N-Phenyl-diethanolamine, reaction products with formaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Positive - OECD 471 (Ames) - Gijsbrechts (2020)

Positive - OECD 487 (MNT) - de Jong (2020)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 17 March 2020 to 16 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was conducted in accordance with the relevant OECD test guideline and in accordance with GLP. All validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9:
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and
2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively
- method of preparation of S9 mix
S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per
10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg
glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.

Test concentrations with justification for top dose:

In the first mutation experiment, the test item was tested up to concentrations of
1600 μg/plate in the strains TA1535, TA1537 and TA98. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.
In the second mutation experiment, the test item was tested up to concentrations of
1600 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and up to 5000 μg/plate in the tester strain WP2uvrA in the pre-incubation assay. The test item did not precipitate on the plates. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (Merck, Darmstadt, Germany)
Saline = physiological saline (Eurovet Animal Health, Bladel, The Netherlands), used for TA1535 without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 4-nitroquinoline N-oxide; 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1E9 cells/mL
- Test substance added in medium; in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 ± 2 minutes (pre-incubation assays only)
- Exposure duration/duration of treatment: 48 ± 4 h
- Harvest time after the end of treatment (sampling/recovery times): Not specified


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- background growth inhibition; decreased in number of revertants and/or presence of microcolonies
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- dose-related increase in the number of revertant colonies compared to the solvent control

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Table 1: Dose-Range Finding Test:  Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

Direct Plate Assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with one Salmonella typhimurium and one Escherichia coli strain.
	TA100	WP2uvrA

Without S9-mix

Positive control	773	±	176		1418	±	112
Solvent control	120	±	10		13	±	1
1.7	112	±	14		17	±	5
5.4	118	±	25		23	±	8
17	123	±	14		20	±	7
52	123	±	23		19	±	5
164	115	±	25		26	±	4
512	113	±	8	^n	20	±	3
1600				^e MC	18	±	4	^n
5000	0	±	0	^a NP				^e NP  MC

With S9-mix

Positive control	1104	±	304		297	±	84
Solvent control	88	±	8		22	±	7
1.7	121	±	30		22	±	6
5.4	119	±	18		20	±	2
17	110	±	30		20	±	9
52	117	±	6		20	±	2
164	93	±	26		26	±	4
512	118	±	34	^n	27	±	6
1600				^e MC	39	±	6	^n
5000	0	±	0	^a NP	25	±	5	^s NP

 

MC	Microcolonies
NP	No precipitate
a	Bacterial background lawn absent
e	Bacterial background lawn extremely reduced
n	Normal bacterial background lawn
s	Bacterial background lawn slightly reduced

 

Table 2          
Experiment 1:  Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay

Direct Plate Assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium.
	TA1535	TA1537	TA98

Without S9-mix

Positive control	1014	±	53		1120	±	9		1292	±	136
Solvent control	9	±	3		6	±	5		10	±	1
5.4	11	±	8		2	±	2		13	±	5
17	8	±	2		6	±	4		9	±	4
52	9	±	5		4	±	4		8	±	2
164	10	±	2		9	±	7		13	±	3
512	11	±	9	^n	6	±	4	^n	23	±	5	^n
1600	5	±	2	^m NP	4	±	1	^m NP				^e NP  MC

With S9-mix

Positive control	354	±	24		311	±	23		1656	±	337
Solvent control	15	±	11		5	±	3		16	±	3
5.4	11	±	4		6	±	6		21	±	3
17	15	±	8		7	±	3		16	±	2
52	14	±	4		6	±	4		17	±	5
164	9	±	4		7	±	5		18	±	3
512	16	±	6	^n	7	±	0	^n	26	±	7	^n
1600	7	±	4	^m NP				^e NP  MC	27	±	13	^s NP

 

MC	Microcolonies
NP	No precipitate
e	Bacterial background lawn extremely reduced
m	Bacterial background lawn moderately reduced
n	Normal bacterial background lawn
s	Bacterial background lawn slightly reduced

Table 3          
Experiment 2:  Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

Pre-incubation Assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain.
	TA1535	TA1537	TA98	TA100	WP2uvrA

Without S9-mix

Positive control	907	±	31		136	±	13		1879	±	59		558	±	68		1818	±	34
Solvent control	4	±	0		7	±	1		12	±	2		92	±	9		15	±	4
5.4	8	±	0		7	±	2		5	±	4		83	±	9			-
17	7	±	6		7	±	4		6	±	3		103	±	13		16	±	7
52	8	±	5		10	±	3		20	±	6		192	±	11		15	±	4
164	12	±	5	^n	5	±	0	^n	40	±	6	^n	274	±	39	^n	23	±	4
512	15	±	4	^s	5	±	2	^s	16	±	3	^s	83	±	30	^s	46	±	8	^n
1600				^a NP				^e NP  MC				^a NP				^a NP	9	±	6	^s
5000		-				-				-				-						^a NP

With S9-mix

Positive control	154	±	14		61	±	15		403	±	20		1088	±	28		536	±	16
Solvent control	7	±	2		4	±	3		13	±	6		56	±	4		18	±	6
5.4	11	±	6		6	±	2		19	±	4		83	±	20			-
17	7	±	4		9	±	2		18	±	10		78	±	8		18	±	2
52	8	±	4		7	±	3		30	±	10		100	±	8		23	±	8
164	13	±	7	^n	4	±	3	^n	58	±	4		184	±	60	^n	31	±	4
512	13	±	2	^s	3	±	1	^s	46	±	15	^n	96	±	6	^s	52	±	4	^n
1600				^e NP  MC				^e NP  MC				^e NP  MC				^a NP	7	±	4	^s
5000		-				-				-				-						^a NP

 

 

MC	Microcolonies
NP	No precipitate
a	Bacterial background lawn absent
e	Bacterial background lawn extremely reduced
n	Normal bacterial background lawn
s	Bacterial background lawn slightly reduced
-	Not tested

 

 

 

 

Conclusions:

In conclusion, based on the results of this study it is concluded that the test item is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of the test item and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonellatyphimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP₂uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). 

