Iron, complexes with chloroacetic acid-formaldehyde-resorcinol reaction products coupled with 2-hydroxy-3,5-dinitrobenzenediazonium hydroxide inner salt and 4-sulfobenzenediazonium hydroxide inner salt, ammonium sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames - OECD TG 471 - nitroreductase deficient strains - key study - read-across - with and without S9 - positive.

OECD TG 476 (In Vitro Mammalian Cell Gene Mutation Test) - key study - read-across - negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Cytotest Cell Research GmbH & Co. KG

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: HAM's F12 (Seromed, D-1000 Berlin, FRG) supplemented with 10 % fetal calf serum (FCS; Seromed)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 microsomal fraction

Test concentrations with justification for top dose:

Experiment 1: 30, 100, 200, 300, 400, 800 µg/ml (without S9 mix); 1, 5, 20, 100, 1000, 2000, 2700, 3420 µg/ml (with S9 mix)
Experiment 2: 80, 300, 600, 800, 1000, 1200 µg/ml (without S9 mix); 342, 1000, 1692, 2000, 2700, 3420 µg/ml (with S9 mix)
Experiment 3: 1, 5, 10, 20, 30, 50 µg/ml (with S9 mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: substance is not soluble in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation Migrated to IUCLID6: 600 µg/ml = 4.8 mM dissolved in medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With metabolic activation Migrated to IUCLID6: 3.85 μg/ml = 15.0 μM dissolved in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 or 16 days

Colony staining with 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: in duplicate per experimental point

NUMBER OF CELLS EVALUATED: 10^6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

Test article is classified as positive if it induces either a significant concentration-related increase in the mutant frequency or a reproducible and significant positive response for at least one of the test points.
A test article producing neither a significant concentration related increase in the mutant frequency nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

Statistics:

Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reduced plating efficiency observed in some plates with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRETEST:
In a pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test article was observed and compared to the controls. Toxicity of the test article was evidenced by a reduction in plating efficiency (PE). The plating efficiency of the CHO cells was reduced after treatment with 1000 µg/ml (without metabolic activation). With metabolic activation toxicity was observed after treatment with 2000 µg/ml and between 40 µg/ml and 300 µg/ml. Therefore, the first experiment was performed with six (without metabolic activation) and eight concentrations (with metabolic activation) ranging from 1 to 3420 µg/ml.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Results:

Experiment	µg/ml	S-9 mix	mean number cells/flask		factor	mean number mutant colonies/flask	mean number mutant colonies/10^6 cells
			seeded	found
1	negative control	-	542	280,5	0,52	2.8 ± 0.8	13,8
	600, EMS	-	530	186	0,35	61.0 ± 6.6	542,9
	30	-	nc
	100	-	618	298,5	0,48	1.4 ± 1.1	7
	200	-	608	362	0,6	2.4 ± 2.1	9,4
	300	-	nc
	400	-	612	459	0,75	5.2 ± 2.9	17,8
	800	-	610	423,5	0,69	3.8 ± 1.3	13,4
	negative control	+	498	289,5	0,58	2.6 ± 1.1	12,1
	solvent control	+	498	260,5	0,52	0.2 ± 0.4	0,8
	3850, DMBA	+	519	123	0,24	109.4 ± 14.2	1215,6
	1	+	nc
	5	+	nc
	20	+	nc
	100	+	508	221	0,44	4.6 ± 2.9	28,6
	1000	+	525	233,5	0,44	1.2 ± 1.1	7,6
	2000	+	nc
	2700	+	454	273	0,6	0.8 ± 0.8	3,1
	3420	+	492	264	0,54	2.6 ± 1.7	13,5
2	negative control	-	519	282,5	0,54	3.2 ± 2.9	13,8
	600, EMS	-	506	148,5	0,29	142.6 ± 3.5	1343,5
	80	-	529	253,5	0,48	1.4 ± 0.9	6,7
	300	-	504	232	0,46	1.4 ± 1.7	7,5
	600	-	nc
	800	-	522	294	0,56	0.2 ± 0.4	0,8
	1000	-	507	282	0,56	0.4 ± 0.5	1,7
	1200	-	nc
	negative control	+	506	267	0,53	2.0 ± 0.7	8,4
	solvent control	+	514	258	0,5	1.8 ± 1.1	8
	3850, DMBA	+	500	159	0,32	153.0 ± 14.8	1048,5
	342	+	511	236,5	0,46	1.2 ± 1.3	5,8
	1000	+	nc
	1692	+	525	211	0,4	0.2 ± 0.4	1,3
	2000	+	nc
	2700	+	515	224,5	0,44	0.2 ± 0.4	1
	3420	+	503	251,5	0,5	2.0 ± 2.0	8,8
3	negative control	+	530	341	0,64	1.2 ± 0.8	4,5
	solvent control	+	506	314	0,62	0.4 ± 0.5	1,7
	3850, DMBA	+	504	298	0,59	77.2 ± 2.9	320,7
	1000	+	531	290,5	0,55	0.4 ± 0.5	2,1
	5000	+	nc
	10000	+	525	320	0,61	1.6 ± 1.5	6,2
	20000	+	504	260,5	0,52	0.2 ± 0.4	0,9
	30000	+	532	313	0,59	0.6 ± 0.5	2,4
	50000	+	519	264	0,51	0.4 ± 0.5	1,9

