New Changes to Chemical Safety Testing

Important updates to OECD Human Health Test Guidelines for the testing of chemicals and how these changes could impact future studies

On June 26, the Organisation for Economic Co-operation and Development (OECD) published 56 updated test guidelines. The full list of updated documents can be found on the OECD website, but they involved updates and clarifications in several areas, including tissue sampling for OMICS, directions for using variant assessments, and histopathology, analytical chemistry, and statistics. 

The most extensive updates were made to Test Guideline 443: Extended One-Generation Reproductive Toxicity Study, which included specific updates to endocrine disrupter-related endpoints and developmental immunotoxicity measurements. Based on the findings of the EOGRTS Review Project conducted by the European Chemicals Agency, several minor clarifications were also included in this version. 

Major Updates to OECD Test Guideline 443

• Adjusted the frequency of moribundity/mortality observations for all animals to once daily on weekends.
• Paragraph 46 on the assessment of preweaning landmarks of development (anogenital distance [AGD] and nipple retention [NR]) was updated significantly. Based on findings of the EOGRTS Review Project, precision of AGD measurement has now been specified – “at least two significant digit numbers and reported in mm” (i.e., 1/100th of a mm). The absolute AGD, the animal’s body weight on the day of assessment and the actual day of assessment should all be reported. As before, the guideline allows for AGD to be measured at least once between Postnatal Day (PND) 0 and 4. In addition, AGD should be normalized to a measure of pup size, preferably the cube root of body weight as described in OECD Guidance Document 151 and published manuscripts by Gallavan et al. (1999). For nipple retention, the guideline now allows assessment of male pups on PND 12, 13 and 14 as long as all pups are evaluated on the same postnatal day. In addition, it now requires a qualitative comment on the fact that female littermates have visible nipples at the same age. 
• Paragraph 47 on the assessment of postweaning landmarks of sexual development was updated to initiate daily evaluation of female pups beginning on PND 24 and male pups beginning on PND 35 for attainment of vaginal patency and preputial separation, respectively. As in multiple paragraphs within the updated guideline, this paragraph also reiterates the importance of assessing up to 3 pups/sex/litter (or a minimum of 50 pups/sex/group) for these landmarks. 
• Paragraph 52 on the assessment of potential developmental immunotoxicity was updated extensively. While the previous versions of the guideline allowed for the use of sheep red blood cells (sRBC) as the antigen for a T-cell dependent antibody response assay (TDAR), this is no longer an option. With this update, only Keyhole Limpet Hemocyanin (KLH) can be used as the immunizing antigen. In addition to an assessment of a primary antibody response, it is now expected that animals selected for Cohort 3 will also receive a secondary immunization to be conducted 2 weeks following the primary immunization, for the assessment of a secondary (IgG) antibody response in a TDAR. Consequently, the age of necropsy for these animals was also updated. 
• Paragraph 58 on sperm parameter assessments was updated to include spermatid (testicular) counts. In addition, the guideline was updated to explicitly state that epididymal weights should include total and cauda epididymal weights. 

Clarifications to Test Guideline 443

• Three (3) pups/sex/litter should be maintained for assessment of landmarks of sexual maturation. Furthermore, the guideline was updated to recommend that the surplus cohort of animals be called Cohort 1C, if not already selected for other cohorts.   
• Splenic immunophenotyping should be conducted for animals selected for Cohort 1A (reproductive toxicity cohort), regardless of whether or not Cohort 3 (developmental immunotoxicity cohort) is included in the study. 
• Histopathology assessments should be conducted for animals selected for Cohort 1B for suspected reproductive and endocrine toxicants. 
• Paragraph 73 was adjusted to separate quantitative ovarian follicle counts from quantitative corpora lutea counts.  The guideline still requires microscopic corpora lutea counts. Furthermore, the sentence was updated to state that ovarian follicle counts should include primary and small growing follicles; however, a reference citation was not provided for how follicles should be classified. 

The Charles River team has already conducted an extensive review of these specific test guideline updates and their associated regulatory impact. Study designs and study plans are being updated to provide the most updated study designs for our clients. For any questions, including specific questions relating to these recent changes, call or email us to speak with one of our subject matter experts today, or check out this helpful link for more information on our reproductive toxicology services.

Pragati Coder, PhD, DABT is the Global Lead for Developmental and Reproductive Toxicology at Charles River.