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP₂uvrA in the direct plate assay.  The test item did not precipitate on the plates at this dose level.  Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in both tester strains in the absence and presence of S9-mix. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested up to concentrations of
1600 µg/plate in the strains TA1535, TA1537 and TA98.  The test item did not precipitate on the plates at this dose level.  Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. 

In the second mutation experiment, the test item was tested up to concentrations of
1600 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and up to 5000 µg/plate in the tester strain WP₂uvrA in the pre-incubation assay.  The test item did not precipitate on the plates. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. 

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate, and that the metabolic activation system functioned properly.

In the second experiment (pre-incubation method), the test item induced dose-related increases in the number of revertant colonies compared to the solvent control in tester strains TA98, TA100 and WP₂uvrA

In tester strain TA98, the increases observed where up to 3.3- fold and 4.5- fold the concurrent control, in the absence and presence of S9-mix, respectively.

In tester strain TA100, the increases observed where up to 3.0- fold and 3.3- fold the concurrent control, in the absence and presence of S9-mix, respectively.

In tester strain WP₂uvrA, the increases observed where up to 3.1- fold and 2.9- fold the concurrent control, in the absence and presence of S9-mix, respectively.

Although the increases in tester strains TA98 and WP₂uvrA were within the laboratory historical control data range, the responses were more than two- and three-fold the concurrent controls in strains WP₂uvrA and TA98, respectively. In addition, the increases in tester strain TA100 were outside the historical control data range and more than two-fold the concurrent controls. Based on these observations, the increases were considered biologically relevant. 

In conclusion, based on the results of this study it is concluded that the test item is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. 

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 March 2020 - 24 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was conducted in accordance with the relevant OECD test guideline and in accordance with GLP. All validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

N/A

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 µmol HEPES (Life Technologies).
The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL) phytohaemagglutinin).
- concentration or volume of S9 mix and S9 in the final culture medium: The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):
WITHOUT metabolic activation: Mitomycin C (MMC-C; CAS No. 50-07-7, Sigma, Zwijndrecht, The Netherlands) was used as a direct acting clastogen at a final concentration of 0.25 and 0.38 µg/mL for a 3 hour exposure period and 0.15 and 0.23 µg/mL for a 24 hour exposure period. Colchicine (Colch; CAS No. 64-86-8, Acros Organics, Geel, Belgium) was used as a direct acting aneugen at a final concentration of 0.1 µg/mL for a 3 hour exposure period and
0.05 µg/mL for a 24 hour exposure period.
WITH metabolic activation: Cyclophosphamide (CP; CAS No. 50-18-0, Sigma) was used as an indirect acting clastogen, requiring metabolic activation, at a final concentration of 15 and 17.5 µg/mL for a 3 hour exposure period.

Test concentrations with justification for top dose:

First cytogenetic assay (Based on the results of the second dose-range finding test):
Without S9-mix: 10, 25, 50, 70, 90 and 110 µg/mL culture medium (3 hours exposure time, 27 hours harvest time).
With S9-mix: 10, 70, 90, 110, 130 and 150 µg/mL culture medium (3 hours exposure time, 27 hours harvest time).
The following dose levels were selected for scoring of micronuclei:
Without S9-mix : 25, 50 and 70 µg/mL culture medium
(3 hours exposure time, 27 hours harvest time).
With S9-mix : 10, 70 and 90 µg/mL culture medium
(3 hours exposure time, 27 hours harvest time).

Second cytogenetic assay:
To obtain more information about the possible clastogenicity and aneugenicity of the test item, a second cytogenetic assay was performed in which human lymphocytes were exposed for 24 hours in the absence of S9-mix. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix: 5, 25, 35, 45, 55, 65 and 75 µg/mL culture medium (24 hours exposure time, 24 hours harvest time).
The following dose levels were selected for the scoring of micronuclei:
Without S9-mix : 5, 45 and 55 µg/mL culture medium
(24 hours exposure time, 24 hours harvest time).

In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose-range finding test. The test item was tested in the absence and presence of S9-mix.
The highest tested concentration was the recommended 5000 µg/mL.
Based on the results of the second dose-range finding test (see 'Details on test system and experimental conditions' for further information on the dose-range finding tests) an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
Vehicle: dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany).
Solvent for positive controls: Hanks’ Balanced Salt Solution (HBSS) (Life Technologies, Bleiswijk, The Netherlands), without calcium and magnesium.
All reference stock solutions were stored in aliquots at ≤-15°C in the dark. These solutions were thawed immediately before use.

- Justification for choice of solvent/vehicle:
DMSO: Recommended in the OECD test guideline.

- Justification for percentage of solvent in the final culture medium: The final concentration of the solvent in the culture medium was 1.0% (v/v). This percentage adheres to the recommendation in the OECD test guideline.

Untreated negative controls:

yes

Remarks:

(vehicle)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Duplicate
- Number of independent experiments: Two cytogenetic assay runs

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): N/A
- Test substance added in medium: Culture medium consisted of RPMI 1640 medium (Life Technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies), L-glutamine (2 mM) (Life Technologies), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) (Life Technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).
No correction was made for the purity/composition of the test item. Test item concentrations were used within 1 hour after preparation.

First cytogenetic assay:
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of the test item for 3 hours in the absence and presence of S9-mix. After 3 hours exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium with Cytochalasin B (5 µg/mL) and incubated for another 24 hours. Appropriate vehicle and positive controls were included in the first cytogenetic assay.

Second cytogenetic assay:
To confirm the results of the first cytogenetic assay a second cytogenetic assay was performed with an extended exposure time of the cells in the absence of S9-mix.
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of the test item with cytochalasin B (5 µg/mL) for 24 hours in the absence of S9-mix. Appropriate vehicle and positive controls were included in the second cytogenetic assay.
 
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: N/A
- Exposure duration/duration of treatment:
First cytogenetic assay: 3 h exposure; followed by 24 h incubation
Second cytogenetic assay: 24 h exposure

- Harvest time after the end of treatment (sampling/recovery times):
First cytogenetic assay: 27 h
Second cytogenetic assay: 24 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): N/A
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure:
Prior to the mitosis (during or after exposure of the test item) the chemical cytochalasin B was added to the cultures to arrest the formation of actin filaments.
First cytogenetic assay: 5 µg/mL, 24 h incubation
Second cytogenetic assay: 5 µg/mL, 24 h incubation

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for
10 - 30 min with 6.7% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): A minimum of 500 cells (with a maximum deviation of 5%) per culture was counted. Three analyzable concentrations were scored for micronuclei.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
To prevent bias, all slides were randomly coded before examination of micronuclei and scored. An adhesive label with Charles River Den Bosch study identification number and code was stuck over the marked slide. At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture were scored for micronuclei separately. Since the lowest concentration of MMC-C and CP resulted in a positive response the highest concentration was not examined for the presence of micronuclei. Due to cytotoxicity the number of examined bi- or mononucleated cells in the positive control groups might be <1000. However, when an expected statistically significant increase was observed, this has no effect on the study integrity.
The following criteria for scoring of binucleated cells were used (1 - 2, 6):
• Main nuclei that were separate and of approximately equal size.
• Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
• Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
• Trinucleated, quadranucleated, or multinucleated cells.
• Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996 (1):
• The diameter of micronuclei should be less than one-third of the main nucleus.
• Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
• Micronuclei should have similar staining as the main nucleus.

- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): N/A
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index.
The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).
%Cytostasis = 100-100{(CBPIt – 1)/(CBPIc –1)}

CBPI = (No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells) / Total number of cells

t = test item or control treatment culture
c = vehicle control culture

- Any supplementary information relevant to cytotoxicity: Three analyzable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded. The highest dose level examined for micronuclei were the cultures that produced 55 ± 5% cytotoxicity. The lowest dose level had little or no cytotoxicity (approximately the same as solvent control). Also, cultures treated with an intermediate dose level were examined.


OTHER:
Dose-range finding test:
Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 ± 2 h and thereafter exposed to selected doses of the test item for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasin B (Sigma; 5 µg/mL) was added to the cells simultaneously with the test item at the 24 hours exposure time. A vehicle control was included at each exposure time.
The highest tested concentration was 5000 µg/mL.
After 3 hours exposure to the test item in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium with Cytochalasin B and incubated for another 24 hours (1.5 times normal cell cycle). The cells that were exposed for 24 hours in the absence of S9-mix were not rinsed after exposure but were fixed immediately.
Cytotoxicity of the test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
As the test item showed steep toxicity, the dose-range finding experiment was repeated. Based on the results of the second dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control.

Rationale for test conditions:

The test conditions were based on the recommendations from the OECD test guideline. The highest tested concentration was the recommended 5000 µg/mL for UVCB substances.
Environmental conditions: All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 39 - 94%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.7 - 38.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.
General test conditions were implemented in accordance with the OECD test guideline.

Evaluation criteria:

Acceptability criteria:
An in vitro micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item colchicine induces a statistically significant increase in the number of mononucleated cells with micronuclei and the positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analyzed by the Chi-square test (one-sided, p < 0.05).

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) and ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) were used for statistical analysis of the data.
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
The Chi-square test showed that there are statistically significant differences between one or more of the test item groups and the vehicle control group. Therefore, a Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.

Key result

Species / strain:

lymphocytes: first assay

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: second assay

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH and the osmolarity of the culture medium containing the highest tested concentration were recorded. No confounding effects reported.
- Data on osmolality: No confounding effects reported.
- Possibility of evaporation from medium: No confounding effects reported.
- Water solubility: A solubility test was performed based on visual assessment at the test item prepration stage. No confounding effects reported.
- Precipitation and time of the determination: No precipitation was seen in the culture medium at the highest concentration tested (5000 µg/L in the dose-range finder)
- Definition of acceptable cells for analysis: Cells with one, two or more nuclei (multinucleated cells)
- Other confounding effects: N/A

RANGE-FINDING/SCREENING STUDIES (if applicable):
A concentration of 5000 µg/mL showed no precipitation in the culture medium and was used as the highest concentration of the test item.
In the dose-range finding test blood cultures were treated with 157, 313, 625, 1250, 2500 and 5000 µg test item/mL culture medium and exposed for 3 and 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.
The pH and osmolarity of a concentration of 5000 µg/mL were 7.4 and 394 mOsm/kg respectively (compared to 7.5 and 439 mOsm/kg in the solvent control).
Due to very steep toxicity after exposure to the test item, the dose-range finding test was repeated. The blood cultures were treated with 4.7, 9.4, 18.8, 37.5, 75 and 150 µg test item/mL culture medium and exposed for 3 and 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.

STUDY RESULTS
- Concurrent vehicle negative and positive control data.
Negative control: The number of mono- and binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control database.
Positive control: The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: A dose related response was observed during the first cytogenetic assay, in the absence of S9-mix (where the test item induced a statistically significant increase in the number of binucleated cells with micronuclei at the highest dose).
- Statistical analysis; p-value if any: Significant trends observed = p < 0.001
- Any other criteria: e.g. GEF for MLA: N/A

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o In the case of the cytokinesis-block method: CBPI or RI; distribution of mono-, bi- and multi-nucleated cells:
o Other observations when applicable (complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells)

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate:


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see Any other information on results incl. tables; Table 1
- Negative (solvent/vehicle) historical control data: see Any other information on results incl. tables; Table 2

Remarks on result:

other: Positive

Table 1. Historical Control Data for in vitro Micronucleus Studies of the Positive Control Substances

	-S9 Mix		-S9 Mix		+S9 mix
	3 hour exposure	24 hour exposure	3 hour exposure	24 hour exposure	3 hour exposure
Mean number of micronucleated cells (per 2000 cells)	49.1	58.0	46.9	42.4	34.1
SD	26.5	29.1	20.1	17.3	13.8
n	89	87	92	94	98
Lower Control Limit (95% Control Limits)	-3	1	7	8	7
Upper Control Limit (95% Control Limits)	101	115	86	76	61

SD = Standard deviation; n = Number of observations; Distribution historical positive control data from experiments performed between June 2017 and June 2020.

Table 2. Historical Control Data for in vitro Micronucleus Studies of the Solvent Control

	Mononucleated			Binucleated
	-S9 Mix		+S9 mix	-S9 Mix		+S9 mix
	3 hour exposure	24 hour exposure	3 hour exposure	3 hour exposure	24 hour exposure	3 hour exposure
Mean number of micronucleated cells (per 2000 cells)	1.4	1.4	1.7	6.1	5.8	6.4
SD	1.4	1.2	2.4	4.1	4.1	4.1
n	89	87	91	89	87	91
Lower Control Limit (95% Control Limits)	-1	-1	-3	-2	-2	-2
Upper Control Limit (95% Control Limits)	4	4	6	14	14	14

SD = Standard deviation; n = Number of observations; Distribution historical negative control data from experiments performed between June 2017 and June 2020.

 

Table 3. Cytokinesis-Block Proliferation Index of Human Lymphocytes Cultures Treated with Test Item the First Cytogenetic Assay

Without metabolic activation (-S9-mix), 3 hours exposure time, 27 hours harvest time
Concentration µg/mL	CBPI	Mean CBPI		% cytostasis
0	1.66 - 1.67	1.66		0
10	1.52 - 1.53	1.53		21
25	1.55 - 1.58	1.57		15
50	1.43 - 1.46	1.44		33
70	1.32 - 1.32	1.32		52
90	1.18 - 1.20	1.19		71
110	1.09 - 1.10	1.10		86
0.25 MMC-C	1.43 - 1.48	1.46		31
0.38 MMC-C	1.31 - 1.31	1.31		53
0.1 Colch	1.18 - 1.20	1.19		71
With metabolic activation (+S9-mix), 3 hours exposure time, 27 hours harvest time
Concentration µg/mL	CBPI	Mean CBPI		% cytostasis
0	1.70 - 1.72	1.71		0
10	1.66 - 1.68	1.67		6
70	1.37 - 1.40	1.39		45
90	1.27- 1.34	1.31		57
110	1.17 - 1.30	1.23		67
130	1.13 - 1.25	1.19		74
150	1.09 - 1.14	1.12		83
15 CP	1.24 - 1.34	1.29		59
17.5 CP	1.15 - 1.26	1.20		72

All calculations were performed without rounding off

Table 4. Number of Mononucleated or Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with Test Item in the First Cytogenetic Assay 

Without metabolic activation (-S9-mix), 3 hours exposure time, 27 hours harvest time
Concentration (µg/mL)	Cytostasis (%)	Number of mononucleated cells with micronuclei^1)			Number of binucleated cells with micronuclei ^1)
		1000	1000	2000	1000		1000	2000
		A	B	A+B	A		B	A+B
0	0	0	1	1	1		2	3
25	15	0	0	0	3		2	5
50	33	1	1	2	4		3	7
70	52	0	0	0	8		12	20^***
0.25 MMC-C	31	1	1	2	13		27	40^***
0.1 Colch	71	12	9	21^***	5^2)		2^2)	7

With metabolic activation (+S9-mix), 3 hours exposure time, 27 hours harvest time
Concentration (µg/mL)	Cytostasis (%)	Number of mononucleated cells with micronuclei^1)			Number of binucleated cells with micronuclei ^1)
		1000	1000	2000	1000		1000	2000
		A	B	A+B	A		B	A+B
0	0	0	0	0	2		3	5
10	6	0	1	1	4		5	9
70	45	0	0	0	5		5	10
90	57	0	1	1	13		14	27^***
15 CP	59	2	1	3^*	17		25	42^***

*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

¹⁾  1000 bi- and mononucleated cells were scored for the presence of micronuclei.

    Duplicate cultures are indicated by A and B.

²⁾ 891 and 723 binucleated cells were scored for the presence of micronuclei, respectively.

 

Table 5. Cytokinesis-Block Proliferation Index of Human Lymphocyte Cultures Treated with Test Item in the Second Cytogenetic Assay

Without metabolic activation (-S9-mix), 24 hours exposure time, 24 hours harvest time
Concentration µg/mL	CBPI	Mean CBPI		% cytostasis
						0	1.72 -1.74	1.73		0
5	1.66 - 1.70	1.68		6
25	1.57 - 1.62	1.59		19
35	1.64 - 1.66	1.65		11
45	1.47 - 1.49	1.48		34
55	1.35 - 1.37	1.36		51
65	1.26 - 1.27	1.27		63
75	1.14 - 1.17	1.16		79
0.15 MMC-C	1.32 - 1.39	1.36		51
0.23 MMC-C	1.23 - 1.28	1.26		64
0.05 Colch	1.04 - 1.04	1.04		95

All calculations were performed without rounding off

 

Table 6. Number of Mononucleated or Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with Test Item in the Second Cytogenetic Assay

Without metabolic activation (-S9-mix), 24 hours exposure time, 24 hours harvest time
Concentration (µg/mL)	Cytostasis (%)	Number of mononucleated cells with micronuclei^1)			Number of binucleated cells with micronuclei ^1)
		1000	1000	2000	1000		1000	2000
		A	B	A+B	A		B	A+B
0	0	0	0	0	2		6	8
5	6	0	0	0	0		3	3
45	34	0	0	0	3		0	3
55	51	1	0	1	3		5	8
0.15 MMC-C	51	2	1	3^*	22		18	40^***
0.05 Colch	95	22	17	39^***	14 ^2)		6 ^2)	20^***

*)   Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

¹⁾    1000 bi- and mononucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.