nc: culture not continued

Conclusions:

The analogue substance was tested for gene mutation in mammalian cells follwoing OECD 476. The tested substance under the reported experimental conditions did not induce gene mutation at the HGRPT locus in CHO cells.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

August 2020 - September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD, 1997, as corrected in 2020

Deviations:

yes

Remarks:

due to the nature of the test article, a modified (reductive Prival) metabolic activation system was employed, and only a single experiment was performed. Use of NR-deficient strains.

Principles of method if other than guideline:

The traditional strains used for OECD TG 471 were checked in parallel with the same strains deficient in the nitroreductase enzyme (present only in bacteria) as to avoid the NO2 group reduction present in the test substance. The reduction of nitro groups in fact produces aromatic amines in the substance that typically give false positive results for the traditional tested strains.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

nitroreductase deficient

Remarks:

TA98NR, TA100 NR

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: reductive (Prival) metabolic activation system.
- Source of S9: uninduced male Golden Syrian hamsters.
- Method of preparation of S9 mix: the S9 was stored frozen at < -50°C, and thawed prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities). Treatments were carried out both in the absence and presence of S9 by addition of either buffer solution or 30% reductive (Prival) S9 mix respectively. The composition of the mix and buffer solutions are reported in the section "Any other information on material and methods incl. tables".
- Concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL of 30% reductive S9 mix.
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).

Test concentrations with justification for top dose:

- Test concentrations: 5, 16, 50, 160, 500, 1600 and 5000 µg/plate (final concentrations). 5000 µg/plate is the maximum recommended concentration according to current regulatory guidelines.

Vehicle / solvent:

Purified water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

congo red

mitomycin C

other: metronidazole (MTZ), 2-aminoanthracene (AAN)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate.
- Number of independent experiments: single experiment.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): not applicable.
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: pre-incubation methodology.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 minutes at 37°C (for the treatments in the absence of S9) or 30°C (for treatments in the presence of S9).
- Exposure duration/duration of treatment: the plates were inverted and incubated protected from light for 3 days in an incubator set to 37°C.
- Harvest time after the end of treatment: not applicable.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: the background lawns of the plates were examined for signs of cytotoxicity. Revertant plate count data were also assessed, as a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response would also be considered as evidence of cytotoxicity.
- Any supplementary information relevant to cytotoxicity: not supplementary information reported.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as intensely coloured agar or bubbles/splits in the agar affected the accuracy of the automated counter.

