Registration Dossier

Administrative data

Endpoint:
extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
See attached justification documents.
Study completion has been delayed. A brief summary follows:
Final ECHA decision was received on 31 August 2018. Requirement was discussed in consortia and approved. In March 2019 Hexion the Lead Registrant was the target of a significant ransomware IT outage. Much of Hexion's REACH tracking software and files were lost or limited for weeks or months. In addition, Hexion also initiated bankruptcy protection in April under it's US affiliate. The contract was developed and signed on 23 July 2019. Pre-funding of the invoice from the consortia members was initiated by the consortia manager. There were significant delays in obtaining and paying the initial invoices. The payment was complete on 23 April 2020. Lab space was allocated and preliminary study start was set for 23 September 2020. Current anticipated completion date and draft report is February 2022.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report Date:
2022

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Principles of method if other than guideline:
See attached justification document
GLP compliance:
yes
Justification for study design:
See attached justification document

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
As per IUCLID5 Sections 1.1 - 1.4.
Specific details on test material used for the study:
Test item: 1,6-hexanediol, reaction products with chloromethyloxirane
Test item identity: HELOXY Modifier HD

EC No. 618-939-5
CAS No 933999-84-9
Batch number D09A50028
Storage conditions Ambient temperature (15 to 25°C)
Purity 100%
Correction factor None
Appearance Yellowish liquid
Stability 07 January 2022

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Age ordered: Males: 22-28 days
Females: 22-28 days

Specification: Males and females unrelated (no male/female siblings)

Number of animals: 108 males and 108 females (includes spares).

Supplier: Charles River (UK) Limited.
Sex:
male/female
Details on test animals and environmental conditions:
Acclimatization
Duration: At least 5 days.
Age at start of treatment: (F0) Nominally 4-5 weeks of age (nominally 14-15 weeks of age at pairing)

Allocation to Treatment Groups (F0 Generation)
Allocation: After a period of acclimatization.
Method: By sex. After exclusion of animals showing signs of ill-health. Animals at the extremes of the body weight range will not be selected if alternatives are available.
At commencement of the study the weight variation should not exceed 20% of the mean weight of each sex.
Cage distribution: Arrangement designed to minimize environmental variables.

Selection of Offspring to form F1 Generation
Formal commencement of the F1 generation is on a nominal Day 28 of age (where possible 28±2 days of age for selected F1 animals).
Selection: Nominally Day 21 of age; selected animals receive direct treatment.
Method: Where possible, two males and two females will be selected from each selected litter and will be allocated to each of the two cohorts. If more are required, up to three males and three females may be selected from each selected litter.
Selected animals will be microchipped on Day 18-21 of age and separated from littermates on Day 21 of age.
Up to 2 male and 2 female F1 offspring per group will be retained as spares, to provide potential replacement in the event of any mortalities. These spares will have body weights and clinical signs monitored weekly and will be terminated after commencement of the F1 generation. The
spares will not receive direct treatment unless used as a replacement animal.

Identification
Numbering: Unique for each F0 animal and selected F1 offspring within the study. All pre-weaning offspring will be numbered individually within each litter on Day 1 of age.
Method: Microchip (F0 and selected F1 generation).
Toe tattoo (pre-weaning offspring).
Cage labels: Uniquely identifying the occupant(s).

Animal Replacement
8 spare males and 8 spare females will be ordered to replace any individuals rejected,
following randomization.
Rejection before treatment: Ill-health.
Body weight range extremes.
Replacement during treatment: None scheduled.
Following Study Director authorization to remove the spare animals from the study, some of the spare animals may be assigned to the Covance sentinel animal program.

Animals - Housing, Diet and Water Supply, and Environmental Enrichment

Environmental Control
Animal facility: Limited access - to minimize entry of external biological and chemical agents.
Air supply: Filtered, not recirculated.
Temperature: Maintained within the range of 20-24ºC.
Relative humidity: Maintained within the range of 40-70%.
Monitored daily. Excursions outside these ranges documented in the study data.
Lighting: 12 hours light: 12 hours dark.
Alarm systems: Activated on ventilation failure and when temperature/humidity limits exceeded.
Electricity supply: Public supply with automatic stand-by generators.

Animal Accommodation
Covance has a policy of improvement of animal welfare which includes permitting social interaction through multiple housing of rats whenever possible and this policy will be followed for this study, except after mating when females will be housed singly to permit collection of food consumption data individually for pregnant females and during lactation, when adult females are singly housed with their litter for good husbandry practice.
Grid bottomed cages will be suspended above absorbent paper which will be changed daily
during pairing. Solid bottomed cages will have bedding which will be changed at appropriate
intervals. Cages, cage-trays, food hoppers and water bottles will be changed at appropriate intervals. Precise details of caging will be included in the study data.