²⁾   485 and 212 binucleated cells were scored for the presence of micronuclei, respectively.

Conclusions:

Positive: the test item induced the formation of micronuclei in human lymphocytes under the experimental conditions described in this study. This effect was confined to the 3 h exposure duration.

Executive summary:

A test was performed in accordance with OECD 487, in order to evaluate the ability of the test item to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix).

The potential for clastogenicity and aneugenicity of the test item was tested in two independent experiments.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the dose range of the test item was 157 to 5000 µg/mL, which was repeated due steep toxicity to 4.7 to 15 µg/mL. The results indicated that the maximum concentration should be limited on toxicity. 

In the first cytogenetic assay, the test item was tested up to for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction, respectively. Appropriate toxicity was reached at these dose levels.

In the second cytogenetic assay, the test item was tested up to 55 µg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The negative/solvent control cultures were within the 95% control limits of the distribution of the historical negative control database. The positive control substances were within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the first cytogenetic assay, the test item induced statistically significant increases in the number of binucleated cells with micronuclei, both in the absence and presence of S9-mix. In addition, dose related trends were observed (p = 0.003 and p < 0.001 in the absence and presence of S9-mix, respectively) and the number of binucleated cells with micronuclei was outside the 95% control limits of the distribution of the historical negative control database. Therefore, these increases were considered biologically relevant. The test item did not induce a statistically significant or biologically relevant increase in the number of mononucleated cells with micronuclei.

In the second cytogenetic assay with a 24 hours continuous exposure time, the test item did not induce a statistically significant and biologically relevant increase in the number of mono- and binucleated cells with micronuclei.

In conclusion, this testing system is valid and the test item induces the formation of micronuclei in human lymphocytes under the experimental conditions described in this study. This effect was confined to the 3 hour exposure duration.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Negative - OECD 474 (MNT in vivo) - de Jong (2021)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 July 2020 - 23 March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Similar to OECD 474 guideline - the study was conducted similar to the guideline study, but incorporated into an OECD 422 study. All validity criteria were met under both guidelines. The study was conducted under GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

This study was combined with the main OECD 422 study.

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

rat

Strain:

other: Crl:WI (Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental and micronucleus (bone marrow) historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Males: 11-12 weeks, Females: 13-14 weeks
- Weight at study initiation: Males: 297 and 352g and females 191 and 243g
- Assigned to test groups randomly: yes
- Fasting period before study: Animals had no access to food for a maximum of 2 hours during activity measurements.
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages (MIII type, height 18 cm).During the lactation phase, females were housed in Makrolon plastic cages (MIII type, height 18 cm).
- Diet : Pelleted roden diet provided ad libitum
- Water: ad libitum
- Acclimation period: The animals were allowed to acclimate for 6 days prior to start of the pretest period (females) or 6 days before the commencement of dosing (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24 degC
- Humidity (%): 40 to 70%
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study.
- Concentration of test material in vehicle: Not stated
- Amount of vehicle (if gavage or dermal): Not stated
- Type and concentration of dispersant aid (if powder): N/A
- Lot/batch no. (if required): Not stated
- Purity: Not stated

Duration of treatment / exposure:

- Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period
- Positive control animals (5 males) were dosed once by oral gavage at 48 (± 1h) hours prior isolation of the bone marrow
- Females that delivered were treated for 50-64 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy
- Females which failed to deliver were treated for 41-44 days

Frequency of treatment:

The test item was administered once daily

Dose / conc.:

19 mg/kg bw/day (nominal)

Remarks:

Cyclophosphamide (positive control)

Dose / conc.:

650 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control: Not stated
- Route of administration: Oral gavage
- Dose: 19 mg/kg/day

Evaluation criteria:

A test item is considered positive in the micronucleus test if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided,
p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

Statistics:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data. Appropriate statistical tests were selected for data evaluation.

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

See Table 2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

See table 2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Dose range finder was completed over 10 days at doses of 500 and 1000 mg/kg/day
- Solubility: Not stated
- Clinical signs of toxicity in test animals: Salivation was noted in animals in the 5000 mg/kg/day dose group (3/3 animals) directly after dosing on days 3 and 5- 10. Animals in the 1000 mg/kg/day group were noted to have hunched posture (3/3 animals, most or all time points on Days 1 to 8), lethargy (2/3 animals, 1 or 3 hours after dosing on Days 1, 3 and/or 5), flat posture (1/3 animals, 1 hour after dosing on Day 3), uncoordinated movements (2/3 animals, seen incidentally but at all time points on Days 2, 3, 4, 5 and/or 6), piloerection (2/3 animals, once 1 hour after dosing on Day 1 for one animal and multiple times at all different time points on Days 2 to 6 and Days 8 and 9 for another animal), hypothermia (1/3 animals, 3 hours after dosing on Day 3), deep respiration and ptosis (1/3 animals at 1 hour after dosing on Day 1), salivation (3/3 animals, directly after dosing on Days 2 to 10).
- Evidence of cytotoxicity in tissue analysed: Not examined
- Rationale for exposure: the dose range finder was conducted to select dose levels for the main study and to determine the peak effect of occurrence of clinical signs following dosing.
- Harvest times: N/A
- High dose with and without activation: N/A
- Other: N/A

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): N/A
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay): The groups that were treated with N-Phenyldiethanolamine, reaction products with formaldehyde (0 mg/kg/day and 650 mg/kg/day) showed no decrease in the ratio of polychromatic compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test item. The group that was treated with cyclophosphamide (positive control) showed an expected decrease in the ratio of polychromatic compared to the vehicle control, demonstrating toxic effects on erythropoiesis
- Appropriateness of dose levels and route: Not specified
- Statistical evaluation: Micronuclei Assessment - A Welch t test was used for inhomoheneous variances. Ratio polychromatic/normochromatic erythrocytes – Student t test for homogeneous variances.

Table 2: Clinical signs of toxicity noted during study period

Sign (max grade) Location		Premating																										Repro period
	Week	1													2													1												2															3
	Day	1	2		3		4		5		6		7		1	2		3		4		5		6		7		1	2		3		4	5		6		7		1		2		3		4		5			6		7		1
Males
Group 1 (control)1
Skin/fur Scabs (3)	G	.		.		.		1		1		1		1			1		.	.	.		.		.		.	.		.		.			.		.		.		.		.		.		.		.	.		.		.	.
	%	.		.		.		1		1		1		1			1		.	.	.		.		.		.	.		.		.			.		.		.		.		.		.		.		.	.		.		.	.
Scabs(3) (neck)	G	.		.		.		.		.		.		.			.		1	1	1		1		1		1	.		.		.			.		.		.		.		.		.		.		.	.		.		.	.
	%	.		.		.		.		.		.		.			.		1	1	1		1		1		1	.		.		.			.		.		.		.		.		.		.		.	.		.		.	.
Group 4 (650 mg/kg/day)
Behaviour lethargy (3)	G	1		.		1		1		.		1		.			.		1	1	1		1		.		1	.		1		.			1		1		1										1	1		1		.	.
	%	2		.		5		2		.		2		.			.		1	1	1		1		.		1	.		1		.			1		2		1		.		.		.		.		1	1		1		.	.

Posture Flat posture  (1)	G	.	.	1	1	.	1	.	.	1	1	1	1	1	.	.	1	.	.	1	1	.	.	.	.	1	1	1	.		.
	%	.	.	5	2	.	2	.	.	1	1	1	3	1	.	.	1	.	.	2	1	.	.	.	.	1	1	1	.		.
Hunched posture (1)	G	.	.	1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.
	%	.	.	1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.
Gait/mobility Uncoordinated movements (3)	G	1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	%	1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.
Breathing Rales (3)	G	.	1	1	1	.	.	.	.	.	.	.	1	.	.	.	.	.	.	.	1	.	.	.	.	.	.	1		1	1
	%	.	1	1	1	.	.	.	.	.	.	.	1	.	.	.	.	.	.	.	1	.	.	.	.	.	.	1		1	1
Skin/fur Piloerection (1)	G	.	1	.	.	.	.	.	.	1	1	1	.	.	1	.	.	.	.	1	1	.	.	.	.	.	.	.		.	.
	%	.	1	.	.	.	.	.	.	1	2	1	.	.	1	.	.	.	.	1	1	.	.	.	.	.	.	.		.	.
Alopecia (3)	G	.	.	.	1	1	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.
	%	.	.	.	1	1	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.
Scabs (3)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	1	1	1	1	1	1	1	1	1	1	1		1	1
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	2	2	2	2	2	2	2	2	2	2	2	2	4		4	4
Secretion/excretion Salivation	G	.	.	.	.	1	1	.	.	.	.	.	1	1	1	.	.	.	1	1	2	1	.	.	.	1	1	1		1	1
	%	.	.	.	.	3	7	.	.	.	.	.	6	8	1	.	.	.	2	8	8	1	.	.	.	8	7	8		6	6

		Premating															Repro period
	Week	1							2							1								2							3							4
	Day	1	2	3	4	5	6	7	1	2	3	4	5	6	7	1		2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3	4	5	6	7
Female
Group 1 (control)
Posture
Hunched posture (1)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	1	.
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	1	.
Skin / fur
Piloerection (1)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	1	.
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	2	2	2	.
Alopecia (3)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	1	1	1	1	1	1	1	1	1	1
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	1	1	1	1	1	1	1	1	1	1
Scabs (3)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
(Back)	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
Scabs (3)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
(Neck)	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
Group 4 (650 mg/kg/day)
Behavior
Lethargy (3)	G	1	1	1	.	.	1	.	.	1	1	1	.	.	.	.		3	.	1	1	.	1	.	1	.	1	1	1	1	1	1	1	1	.	.	.	1	.	1	1	1	.	1
	%	3	2	2	.	.	1	.	.	1	1	1	.	.	.	.		1	.	1	1	.	2	.	1	.	1	1	2	3	2	1	2	1	.	.	.	3	.	1	1	1	.	2
Posture
Flat posture (1)	G	1	1	1	.	.	1	.	.	1	1	1	.	1	.	.		1	.	.	.	.	1	.	.	.	1	1	1	1	1	1	1	1	.	.	.	1	.	1	1	.	.	1
	%	2	2	2	.	.	1	.	.	1	1	1	.	1	.	.		1	.	.	.	.	2	.	.	.	2	1	1	3	2	1	2	1	.	.	.	2	.	1	1	.	.	1
Hunched posture (1)	G	.	.	.	.	.	.	1	1	1	1	.	.	.	.	.		.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	.	.	.
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	.	.	.
Gait / motility
Uncoordinated movements (3)	G	1	1	.	.	.	.	.	.	.	.	1	.	.	.	.		.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	%	2	1	.	.	.	.	.	.	.	.	1	.	.	.	.		.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
Breathing
Rales	G	.	.	.	.	.	1	.	.	.	.	.	.	1	.	.		.	.	.	.	.	.	.	.	.	.	.	1	1	.	.	.	.	.	.	.	.	.	.	.	.	.
	%	.	.	.	.	.	1	.	.	.	.	.	.	1	.	.		.	.	.	.	.	.	.	.	.	.	.	1	2	.	.	.	.	.	.	.	.	.	.	.	.	.
Skin/fur
Piloerection	G	1	1	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	.	.	.	1	1	1	.	.	.	.	.	.	1	.	.	.	.	.	.	.	1	1	1	1	.
	%	2	1	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	.	.	.	1	2	2	.	.	.	.	.	.	1	.	.	.	.	.	.	.	1	1	4	4
Alopecia (3)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	1	1	1	1	1	1
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	1	1	1	1	1
Secretion/excretion
Salivation (3)	G	.	.	.	.	1	1	.	.	.	.	.	1	1	1	1		1	.	1	1	1	1	1	1	1	1	1	1	1	1	1	1	.	.	.	1	1	1	1	1	.	1	1
	%	.	.	.	.	1	1	.	.	.	.	.	2	3	1	1		1	.	1	3	4	2	2	3	2	3	5	5	5	4	3	A	.	.	.	5	4	1	4	1	.	1	4
Various
Ptosis (3)	G	.	1	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	%	.	1	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.

		Repro Period
	Week	5							6							7
	Day	1	2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3	4	5	6	7
Posture		.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
Hunched posture (1)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
Skin / fur
Piloerection (1)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
Alopecia (3)	G	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.
	%	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.
Scabs (3)	G	.	.	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.
(Back)	%	.	.	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.
Scabs (3) (Neck)	G	.	.	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.
	%	.	.	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.
Group 4 (650 mg/kg/day)
Behavior
Lethargy (3)	G	1	1	.	1	1	1	1	.	1	.	.	.	.	.	.	.	.	.
	%	2	1	.	1	2	2	1	.	2	.	.	.	.	.	.	.	.	.
Posture
Flat posture (1)	G	1	.	1	1	1	1	1	.	1	.	.	.
	%	2	.	1	1	2	1	1	.	2	.	.	.
Hunched posture (1)	G	.	.	.	.	.	.	.	.	.	.	.	.
	%	.	.	.	.	.	.	.	.	.	.	.	.
Gait / motility
Uncoordinated movements (3)	G	.	.	.	.	.	.	.	.	.	.	.	.
	%	.	.	.	.	.	.	.	.	.	.	.	.
Breathing
Rales	G	.	1	1	.	.	.	1	1	.	.	.	.
	%	.	1	1	.	.	.	1	1	.	.	.	.
Skin/fur
Piloerection	G	.	1	.	.	.	.	.	.	.	.	.	.
	%	.	2	.	.	.	.	.	.	.	.	.	.
Alopecia (3)	G	1	1	1	1	1	1	1	1	1	.	.	.
	%	1	1	1	1	1	1	1	1	1	.	.	.
Secretion/excretion
Salivation (3)	G	1	1	1	1	1	1	1	1	1	.	.	.
	%	7	4	1	9	A	9	2	6	2	.	.	.
Various
Ptosis (3)	G	.	.	.	.	.	.	.	.	.	.	.	.
	%	.	.	.	.	.	.	.	.	.	.	.	.

 

A number of clinical signs were noted during the study period in both males and females. In group 4, 650 mg/kg/day, lethargy and flat posture were recorded for 8 out of 10 males and all females. For each of these animals these signs occurred on a number of days during the treatment period.  Generally, these animals also showed hunched posture, rales, piloerection, uncoordinated movements and/or ptosis on a number of days during treatment, this is noted in table 2.

Additionally, no findings were noted during the weekly arena observations in this study.

Salivation seen after dosing among animals in group 4 and in the control groups. This was not determined to be a dose-related effect and was not considered toxicologically relevant. This was considered to be a physiological response rather than a sign of systemic toxicity and it was noted by the lab to be a common occurrence during oral gavage studies.

Other clinical signs outlined in Table 2 occurred within the range of background findings and is described as to be expected for rats of this age and strain which are housed and treated under the conditions in this study and it was concluded that it did not represent any dose-related trend.

Table 3: Mean number of micronucleated polychromatic erythrocytes and ratio of polychromatic/normochromatic erythrocytes

 

Group	Treatment	Dose Level (mg/kg/day)	Number of micronucleated polychromatic erythrocytes			Ratio polychromatic/ normochromatic erythrocytes
			(mean ± S.D.) ^(1,2)			(mean ± S.D.) ^(1,3)
	MALES
1	Vehicle Control	0	4.2	±	2.3	0.98	±	0.30
4	Test Item	650	4.1	±	1.3	1.30	±	0.24
5	CP	19	96.8	±	11.9 ^(4)	0.19	±	0.05 ^(5)
	FEMALES
1	Vehicle Control	0	4.7	±	2.1	1.09	±	0.27
4	Test Item	650	4.5	±	2.9	1.03	±	0.16

Vehicle control = DMSO

CP = Cyclophosphamide.

 

(1)        10 animals per treatment group, 5 animals in treatment group 5.

(2)        At least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of ±5%.

(3)        The ratio was determined from at least the first 1000 erythrocytes counted.

(4)        Significantly different from corresponding control group (Welch t test for inhomogeneous variances,

     P < 0.001).

(5)        Significantly different from corresponding control group (Students-t test for homogeneous variances,

     P < 0.001).

 

Table 4: Statistical Analysis 

Micronuclei Assessment:

Welch t test for inhomogeneous variances

Number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes; treatment/control comparison ¹⁾.

Group	Treatment	Dose Level	Test	P-value (one-sided)	Decision at 95% confidence level
5	CP	19 mg/kg (single dose)	Welch t test	< 0.001	significant

1) Only statistically significant results are presented.

 

 

Ratio polychromatic/normochromatic erythrocytes:

Students t test for homogeneous variances

Number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes; treatment/control comparison ¹⁾.

Group	Treatment	Dose Level	Test	P-value (one-sided)	Decision at 95% confidence level
5	CP	19 mg/kg (single dose)	Student’s t test	< 0.001	significant

1) Only statistically significant results are presented.

 

Table 5: historical negative control data for Micronucleus Studies 

 

 

	Male	Female	Male & Female
Mean Number of Micronucleated cells per 4000 cells	4.8	6.0	5.2
SD	2.6	2.3	2.5
n	26	13	39
Lower Control Limit (95% Control Limits)	0	1	0
Upper Control Limit (95% Control Limits)	10	11	10

 

SD = Standard deviation

n = Number of observations

Distribution historical negative control data from experiments performed between March 2018 and November 2020.