- OTHER: it should be noted that data from the initial treatments of strain TA98 and TA98NR in the absence and presence of S9, and data from initial repeat treatments of strain TA98NR, were invalidated due to a combination of unacceptable vehicle control counts and confounding contaminating colonies. A further repeat of these strain treatments was therefore performed in order to provide the Mutation Experiment data presented in this study. As comparisons between concurrent strain TA98 and TA98NR data are required in this study, strain TA98 and TA98NR treatments were performed in the initial repeat treatments, and data from these strain TA98 treatments were valid, but strain TA98 was also treated alongside strain TA98NR in the further repeat treatments. The strain TA98 data from the initial repeat treatments are therefore presented as further Mutation Experiment in this study.
As data from the Mutation Experiment showed clear positive responses in most tester strains, then further testing in a second experiment was not considered necessary, in accordance with current regulatory guidance.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
A concentration related increase in revertant numbers was ≥1.5-fold (in strain
TA102), ≥2-fold (in strains TA98, TA98NR, TA100 or TA100NR) or ≥3-fold (in
strains TA1535 or TA1537) the concurrent vehicle control values.
The test article was considered positive in this assay if the above criterion was met.
The test article was considered negative in this assay if the above criterion was not
met.
Results which only partially satisfied the above criteria were dealt with on a case-bycase
basis. Biological relevance was taken into account, for example consistency of
response within and between concentrations.
Data from strain TA98 were compared (non-statistically) with that from TA98NR,
and data from strain TA100 were compared with that from TA100NR. Where a
mutagenic response was seen in one or both parent strains but was absent or much
reduced in the corresponding NR variant strain(s), this was considered to be indicative
that bacterial nitroreduction enzymes play a significant role in the mutagenicity of the
test compound as observed in this study.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

bacteria, other: TA 98NR

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

bacteria, other: TA 100NR

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Cytotoxicity, Solubility and Concentration Selection:
Mutation Experiment treatments of all the tester strains were performed using a pre-incubation methodology in the absence and presence of a modified (reductive) S9 mix using final concentrations of the test substance at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus vehicle and positive controls. Following these treatments, no evidence of cytotoxicity was observed, as would usually be indicated by a diminution of the background bacterial lawn and/or a marked reduction in revertant numbers.

Data Acceptability and Validity:
The individual mutagenicity plate counts were averaged to give mean values. From the data it can be seen that vehicle control counts fell within the laboratory’s historical ranges, with the exception of a few isolated vehicle control counts that fell slightly outside the laboratory control ranges. In each case, these counts were comparable to the other vehicle control replicate counts and the laboratory historical control ranges, and therefore these data were accepted as characteristic and valid. The positive control chemicals all induced increases in revertant numbers of ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle controls, and ≥2-fold with NQO, B[a]P and CR in strain TA98NR and NaN3, AAN and CR in strain TA100NR. The positive control treatments with 2NF in TA98NR and with MTZ in TA100NR each demonstrated a reduced response compared to the equivalent treatments in the parent strains TA98 and TA100, confirming the nitroreductase deficient status of strain TA98NR and TA100NR. The control treatments therefore confirmed discrimination between different strains, and an active S9 preparation, and the correct strain and assay functioning was demonstrated. The study data were therefore accepted as valid.

Mutation:
Following test substance treatments of all the test strains apart from TA100NR, clear and concentration-related increases in revertant numbers were observed in the absence and presence of S9.
These increases were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98, TA98NR or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control, although it should be noted that in strain TA98NR in the absence of S9, the 2-fold threshold level was only achieved at the maximum treatment concentration of 5000 μg/plate. These increases were however all sufficient to be considered as clear evidence of the test substance mutagenic activity in this assay system. In strain TA100NR, a notable and concentration-related increase in revertant numbers was observed in the absence of S9 only, but this increase fell just below the 2-fold threshold for an increase to be considered as clear evidence of mutagenic activity in this strain. No notable increase in revertant numbers was observed following strain TA100NR treatments in the presence of S9. The mutagenic responses in strains TA98NR and TA100NR in the absence of S9 were markedly reduced when compared to the responses with their respective parent strain, both in terms of magnitude of increase in revertants, and lowest responding treatment concentration. In the presence of S9 this difference was again observed, particularly between strain TA100 and TA100NR, as no response was observed in the latter strain, but the difference was somewhat less pronounced between strains TA98 and TA98NR. Strains TA98NR and TA100NR still retain some low level residual nitroreductase activity (Rosenkranz and Mermelstein, 1983), and therefore complete elimination of any mutagenic response was not expected in these strains. Therefore, overall the data from this study with the nitroreductase deficient strains provided reduced responses compared to their nitroreductase proficient parent strains, indicating that nitroreduction plays a significant role in the mutagenic activity of the test substance.

Conclusions:

It was concluded that the test substance induced mutation in histidine-requiring strains TA98, TA98NR, TA100, TA1535, TA1537 and TA102 of Salmonella typhimurium in the absence and in the presence of a reductive hamster liver metabolic activation system (S9), when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines). The relative reduction in (or absence of) mutagenic response with the nitroreductase deficient strains compared to those with the parent strains indicate that nitroreduction plays a significant role in the mutagenic activity of the test substance.

Executive summary:

The test substance was assayed for mutation in seven histidine-requiring strains (TA98, TA100, TA1535, TA1537, TA102, TA98NR and TA100NR) of Salmonella typhimurium, both in the absence and in the presence of a reductive hamster liver metabolising system (S9) in a single experiment.
All treatments in this study were performed using formulations prepared in purified water. As the test substance is an azo compound, testing in the presence of S9 in this study was performed using a modified reductive (Prival) S9 pre-incubation methodology, as it is known that azo compounds can be reduced to free aromatic amines, which can be mutagenic.
Mutation Experiment treatments of all the tester strains were performed using a pre-incubation methodology in the absence and presence of a modified (reductive) S9 mix using final concentrations of the test substance at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate. Following these treatments, no clear evidence of cytotoxicity was observed, as would usually be indicated by a diminution of the background bacterial lawn and/or a marked reduction in revertant numbers.
It should be noted that due to a combination of uncharacteristic vehicle control data and confounding contaminant colonies on the test plates following the initial Mutation Experiment treatments of strain TA98 and TA98NR in the absence and presence of S9, these treatments were repeated to provide valid mutation data for these strain treatments. No valid strain TA98NR data were available from these initial repeat treatments due to confounding contaminant colonies on these test plates, but the strain TA98 mutation data were valid. In order that concurrent mutation data were available for direct comparison of any mutagenic response in the nitroreductase deficient strain and the parent strain, strain TA98 treatments were also included alongside the strain TA98NR repeat treatments, and the data from these further repeat treatments are presented in this report as the Mutation Experiment data for these strains. As mutation data from the initial repeat treatments in strain TA98 in the absence and presence of S9 were valid, these are reported as further Mutation Experiment data.

Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies were comparable with acceptable ranges for vehicle control treatments, and were elevated by positive control treatments. Following the test substance treatments of all the test strains apart from TA100NR, clear and concentration-related increases in revertant numbers were observed in the absence and presence of S9. These increases were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98, TA98NR or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control, although it should be noted that in strain TA98NR in the absence of S9, the 2-fold threshold level was only achieved at the
maximum treatment concentration of 5000 µg/plate. These increase were however sufficient to be considered as clear evidence of the test substance mutagenic activity in this assay system. In strain TA100NR, a notable and concentration-related increase in revertant numbers was observed in the absence of S9 only, but this increase fell just below the 2-fold threshold for an increase to be considered as clear evidence of mutagenic activity in this strain.

The mutagenic responses in strains TA98NR and TA100NR in the absence of S9 were markedly reduced when compared to the responses with their respective parent strain, both in terms of magnitude of increase in revertants, and lowest responding treatment concentration. In the presence of S9 this difference was again observed, particularly between strain TA100 and TA100NR, as no response was observed in the latter strain, but the difference was somewhat less pronounced between strains TA98 and TA98NR. Strains TA98NR and TA100NR still retain some low level residual nitroreductase activity (Rosenkranz and Mermelstein, 1983), and therefore complete elimination of any mutagenic response was not expected in these strains. Therefore, overall the data from this study with the nitroreductase deficient strains provided reduced responses compared to their nitroreductase proficient parent strains, indicating that nitroreduction plays a significant role in the mutagenic activity of the test substance.

It was concluded that the test substance induced mutation in histidine-requiring strains TA98, TA98NR, TA100, TA1535, TA1537 and TA102 of Salmonella typhimurium in the absence and in the presence of a reductive hamster liver metabolic activation system (S9), when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines). The relative reduction in (or absence of) mutagenic response with the nitroreductase deficient strains compared to those with the parent strains indicate that nitroreduction plays a significant role in the mutagenic activity of the test substance.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

OECD TG 474 (Mammalian Erythrocyte Micronucleus Test) - read-across - negative.
OECD TG 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo) - read-across - negative.
OECD TG 489 (In vivo Mammalian Alkaline Comet Assay) - read-across - TPE.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

other: read across from supproting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

not yet defined

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

NON-CONFIDENTIAL NAME OF SUBSTANCE:
Name of the substance on which testing is proposed to be carried out: analogue substance 03

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
Available GLP studies: not available.
Available non-GLP studies: not available.
Historical human data: not available.
(Q)SAR: not available.
Weight of evidence: not available.
Grouping and read-across:
- OECD TG 476 (In Vitro Mammalian Cell Gene Mutation Test) – Key study – Read-Across on analogue substance 02.
- OECD TG 471 – NR-deficient strains – Key study – Read-Across on analogue substance 02.
- OECD TG 471 (IHMA) – Supporting study – Read-Across on analogue substance 02.
- OECD TG 471 – Only strain TA100 – Disregarded study – Read-Across on analogue substance 02.
- OECD TG 474 (Mammalian Erythrocyte Micronucleus Test) – Key study – Read-Across on analogue substance 02.
- OECD TG 486 (Unscheduled DNA Synthesis) – Key study – Read-Across on analogue substance 02.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
Under Annex VIII Section 8.4., column 2 of REACH, further mutagenicity studies must be considered in case of a positive result in an in vitro gene mutation study in bacteria.

Guidance on information requirements R7a, section 7.7.6 (2017), states that regarding Annex VIII, when both the mammalian cell tests are negative but there was a positive result in the bacterial test, it will be necessary to decide whether any further testing is needed on a case-by-case basis. For example, suspicion that a unique positive response observed in the bacterial test was due to a specific bacterial metabolism of the test substance could be explored further by investigation in vitro. Alternatively, an in vivo test may be required.

The present dossier contains positive results for the in vitro gene mutation study in bacteria in read across from analogue substance 02, following OECD TG 471 which raises the concern for gene mutation.

The submitted dossier on Acid Brown 332 contains positive results for the in vitro gene mutation study in bacteria, following OECD TG 471 on the similar substance 02, which raises the concern for gene mutation.

As presented in the attached document regarding genotoxicity, a fist attempt to explain the mechanism of the nitroreductase in the positivity of the Ames test was evaluated on the same read across substance. The test substance was tested in a modified Ames test following OECD TG 471 with NR deficient strains, namely TA 98 and TA 100, using the Prival method. Results really support the major role played by the nitroreductase and a great decrease on the number of revertants with respect to the control was obtained for TA 98NR and TA 100NR. However, the modified Ames test cannot be finalised as totally negative.

Annex VIII, Column 2, requires the registrant to consider appropriate mutagenicity in vivo studies already at the Annex VIII tonnage level, in cases where positive results in genotoxicity studies have been obtained, which involves studies mentioned in Annex IX (as first step OECD TG 474 – Mammalian Erythrocyte Micronucleus Test, OECD TG 488 – Transgenic Rodent Mutation Assay, OECD TG 489 – In Vivo Mammalian Alkaline Comet Assay and OECD TG 486 – Unscheduled DNA Synthesis).

CONSIDERATIONS ON THE STUDIES INSERTED IN THE PRESENT DOSSIER AND EXPERT ASSESSMENT ON TESTING PROPOSAL:
The submitted dossier on Acid Brown 332 contains an OECD TG 474 (Mammalian Erythrocyte Micronucleus Test) in vivo study, conducted on the analogue substance 02, with negative results, which is adequate to cover the chromosomal aberration potential of the two substances and to waive the performance of an in vitro cytogenicity in mammalian cells, as laid down in Column II of Annex VIII of the REACH Regulation.

Moreover, an OECD TG 486 (in vivo UDS assay) is also present and conducted in read across on the analogue substance 02, which resulted negative and can be used as supporting information for the gene mutation properties, since cells analysed in the UDS assay involve only those of the liver.

Therefore, in order to further and completely assess the gene mutation properties of the substance in different tissues of the animal, a Comet Assay, OECD TG 489, on the read across substance 03 is presented as testing proposal and it will be also used in read across for assessing the in vivo potential gene mutation properties of Acid Brown 322.

Analogue substance 03 is, in fact, considered as representative of the mutagenic behaviour of Acid Brown 322 in a worst-case scenario approach, showing a positivity also in the in vitro gene mutation test, and considering that the metabolites for the target substance are covered by analogue substance 03 structure.
OECD TG 489 allows to measure DNA strand breaks, that may result from direct interactions with DNA, alkali labile sites or as a consequence of incomplete excision repair. Therefore, the alkaline comet assay recognises primary DNA damage that would lead to gene mutations and/or chromosome aberrations, but will also detect DNA damage that may be effectively repaired or lead to cell death. The comet assay can be applied to almost every tissue of an animal from which single cell or nuclei suspensions can be made, including specific site of contact tissues.

OECD TG 488 is not considered the first choice for assessing the gene mutation in vivo for this substance, since preliminary data for gene mutation in vivo (OECD TG 486) already indicates negativity in the somatic cells of the liver. A confirmation by the Comet assay performed over other tissues (and for azo dyes the intestinal tract is the site of major metabolism and dye/metabolites absorption) would be sufficient to assess the genotoxic potential of the substance.

Finally, as reported in literature, from the analysis of 91 chemicals with published data from Comet Assay and Transgenic rodent mutation assay (TGR), the comet assay appears to yield similar results to the TGR assay in liver and gastrointestinal tract (predominantly stomach and colon data) and, hence, can be confidently performed to confirm in vivo gene mutation activity in terms of genotoxicity in general.

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes

Type of assay:

mammalian comet assay

Route of administration:

oral: gavage

Sex:

not specified

Genotoxicity:

other: to be performed

Remarks on result:

other: the test is in read across from a submitted testing proposal still under evaluation

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

other: read across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, WIGA, D-8741 Sulzfeld, FRG
- Weight at study initiation: mean 26.7 g
- Assigned to test groups randomly: yes
- Housing: individually in Makrolon cages, type M I
- Diet (e.g. ad libitum): Standardized pelleted feed (Kliba Haltungsdiät, Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland); ad libitum
- Water (e.g. ad libitum): drinking water from bottles; ad libitum
- Acclimation period: about one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
- Concentration of test material in vehicle: 8.5, 17, 34 g/100 ml
- Amount of vehicle (if gavage or dermal): 20 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
All test substance formulations were prepared immediately before administration. The amount of substance or volume to be administered was related to the specific weight of the individual animals on the day of the experiment.

Duration of treatment / exposure:

16, 24, 48 h for the highest dose of test material; 24 h for all other dose groups and controls

Frequency of treatment:

single application

Post exposure period:

16, 24, 48 h for the highest dose of test material; 24 h for all other dose groups and controls

Remarks:

Doses / Concentrations:
1700, 3400, 6800 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide;
- Route of administration: oral by gavage
- Doses / concentrations: 40 mg/kg bw

Tissues and cell types examined:

bone marrow of the two femora

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In the determination of the acute oral toxicity all animals survived the high dose of 6810 mg/kg body weight without any clinical signs or symptoms. A volume as high as 20 ml/kg body weight had to be selected in order to be able do administer this amount. Higher doses suspended in an 0.5 % aqueous CMC formulation led to a viscous mass which could no longer be administered.


DETAILS OF SLIDE PREPARATION:
Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained. Staining in eosin and methylene blue solution for 5 minutes. Rinsed in aqua dest., then placed in fresh aqua dest. for 2 or 3 minutes. Staining in Giemsa solution for 12 minutes. After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Entellan.


METHOD OF ANALYSIS:
As a rule, 1000 polychromatic erythrocytes from each of the male and female animals of every test group are evaluated and investigated for micronuclei. The normochromatic erythrocytes (= normocytes), which occur, are also scored.

Evaluation criteria:

The increase in the micronucleus rate in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome breaking (clastogenic) effect or of a spindle activity of the substance tested.

Statistics:

Two statistical tests were used to answer the questions of whether there are significant differences between control group and dose group or between the individual dose groups concerning the rate of micronuclei in polychromatic erythrocytes: first, the exact test according to FISHER, which was applied to register significant differences between the relative frequencies of a characteristic of two groups, and, second, the asymptotic U test according to MANN-WHITNEY (rank test modified according to WILCOXON). The relative frequencies of cells with micronuclei per animal were use d
as a criterion of the rank determination for the U test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: The single oral administration in doses of 6800 mg/kg, 3400 mg/kg or 1700 mg/kg body weight was tolerated by all animals without any signs of toxicity.
-Necropsy: The gross-pathological examination of the animals sacrificed at the end of the study did not reveal any changes of the internal organs which could the attributed to the test substance administered.

Results:

Substance	Dose (mg/kg bw)	Interval: 16 hours				Interval: 24 hours				Interval: 48 hours
		Polychromatic erythrocytes investigated	Normocytes / 10000 polychromatic erythrocytes	Cells with micronuclei		Polychromatic erythrocytes investigated	Normocytes / 10000 polychromatic erythrocytes	Cells with micronuclei		Polychromatic erythrocytes investigated	Normocytes / 10000 polychromatic erythrocytes	Cells with micronuclei
				per 1000 polychromatic erythrocytes	per 1000 normochromatic erythrocytes			per 1000 polychromatic erythrocytes	per 1000 normochromatic erythrocytes			per 1000 polychromatic erythrocytes	per 1000 normochromatic erythrocytes
vehicle control, 0.5% CMC		-				10000	3485	1.8	0
Säurebraun 6229	6800	10000	2988	1.4	1.34	10000	4349	1.4	1.61	10000	4359	1.3	1.61
Säurebraun 6229	3400	-				10000	3506	1.4	0.86
Säurebraun 6229	1700	-				10000	4307	1.9	0.46
Cyclophospamide	40	-				10000	5623	23.4	1.78

Conclusions:

The analogue substance was tested for chromosome aberration potential following OECD 476, by oral administration. The tested sample under the experimental conditions, did not induce chromosome breaking (clastogenic) effect or a spindle activity in polychromatic erythrocytes of the bone marrow of the femora mice.

Reference 3

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

other: read across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

yes

Remarks:

limited given data

GLP compliance:

yes

Remarks:

HAZLETON LABORATORIES AMERICA, INC.

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc.
- Age at study initiation: adult
- Weight at study initiation: 150 - 300 g
- Assigned to test groups randomly: yes
- Diet (e.g. ad libitum): Purina Certified Rodent Chow (Formula 5002); ad libitum
- Water (e.g. ad libitum): water; ad libitum
- Acclimation period: minimum of 5 days prior to use

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Amount of vehicle (if gavage or dermal): 9 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Fresh preparations of test article in vehicle were used for any testing purpose.

