ANNEX B. EFFECT-BIOMONITORING

 91

Annex B. Effect-biomonitoring

Annex Table B.1. Potential suitable effect biomarkers characterized for occupational use or already applied in other contexts. Grey marked effect-biomarkers were not characterized

No Covered MoA or endpoint Name of the assay or effect biomarker Biomarker categorization Measured endpoint

1a C including genotox DIN EN ISO 21427 is for the application of the Micronuclei (MN) assay in water quality assessment. The MN assay is better referred to OECD 487. Dividing cells are needed to apply the MN assay

1b C including genotox

1c C including genotox

1d C including genotox peripheral blood lymphocyte micronucleus test (OECD 474) and buccal mucosa micronucleus test

1e Oxidative stress level indicative for C and genotox 2a M

3a R

3b R

4a ED- ER receptor activation in vitro

buccal micronucleus approach ex vivo induction rate of micronuclei

induction rate of micronuclei in buccal cells micronuclei frequencies epithelial buccal cells

cytokinesis-block micronucleus assay (CBMN-Assay) (similar to OECD 487) in vitro or ex vivo induction rate of micronuclei in mammalian cells

ex vivo micronuclei frequencies in lymphocytes and epithelial buccal cells

reduced/oxidized glutathione (GSH/GSSG) ratio Ames Test/Bacterial Reverse Mutation Test in vitro or ex vivo increase or decrease of the GSH/GSSG ratio

in vitro measuring reverse mutation in bacterial cells

reproductive Hormones - female hormones ex vivo measuring estradiol levels in serum

reproductive Hormones – male hormones ER CALUX ex vivo

in vitro measuring testosterone or 2-DHT levels in serum

measuring the receptor activation of the human estrogen receptor

OCCUPATIONAL BIOMONITORING GUIDANCE DOCUMENT © OECD 2022

92 

No Covered MoA or endpoint Name of the assay or effect biomarker Biomarker categorization Measured endpoint

4b 4c ED- AR receptor activation ED- TR receptor activation

AR CALUX TR CALUX, anti-TR CALUX, TTRTR CALUX, TTR-FITC assay, TPO assay 4d ED-steroidogenesis modulation H295-R-steroidogenesis modulation assay 5a inhibition of acetyl-choline-esterase acetylcholine-esterase-inhibition assay

5b and 6b

Brain Derived Neurotrophic Factor (BDNF) (indicative of neuronal survival, development and synaptic plasticity) 5c neuroaxonal damage/ scaffolding proteins, neurofilament light-chain (NF-L) in serum 5d neuroaxonal damage/ scaffolding proteins (Glial fibrillary acidic protein (GFAP), neurofilament light (NFL), medium and heavy chains (NFH), S 100 beta (a Ca2+-binding protein and is expressed primarily by astrocytes) BDNF Assay

neuroaxonal damage/ scaffolding proteins (small parameter selection)

neuroaxonal damage/ scaffolding proteins (enlarged parmeter selection) in vitro in vitro

in vitro measuring the receptor activation of the human androgen receptor measuring the receptor activation of the human thyroid receptor measuring steroidgenesis modulation

biochemical/biological measuring inhibition of acetyl-choline- esterase

biochemical/biological peripheral BDNF levels in blood cells (gene expression and protein) are used as potential biomarker for psychiatric disorders, Parkinson (PD) and Alzheimer’s disease (AD). biochemical/biological Neurofilament-light chain (NF-L) in serum

biochemical/biological neurofilament light chain (NfL), neurofilament medium chain (NfM), neurofilament heavy chain (NfH), a-internexin and peripherin in serum/ blood

6a Thyroid stimulating hormone (TSH), free triiodothyronine (T3) and thyroxine (T4) and antithyroperoxidase (TPO) antibodies in serum for addressing developmental neurotoxicity TSH assay biochemical/biological levels in serum during pregnancy

6b and 5b Brain Derived Neurotrophic Factor (BDNF) (indicative of neuronal survival, development and synaptic plasticity) BDNF Assay biochemical/biological peripheral BDNF levels in blood cells (gene expression and protein) are used as potential biomarker for psychiatric disorders, Parkinson (PD) and Alzheimer’s disease (AD).

7a The murine embryonic stem cell test Sarcomeric myosin heavy chain and in vitro Quantitative expression of sarcomeric myosin heavy

OCCUPATIONAL BIOMONITORING GUIDANCE DOCUMENT © OECD 2022

 93

No Covered MoA or endpoint Name of the assay or effect biomarker Biomarker categorization Measured endpoint

(EST) – to assessembryo-toxicity (teratogenicity) potential of chemicals* alpha-actinin proteins chain and alpha-actinin proteins in beating cardiomyocyte’s as well as counting of contracting cardiomyocyte agglomerates. The morphological analysis of beating cardiomyocytes in embryoid body outgrowths compared to cytotoxic effects on murine ES cells and differentiated 3T3 fibroblasts.