 

Table 6: Historical positive control data for micronucleus studies 

 

	Male	Female	Male & Female
Mean Number of Micronucleated cells per 4000 cells	61.7	82.2	66.6
SD	35.1	44.8	37.9
n	22	7	29
Lower Control Limit (95% Control Limits)	-7	-6	-8
Upper Control Limit (95% Control Limits)	130	170	141

 

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between March 2018 and November 2020

Conclusions:

Under the condition of the Mammalian Erythrocyte Micronucleus test method (474) the test substance did not show clastogenic or aneugenic in the bone marrow micronucleus test of rats up to a dose of 650 mg/kg/day. Therefore, the test substance cannot be classified according to CLP Regulation Reg. (EC) No 1907/2006.

Executive summary:

The study was performed according to OECD TG 474 to assess the clastogenicity and aneugenicity of the test item when administered to rats by measuring the increase in the number of micronucleated polychromatic erythrocytes in rat bone marrow. This study was completed as part of a wider OECD 422 Combined Repeat Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test which was conducted under GLP conditions. Both the 650 mg/kg/day group and control group, containing 10 males and 10 females, was administered the dose by oral gavage once daily for a minimum of 28 days. The positive control group (5 males) received 19 mg/kg/day cyclophosphamide while the control group received only the vehicle, Dimethyl sulfoxide at a dose volume of 1mL/kg. Clinical observations noted during the testing period are noted above in Table 2. The positive control males were dosed only by oral gavage at 48 (± 1h) hours prior to isolation of the bone marrow. Bone marrow was sampled the day after the last dosing time point where the right femur was removed and prepared for analysis. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. The mean number of micronucleated polychromatic erythrocytes scored in the 6650 mg/kg/day group were compared with the corresponding vehicle control group. It was concluded that the test item, N-Phenyldiethanolamine, reaction products with formaldehyde is not clastogenic or aneugenic in the bone marrow micronucleus test of rats up to a dose of 650 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

OECD 471 - bacterial reverse mutation test, Gisbrechts (2020):

The objective of this study was to determine the potential of the test item and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonellatyphimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP₂uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). 

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP₂uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level.  Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in both tester strains in the absence and presence of S9-mix. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested up to concentrations of
1600 µg/plate in the strains TA1535, TA1537 and TA98.  The test item did not precipitate on the plates at this dose level.  Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.  In the second mutation experiment, the test item was tested up to concentrations of
1600 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and up to 5000 µg/plate in the tester strain WP₂uvrA in the pre-incubation assay.  The test item did not precipitate on the plates. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. 

The negative and strain-specific positive control items were valid.

In the second experiment (pre-incubation method), the test item induced dose-related increases in the number of revertant colonies compared to the solvent control in tester strains TA98, TA100 and WP₂uvrA

In tester strain TA98, the increases observed where up to 3.3- fold and 4.5- fold the concurrent control, in the absence and presence of S9-mix, respectively. In tester strain TA100, the increases observed where up to 3.0- fold and 3.3- fold the concurrent control, in the absence and presence of S9-mix, respectively. In tester strain WP₂uvrA, the increases observed where up to 3.1- fold and 2.9- fold the concurrent control, in the absence and presence of S9-mix, respectively.

Although the increases in tester strains TA98 and WP₂uvrA were within the laboratory historical control data range, the responses were more than two- and three-fold the concurrent controls in strains WP₂uvrA and TA98, respectively. In addition, the increases in tester strain TA100 were outside the historical control data range and more than two-fold the concurrent controls. Based on these observations, the increases were considered biologically relevant. 

Based on the results of this study it is concluded that the test item is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. 

 

OECD 487 - MNT in vitro, de Jong (2020):

A test was performed in accordance with OECD 487, in order to evaluate the ability of the test item to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix).

The potential for clastogenicity and aneugenicity of the test item was tested in two independent experiments. The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the dose range of the test item was 157 to 5000 µg/mL, which was repeated due steep toxicity to 4.7 to 15 µg/mL. The results indicated that the maximum concentration should be limited on toxicity.  In the first cytogenetic assay, the test item was tested up to for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction, respectively. Appropriate toxicity was reached at these dose levels. In the second cytogenetic assay, the test item was tested up to 55 µg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The negative/solvent control cultures and positive control substances were valid.

In the first cytogenetic assay, the test item induced statistically significant increases in the number of binucleated cells with micronuclei, both in the absence and presence of S9-mix. In addition, dose related trends were observed (p = 0.003 and p < 0.001 in the absence and presence of S9-mix, respectively) and the number of binucleated cells with micronuclei was outside the 95% control limits of the distribution of the historical negative control database. Therefore, these increases were considered biologically relevant. The test item did not induce a statistically significant or biologically relevant increase in the number of mononucleated cells with micronuclei. In the second cytogenetic assay with a 24 hours continuous exposure time, the test item did not induce a statistically significant and biologically relevant increase in the number of mono- and binucleated cells with micronuclei.

In conclusion, the test item induces the formation of micronuclei in human lymphocytes under the experimental conditions following 3 hours exposure.

Justification for classification or non-classification

Two in vitro tests undertaken were positive (OECD 471, gene mutation test in bacteria, and OECD 487 micronucleus test in cultured human lymphocytes). Therefore according to the requirements of the ITS for mutagenicity under REACH guidance document R7a (v6, 2017), an in vivo test is required. 

 

An in vivo micronuclus screen (OECD 474) was incorporated into the combined repeated dose toxicity test with reproductive and developmental screening (OECD 422). According to the conditions of the study, the test item was considered non-clastogenic and does not meet the criteria for classification in accordance with Regulation (EC) No 1272/2008 (CLP).

 

However, data on mutagenicity is currently inconclusive based on the positive in vitro gene mutation and as such the overall classification on genotoxicity is also considered inconclusive. A testing proposal for a 2nd in vivo test (TG489, COMET assay) has been submitted.