Table 5.

In vitro chromosomal aberration results

Type of test	Experimental test system	Test substance	Exposure conditions	Result	WG considerations and conclusion	Reference
In vitro chromosomal aberration	Human lymphocytes (pooled blood from healthy male non‐smoking donors)	Triazine amine IN‐A4098 (97%) Negative control: solvent DMSO (2% v/v)	1st exp.: −S9: 3h expo., harvesting after 18 h, 0 (DMSO), 110.88, 184.8 and 308 μg/mL; +S9 (2% v/v): 3 h expo., harvesting after 18 h, 0 (DMSO), 110.88, 184.8 and 308 μg/mL 2nd exp.: −S9: 21 h expo., harvesting at end of treatment, 0 (DMSO), 66.33, 184.8 and 308 μg/mL; +S9 (5% v/v): 3h expo., harvesting after 18 h, 0 (DMSO), 110.88, 184.8 and 308 μg/mL Duplicate cultures	No stat. significant increase in % aberrant metaphases No stat. significant increase in proportion of polyploid cells 1st exp: No reduction in MI at 308 μg/ml with or without S9 2nd exp: decrease of MI at the two high doses w/o S9 (MI: 75% and 59% of neg. control, respectively)	GLP compliant OECD TG 473 (1997) Solubility of IN‐A4098 in DMSO: up to 15.4 mg/mL (308 μg/mL = limit solubility) 2% v/v DMSO added to the culture medium instead of 1% v/v No precipitation, no fluctuation in osmolality or change in pH Cytotoxicity: calculation of MI (not for pos. control treated cultures or cultures where no signs of cytotoxicity) 200 metaphases analysed/dose (according to OECD TG 473, 2016: at least 300 metaphases should be analysed) Minor deviations from current OECD requirements which do not impact the conclusion Negative	Woods (2011b)
In vitro chromosomal aberration	Human lymphocytes (1 healthy non‐smoking 23‐year‐old female donor)	Triazine amine IN‐A4098 (98.7%) Negative control: solvent DMSO	Preliminary toxicity test: max. dose tested: 1,400 μg/mL, with or without S9 −S9: expo. 4 h and 20 h, +S9: 4 h Main test: 1st exp.: −S9: 4 h expo., harvesting 20 h after exposure, 0 (DMSO), 500, 1,000 and 1,400 μg/mL; +S9 (2% v/v): 4 h expo., harvesting 20 h after exposure, 0 (DMSO), 500, 1,000 and 1,400 μg/mL 2nd exp.: −S9: 20 h expo., harvesting at end of treatment, 0 (DMSO), 500, 1,000 and 1,400 μg/mL; Duplicate cultures	Visible precipitate at ≥ 420 μg/mL. Dose levels ≤ 140 μg/mL were soluble in treatment medium. Absence of toxicity No stat. significant increase in the % of cells with structural or numerical aberrations Visible precipitate at ≥ 250 μg/mL, dose level of 125 μg/mL was soluble in treatment medium at beginning of treatment, visible; precipitate at conclusion of treatment at ≥ 500 μg/mL, doses ≤ 250 μg/mL were soluble MI: decrease up to 53% in 1st exp. ‐S9, up to 61% in 1st exp +S9, up to 57% in 2nd exp. –S9	GLP compliant OECD TG 473 (1997) Solubility of IN‐A4098 in DMSO: at 150 mg/mL Only conc. above the solubility limit of TA in the culture medium have been tested 200 metaphases analysed/dose (according to OECD 473, 2016: at least 300 metaphases should be analysed) Minor deviations from current OECD requirements which do not impact the conclusion Negative	Gudi and Rao (2009)