7b Male-mediated developmental toxicity and mutagenicity* dominant lethal and specific locus mutation tests: in vivo; DNA methylation: in vitro Different biomarkers in battery

Battery of tests to identify germ cell mutations, such as dominant lethal and specific locus mutation tests, epigenetics (DNA methylation e.g. acrylamide, lead) 8a Methemoglobin respiratory toxicity Methemoglobin binding assay ex vivo / biochemical measurement of building of methemoglobin which is functional inactive

*Were not characterized further due to expected sensitivity coverage under DNT.

OCCUPATIONAL BIOMONITORING GUIDANCE DOCUMENT © OECD 2022

94 

Annex Table B.2. Scoring method for effect-biomarker characterisation questions & answers.

Question No Questions for assessing relevance and invasiveness (score 0-16) 1) Has the biomarker been assessed in easy accessible human biological matrices?

2) Is there a plausible MoA?

3) Is an Adverse Outcome Pathway AOP reported for this effectbiomarker? 4) Is the biomarker able to detect relevant (adverse and severe) effects in workers during a long-term exposure?

Questions for assessing applicability (score 0-16) 5) Has the effect biomarker been applied in occupational or epidemiological studies and resulted in meaningful results for a workplace or chemical exposure? 6) Has the biomarker been applied in environmental risk assessment or other studies with regulatory relevance (e.g. drinking water, food regulation)? 7) How would you define workload and applicability for occupational settings? Answer Options

Non-invasive: urine (5 points) saliva (4 points) buccal or nasal cells (3 points) Invasive: blood (2 points) other (1 point) None (0 points) 0-5

Yes - please report your MoA in the comment field (3 points) No (0 points) Yes - please report it in the comment field, add the link (3 points) No (0 points) 0-3

High - please provide proof of evidence via DOI or ref. in the comment field (5 points) Medium (3 points) Low (1 point) No adversity expected (0 points) 0-5

Yes - please report it, provide the DOI in the comment field (5 points) Can be applied in a modified form (3 points) No (0 points) 0-5

Yes - please report it, provide the DOI (3 points) No (0 points)

High - minimal work load and training necessary (5 points) Medium - moderate work load and training necessary (3 points) Low - high work load and expert judgement necessary (0 points)

Certified commercial labs offer this bioanalysis (3 points) 0-8

Questions for assessing validation and cost (score 0-16) Score

0-3

0-3

OCCUPATIONAL BIOMONITORING GUIDANCE DOCUMENT © OECD 2022

 95

Question No Answer Options

8) Does the biomarker have a well-described standard operating procedure (SOP)?

9) Does an OECD guideline or a standardized DIN EN ISO exist for the effect-biomarker?

10) What is the cost per sample?

Yes - publically available in a peer reviewed journal. Please provide DOI in the comment field (4 points) Partially - as internal or non peer reviewed SOP (2 points) No (0 points) 0-4

Yes - OECD guideline. Please provide links in the comment field (3 points) Yes - standardized DIN EN ISO. Please provide links in the comment field (3 points) No (0 points) 0-6

Very low - <100 EURO/sample (6 points) Low - 100-250 EURO/sample (4 points) Medium - 250-400 EURO/sample (2 points) High - 400-750 EURO/sample (1 points) Very high - >750 EURO/sample (0 points) 0-6

Questions for assessing sensitivity & specificity & robustness (score 0-12) 11) Is the Limit Of Quantification (LOQ) below an accepted occupational exposure limit for a relevant reference substance?

Yes - please provide reference or DOI and LOQ in the comment field (4 points) Partially (2 points) No or unknown (0 points) 0-4

13) Is the specificity* of the biomarker sufficient for the

substances or effects of concern?

Yes - please provide reference or DOI and LOQ in the comment field (4 points) Partially (2 points) No or unknown (0 points) 0-4

13+14) What are geometric mean concentrations and geometric standard deviations of the biomarker in the general population? Is the effect-biomarker sufficiently robust to compare different levels of exposure risks (e.g. does it have age dependent variations, body mass index or smoking dependency)?

Yes - please provide reference or DOI and LOQ in the comment field (4 points) Partially (2 points) No or unknown (0 points) 0-4

Score

* specificity means the method is less prone to detect false positive results , e.g. specificty of 95% means only in 5 % of cases the biomarker detects false positive results. Weak point: We have currently no agreement which level of specificity is sufficient. It can be 90% or lower or higher. Preliminary we can work with 80% specificity as preliminary treshold.

OCCUPATIONAL BIOMONITORING GUIDANCE DOCUMENT © OECD 2022

96 

Annex Table B.3. Similar Exposure Groups (SEG) or as Working Contributing Scenario (WCS) approach template adapted to effect-biomarker reporting

RAC Template adapted V1.1_SV.xlsx

OCCUPATIONAL BIOMONITORING GUIDANCE DOCUMENT © OECD 2022