Bedding, Diet and Water Supply
Bedding type: Softwood based bark-free fiber, sterilized by autoclaving.
Certification: Certificates of analysis are routinely received from the supplier.
Diet supply
Diet name: SDS VRF1 Certified, pelleted diet.
Availability: Non-restricted (except overnight for blood sampling for hematology or blood chemistry or during urine collection).
Certification: Before delivery, each batch of diet is analyzed by the supplier for various nutritional components and chemical and microbiological contaminants.
Supplier's analytical certificates are scrutinized and approved before any batch of diet is released for use.
The content of phytoestrogens in the diet is controlled. This diet contains no added antibiotic
or other chemotherapeutic or prophylactic agent. This diet contains no added antibiotic or
other chemotherapeutic or prophylactic agent.

Water supply
Supply: Potable water from the public supply.
Availability: Non-restricted via polycarbonate bottles with sipper tubes.
Certification: Certificates of analysis are routinely received from the supplier.

Environmental enrichment
During the acclimatization and appropriate study periods environmental enrichment in the form of Aspen wood based products (soft white untreated wood product) and a plastic shelter will be available in each home cage.
Plastic shelters are not provided during pairing. Aspen wood based products and plastic shelters are not provided during lactation (from Day 20 after mating).
From Day 20 after mating and throughout lactation, approximately two handfuls of paper shavings will be provided to each cage as nesting material; this nesting material will be changed at the same frequency as the cage bedding.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Administration

Route: Oral gavage.
Treated at: Constant doses in mg/kg/day.
Volume dose: 4 mL/kg/day.
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Controls (Group 1): Vehicle at the same volume dose as treated groups.
Frequency: Once daily, at approximately the same time each day.
Sequence: Groups dosed in ascending order.
Formulation: A daily record of the usage of formulation will be maintained based on weights before and after dosing. The difference between actual and expected usage will be monitored as a check of correct administration.
Doses will be allowed to reach room temperature for a short time prior to dosing (actual temperature not measured).
Formulations are stirred using a magnetic stirrer before and throughout the dosing procedure.
Expected appearance: Clear to white opaque suspension.
Storage of formulation: 21 days when stored refrigerated (2 to 8°C).
Storage of formulations at ambient temperature (15 to 25C) will be added in an Amendment.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Liquid formulation: Before commencement of treatment, specimen formulations will be analyzed to assess the stability and homogeneity of the test item in the vehicle.
At specified intervals during treatment, the test formulations will be analyzed for achieved concentration of the test item.

Stability and homogeneity: Homogeneity and stability of the dose formulation at 7.5 mg/mL to 75 mg/mL was established for 21 days when stored refrigerated (2 to 8°C) as part of another study at Envigo Research Limited, Shardlow, UK, Analytical Services (Envigo Study no. WG45RN; stability of formulations at ambient temperature (15 to 25°C) will be determined as part
of this study.
Stability will be assessed following storage at ambient temperature (15 to 25ºC) for 0 hour, 1 hour, 2 hours (continuously stirred) and 1 day. Prior to the initial sampling time point each day, the formulation will be mixed by 20-fold inversion and magnetic stirring for at least 20 minutes or until visibly homogeneous. Samples will be taken for assay from the top, middle and bottom of the formulation.
• Mean analyzed concentrations of the freshly-prepared formulations are expected to be within +10/-15% of the nominal values.
• Coefficient of variation (n=3) is expected to be ≤5%.
The stability will be determined from the mean concentration of test item in the vehicle at each time point. Results are expected to be within 10% of the initial time zero concentration value.
Analysis: The method was developed as part of another study at Envigo Research Limited, Shardlow, UK, Analytical Services (Envigo Study no. WG45RN).
Transfer of the analytical method, including specificity, linearity, system precision and method accuracy and precision will be assessed as part of this study.
The formulated samples will be analyzed using the method, validated with respect to the determination of the specificity of analysis, limits of detection, linearity of detector response,
repeatability, method accuracy and precision.

Achieved concentration
Assay sampling: Test formulations will be analyzed for the occasions given below.
Sample storage: Samples will be retained refrigerated (2 to 8°C) pending analysis
Sample Disposal: Samples (contingency/residual) will be disposed of once satisfactory results are obtained.
Frequency of treatment:
Once per day
Details on study schedule:
F0 animals: For 10 weeks before pairing until termination after litters are weaned.
F1 animals: From weaning until termination of respective cohort.
Oral gavage - Direct treatment of F1 offspring commences at weaning (Day 21 of age). Although direct treatment starts at weaning, all offspring have potential for exposure in-utero and via the milk during lactation.
Cohort 1A : General toxicity and pathology of the tissues of the male and female reproductive systems.
Treated from weaning to 13 weeks of age
Cohort 1B : Animals for potential follow-up assessment of reproductive performance.
Treated from weaning to approximately13 weeks of age.

No. of animals per sex per dose:
F0: 25
F1: 20
Control animals:
yes

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Sex:
male/female
Remarks on result:
other: Study is currently underway. Draft report is anticipated in February 2022 with a submission date of April 2022.

Results: F1 generation

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Study is currently underway. Draft report is anticipated in February 2022 with a submission date of April 2022.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion