "attached justification" field.

HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
VHGOs are UVCB substances grouped within established production boundaries based on phys-chem properties and hydrocarbon type (a full justification for this grouping is given in a separate document); substances within the category have qualitatively similar properties. The prediction for read-across in the category will be based on what is considered to be the worst-case substance.

It is hypothesised that the reproductive toxicity of VHGO will be related to the types and levels of aromatics present, and will generally follow a pattern of increasing severity with increasing ring number. Any trend for the developmental toxicity of gas oils would thus be hypothetically described in terms of increasing aromatic content and number of fused aromatic rings.

The predominant PACs in VHGOs are 2- and 3-ring aromatic compounds but some VHGOs may contain very low levels of PACs with four and more aromatic rings. It is hypothesised therefore that there is low potential for adverse effects in developmental reproductive toxicity tests from exposure to VHGOs.

In conclusion, there is a hypothetical case to suggest that any developmental reproductive effects observed in petroleum substances are associated with 4 – 7 ring (possible 3 – 7 ring) PACs, and there is in-vitro and in-vivo toxicity data to support this hypothesis. However, there is no comprehensive investigation of fertility and the implications of interaction with the AhR receptor are not yet fully evaluated (work in progress, as explained above). It is therefore proposed to complement the ongoing work through fully investigating the potential reproductive effects in this study by including cohort 1B.

CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL)
See detailed discussion of available in vitro and in vivo data on UVCB hydrocarbon classes as well as work currently in progress to help underpin the justifications for category read-across for toxicity to reproduction in "VHGO EOGRTs study proposal v9" document attached below.
• <3 ring polycyclic aromatics: no effects on reproductive organs and no selective developmental effects.
• ≥4 ring polycyclic aromatics (mainly 4-7 ring PACs) with specific structures (not necessarily present in gas oils) are associated with systemic toxicity (effects on liver, thymus and blood forming organs but not reproductive organs) and are potentially mutagenic and dermal carcinogens. In developmental studies they produce foetal death and resorption. Recent in vitro work suggests that ≥3 ring polycyclic aromatics can alter embryo development.

Based on recent analytical research work on representative samples of category members; the following substance has been chosen as a worst-case based on having the highest level of 4-7 ring PAH

- Name of the substance on which testing is proposed to be carried out
Gas oils (petroleum), light vacuum (CAS 64741-58-8, EC 265-059-9)

- Name of the substances for which the testing proposal will be used [if different from tested substance]
Condensates (petroleum), vacuum tower (CAS 64741-49-7, EC 265-049-4)
Gas oils (petroleum), light vacuum (CAS 64741-58-8, EC 265-059-9)
Distillates (petroleum), light hydrocracked (CAS 64741-77-1, EC 265-078-2)
Gas oils (petroleum), hydrodesulfurized light vacuum (CAS 64742-87-6, EC 265-190-1)
Fuels, diesel (CAS 68334-30-5, EC 269-822-7)
Fuel oil, no. 2 (CAS 68476-30-2, EC 270-671-4)
Fuel oil, no. 4 (CAS 68476-31-3, EC 270-673-5)
Fuels, diesel, no. 2 (CAS 68476-34-6, EC 270-676-1)
Gas oils (petroleum), hydrotreated light vacuum (CAS 92045-24-4, EC 295-407-5)

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
· available GLP studies
There are no available GLP studies within the vacuum gas oils, hydrocracked gas oils, and distillate fuels (VHGO) group,that fully address the reproductive toxicity endpoints needed for classification.

· available non-GLP studies
There are no non-GLP studies available for the endpoint toxicity to reproduction.

· historical human data
No relevant human health data are available.

· (Q)SAR
There are no QSAR’s which are adequate to fully evaluate a UVCB substance (for more detailed explanation on [the complexity of] petroleum UVCBs see Section 2 of the document "VHGO EOGRTs study proposal v9" attached below).

· in vitro methods
There are no validated in vitro methods that fulfil this endpoint. An academic research programme applying an in vitro battery of developmental toxicity screening assays has been launched to underpin the reprotoxicity testing hypothesis that 4-7 ring Poly Aromatic Compounds (PAC) are the only constituents in petroleum substances that are associated with prenatal developmental toxicity (see section 2 of the document "VHGO EOGRTs study proposal v9" attached below for the testing hypothesis, a summary of this in-vitro project and some first published data). These data will be used in combination with in vivo data to further underpin grouping of these substances, in order to minimize animal testing as it supports the selection of the most representative worst case group member for testing and read-across of this test outcome to the other group members.

· weight of evidence
Not available according to ECHA guidance document (Chapter R.7a: Endpoint specific guidance Version 4.1 – October 2015). Concawe are undertaking a multi-year research project to support the grouping of petroleum substances for human health risk assessment (see below) and the data being generated from the various assays applied in this- and other ongoing projects may allow more substantial weight of evidence arguments to be developed in the future.

· grouping and read-across
This EOGRT will be conducted on one representative worst-case sample from the VHGO group (based on PAC content, see Section 2 of this testing proposal for the testing hypothesis and Section 4 for selection of the worst-case test sample). An ongoing research programme, in which high-content in-vitro screening assays and transcriptomic data in combination with phys/chem, analytical chemistry and already available in-vivo data are applied in an integrative analysis to further underpin grouping (www.concawe.eu/cat-app-project), will strengthen the read across assessment from the tested worst-case substance to the other group members. This is expected to significantly reduce the number of tests in vertebrate animals compared to conducting Individual substance evaluations.

There are 9 CAS numbers in the VHGO group. Historically data for toxicity assessment has been generated via the dermal and inhalation route to replicate the route of human exposure to petroleum substances. However, to comply with regulatory requirements for hazard identification, oral exposure is required. Therefore, part of Concawe’s informed testing strategy is to supplement these historical data with targeted oral studies: the range-finding work to be conducted for this EOGRTS will be conducted as an OECD 422 guideline study (oral) which, together with OECD 422 data from other category members, will be used to inform repeated dose toxicity endpoints via the oral route (also underpinning the exposure route / dermal database) and further support grouping & read-across in the group.

· substance-tailored exposure driven testing [if applicable]
Not applicable

· [approaches in addition to above [if applicable]
Not applicable

· other reasons [if applicable]
Not applicable

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
Adaptation options as defined in Annexes VI to X were not applicable for this group and this endpoint. Regarding the column 2 rules for adaption, the data base was fully evaluated according to ‘Guidance on information requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance’.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
VHGO’s are widely used as fuels, but do not have significant nonfuel widespread use. Some VHGO’s contain 4 – 7 ring PACs which are believed to interact with the Aryl Hydrocarbon (Ah) receptor which may then lead to modifications of the estrogen receptor pathway; the rationale for this hypothesis and the data to support it are described in detail in section 2 of this testing proposal. As the available data base is limited and because the potential role of PAC’s in effects on fertility is not fully understood it is proposed that the study includes cohort 1B (second generation) to fully evaluate the effects on the developing offspring.

There are no pathological changes in the nervous system or CNS type clinical signs in the current database of dermal studies. However, in one inhalation study on a similar petroleum substance a minor effect on startle response was observed which may be indicative of neurotoxic effects. Due to uncertainties in the data base it is proposed that cohort 2 is included.

VHGOs are classified as H373: May cause damage to thymus, liver, and bone marrow through prolonged or repeated exposure, suggesting organs associated with the immune system are a target for toxicity. It is therefore proposed that cohort 3 for immunotoxicity is also included.

-Overall
With the aim to minimize -and avoid unnecessary- animal testing (on all 192 petroleum substances) while not underestimating the potential human health risks, a worst case testing approach and overall informed tiered testing strategy is proposed holistically across the entire portfolio of petroleum substances. Targeted CAS numbers will be subject to in-vivo testing covering all Concawe petroleum categories.

The sample selected for this test to cover the VHGO category is the CAS number with the highest % (w/w) of 4 – 7 ring PAC’s, as it is considered that this has the greatest potential for reproductive and developmental toxicity (see grouping of VHGO substances and testing hypothesis in section 2). The results of this test, supported with other targeted oral studies, historical data and mechanistic data from in-vitro assays, will be used as read-across for the remaining VHGO group members therefore avoiding additional high animal consuming in vivo studies.

The proposed EOGRTS on VHGOs is part of the overall informed testing strategy (see section 13 of the dossier). The results of this study is expected to help inform the decision making on potential further testing needs and design of similar studies which are required for other petroleum categories. The basic types of hydrocarbon molecules found in this substance and related petroleum category (ie straight and branched alkanes and alkenes, cycloalkanes and cycloalkenes, aromatic and aromatic cycloalkanes) are found in other petroleum categories and, although petroleum substances are UVCB substances of limited variability within product specifications, their similarities can help define a targeted approach to minimize animal use in further testing.
Cross-reference
Reason / purpose:
other: range finding study

Data source

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals - 10 weeks

- Basis for dose level selection – an OECD 422 study by the oral (dietary) route is scheduled and results from this will be used as a basis for dose level selection.

- Inclusion/exclusion of extension of Cohort 1B – some of the 4 – 7 ring aromatic substances present in the VHGO may lead to interactions with the estrogen receptor and may be associated with foetal death and resorption. There are no indications of developmental effects in the absence of maternal toxicity nor robust evidence of endocrine disruptor activity. However, it is recognized that the data base is limited and the potential role of PACs is not fully understood and hence it is requested that the study includes a cohort to fully evaluate the effects on the developing offspring.

- Termination time for F2 – post natal day 4

- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B – Based on a minor effect on startle response, cohort 2 is required

- Inclusion/exclusion of developmental immunotoxicity Cohort 3 – VHGO’s have a category 2 classification for repeated dose toxicity of the liver, thymus and bone marrow and therefore there is potentially risk to the developing immune system. Cohort 3 for the EOGRTS is requested

- Route of administration – It is considered that the most likely routes of exposure to VHGO are dermal and inhalation however, ECHA have previous indicated to Concawe that the route of administration for the study addressing this endpoint in Annex X would be oral. It should be noted that the oral route may result in a pattern and degree of exposure that is different to that observed following dermal application.
Concawe propose to conduct the range-finding and study and EOGRT’s via the dietary route. An OECD 422 will be conducted as the range-finding study.
This substance is an aspiration hazard and this is of concern, especially regarding the dose administration to neonates. In addition, VHGO’s are classified as irritating to the skin and petroleum substances are associated with the de-fatting of dermal tissue. Therefore the dietary route may ameliorate any possible gastrointestinal disturbances and irritancy.
Dietary administration is less invasive for the animals, therefore promoting animal welfare. In addition, the procedure of gavage administration may result in mild stress, which may influence some of the sensitive outcomes in this type of study.
The diet will be administered to achieve a constant mg/kg/day dose, the dietary concentration being adjusted to the weekly bodyweight. However, it is necessary to first ensure that homogeneous mixing with the diet and adequate concentration analysis and stability can be achieved and that the treated diet is palatable at sufficiently high concentrations; if this is not possible then the route of administration will revert to gavage. The following approach is suggested to the final design of the EOGRT’s.

- Other considerations, e.g. on choice of species, strain, vehicle and number of animals [if applicable]-

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Sex:
male/female

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
This substance is an aspiration hazard and this is of concern, especially regarding the dose administration to neonates. In addition, VHGO’s are classified as irritating to the skin and petroleum substances are associated with the de-fatting of dermal tissue. Therefore the dietary route may ameliorate any possible gastrointestinal disturbances and irritancy.
Dietary administration is less invasive for the animals, therefore promoting animal welfare. In addition, the procedure of gavage administration may result in mild stress, which may influence some of the sensitive outcomes in this type of study.
The diet will be administered to achieve a constant mg/kg/day dose, the dietary concentration being adjusted to the weekly bodyweight. However, it is necessary to first ensure that homogeneous mixing with the diet and adequate concentration analysis and stability can be achieved and that the treated diet is palatable at sufficiently high concentrations; if this is not possible then the route of administration will revert to gavage. The following approach is suggested to the final design of the EOGRT’s.
Details on study design:
an OECD 422 study by the oral (dietary) route is scheduled and results from this will be used as a basis for dose level selection.

Results and discussion

Any other information on results incl. tables

Interpretation of results will be in accord with that described in OECD Testing Guideline 443.

Applicant's summary and conclusion