In vitro chromosomal aberration	Human lymphocytes (healthy donors)	Triazine amine IN‐A4098 (99.5%) Negative control: solvent DMSO	Preliminary cytotoxicity: 0.1–100 μg/mL Main test: 1st exp.: −S9: 4 h expo., harvesting 20 h later, 0 (DMSO), 25, 50, 100 and 200 μg/mL; + S9 (10% v/v): 4 h expo., harvesting 20 h later, 0 (DMSO), 25, 50, 100 and 200 μg/mL 2nd exp.: −S9: 24 h expo., harvesting at end of treatment, 0 (DMSO), 25, 50, 100 and 200 μg/mL; + S9 (10% v/v): 4 h expo., harvesting 20 h later, 0 (DMSO), 25, 50, 100 and 200 μg/mL Duplicate cultures	MI: −S9: 65% of negative control at HD, +S9: 85% of negative control at HD No stat. significant increase in the % of cells with aberrations excluding gaps No test item‐related polyploidy or endoreduplication Precipitation at the HD 200 μg/mL, no signs of cytotoxicity 1st exp: −S9 MI: 134% of negative control at HD, +S9: 90% of negative control at HD; 2nd exp: −S9 MI: 98% of negative control at HD, +S9: 107% of negative control at HD	GLP compliant OECD TG 473 (1997) Solubility of IN‐A4098 in DMSO: 10 mg/mL Max. conc. Achieved: 200 μg/mL 200 metaphases analysed/dose (according to OECD 473, 2016: at least 300 metaphases should be analysed) Minor deviations from current OECD requirements which do not impact the conclusion Negative	Flügge (2011c)
In vitro chromosomal aberration	Human lymphocytes	Triazine amine CGA 150 829 tech. (97%) Negative control: solvent DMSO (1% v/v)	Preliminary cytotoxicity: 0.0122–100 μg/mL Main test: − S9: 3 h expo., harvesting 44 h later, 0 (DMSO), 0.625, 1.25, 2.5, 5 & 10 μg/mL; +S9 (10% v/v): 3 h expo., harvesting 44 h later, 0 (DMSO), 6.25, 12.5, 25, 50 & 100 μg/mL	−S9 MI: 37% of negative control at HD, +S9 MI: 75% of negative control at HD −S9: No increase in the % of aberrant metaphases +S9: no dose‐related increase in the % of aberrant cells No increase in polyploidy or aneuploid metaphases Positive control: no clear increase in the % aberrant cells in test +S9	Solubility in DMSO: 10 mg/mL No determination of solubility limit in culture medium Cytotoxicity not concurrently measured 100 metaphases analysed/dose (according to OECD TG 473, 2016: at least 300 metaphases should be analysed) No repetition of the experiment No experiment with an extended treatment period No statistical evaluation Major deviations from current OECD requirements which impact the conclusion Negative but not reliable	Dollenmeier (1987)
In vitro chromosomal aberration	CHO cells (CCL 61)	Triazine amine CGA 150 829 tech. (99.4%) Negative control: solvent DMSO (1% v/v)	Cytotoxicity: 0.78–100 μg/mL Main test: Original study: Exp 1 −S9: 25, 50 & 100 μg/mL, expo: 18 h, harvesting at end of treatment; Exp 2 +S9 (1.5%): 25, 50 & 100 μg/mL, expo: 3 h, recovery: 15 h later Confirmatory study: Exp 1 −S9: 25, 50 & 100 μg/mL, expo: 18 h, harvesting at end of treatment; Exp 2 +S9 (1.5%): 25, 50 & 100 μg/mL, expo: 3 h, recovery: 15 h later Exp 3 −S9: 25, 50 & 100 μg/mL, expo: 42 h, harvesting at end of treatment; Exp 4 +S9 (1.5%): 25, 50 & 100 μg/mL, expo: 3 h, recovery: 39 h later	No increase in the % of cells with aberrations Original study: Exp 1: HD: MI = 97% of negative control Exp 2: HD: MI = 73% of negative control Confirmatory study: Exp 3 HD: MI = 35% of negative control Exp 4 HD: MI = 98% of negative control	GLP compliant OECD TG 473 (1983) Substance dissolved in DMSO for 2 h by ultrasonic treatment. Neither the solubility limit of triazine amine in DMSO nor the solubility limit of triazine amine in culture medium were determined 200 metaphases analysed/dose and only 50 metaphases in the positive control (according to OECD TG 473, 2016: at least 300 metaphases should be analysed) No statistical evaluation Absence of data allowing to determine if the concentrations used were sufficiently high or not Major deviations from current OECD requirements which impact the conclusion Negative but not reliable	Meyer (1991)

DMSO: dimethyl sulfoxide; S9: metabolic activation; MI: mitotic index; HD: high dose.