Duration of treatment / exposure:

4 h

Frequency of treatment:

single application

Post exposure period:

4 h

Remarks:

Doses / Concentrations:
500, 1000, 2000, 4000 mg/kg bw
Basis:
nominal in water

No. of animals per sex per dose:

3 animals/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Dimethylnitrosamine (DMN)
- Justification for choice of positive control(s): known to induce UDS in vivo in rat hepatocytes
- Route of administration: intraperitoneal
- Doses / concentrations: ca. 10 mg/kg bw

Tissues and cell types examined:

hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In the preliminary study to determine dose and perfusion time, an attempt was made to gavage two animals with 5000 mg/kg, but the test material formed a sludge in CMC that could not be forced through a syringe. The highest dose that could be administered was between approximately 3200 mg/kg and 3400 mg/kg although one animal died due to gavage back up. The amount administered was not exact because the sludge was difficult to measure. One rat was sacrificed (liver perfusion) approximately 4.5 hours later and the other approximately 15 hours later and slides were prepared for UDS. Microscopic examination of the slides prepared at the two time points indicated that they were similar. It was decided that the UDS assay would be performed approximately 4 hours after administration of a single dose of the test material.


DETAILS OF SLIDE PREPARATION:
UDS based on the procedures in rats described by Williams (1980) and Mirsalis, Tyson and Butterworth (1982): Briefly, isolated hepatocytes were cultured for 1.5 to 2 hours at 37°C in a humidified atmosphere containing 5 % C02. Three of the replicate cultures from each animal were used for the UDS assay, thus labeld and fixed with acetic acid : ethanol (1:3) and dried for at least 24 hours.

METHOD OF ANALYSIS:
The cells were examined microscopically at approximately 1500x magnification under oil immersion and the field was displayed on the video screen of an automatic counter. UDS was measured by counting nuclear grains and subtracting the average number of grains in three nuclear-sized areas adjacent to each nucleus (background count). This value is referred to as the net nuclear grain count. The coverslips were coded to prevent bias in grain counting. The net nuclear grain count was determined for 50 randomly selected cells on each coverslip. Only nuclei with normal morphologies were scored, and any occasional nuclei blackened by grains too numerous to count were excluded as cells in which replicative DNA synthesis occurred rather than repair synthesis. The mean net nuclear grain count was determined from the triplicate coverslips (150 total nuclei) for each treatment condition. Occasionally, a coverslip is recounted at a later date or by a different technician. Since a different cell population will generally be scored, the average count for 50 cells was used in the calculation of the mean for the triplicate treatment.

Evaluation criteria:

The test material is considered active in the UDS assay at applied concentrations that cause:
- an increase in the mean net nuclear grain count to at least six grains per nucleus after subtraction of the concurrent negative control value, and/or
- an increase in the percent of nuclei having 6 or more net grains to at least 10% of the analyzed population after subtraction of the concurrent negative control value, and/or
- the percent of nuclei with 20 or more grains to reach or exceed 2% of the analyzed population

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 3200 - 5000 mg/kg bw
- Solubility: material formed a sludge in CMC that could not be forced through a syringe at 5000 mg/kg bw

Results:

Test condition	Dose Level	Animal	UDS grains/nucleus	Average* % nuclei with
				>= 6 grains	>= 20 grains
water	-	1	0.45 ± 0.18	0.7	0.0
		2	-0.43 ± 0.10	0.7	0.0
		3	-0.163 ± 1.59	0.7	0.0
DMN	10 mg/kg bw	1	25.22 ± 8.79	94.7	64.7
		2	15.47 ± 2.65	83.3	41.3
		3	17.2 ± 7.8	84.0	41.3
Saeurebraun 6229	4000 mg/kg bw	1	0.36 ± 0.52	2.0	0.0
		2	-0.08 ± 0.73	0.0	0.0
		3	0.08 ± 0.66	0.7	0.0
	2000 mg/kg bw	1	-1.32 ± 0.50	0.7	0.0
		2	-1.00 ± 0.76	0.0	0.0
		3	-1.03 ± 1.02	0.7	0.0
	1000 mg/kg bw	1	-1.49 ± 0.57	0.0	0.0
		2	-1.11 ± 0.88	5.3	0.0
		3	-0.57 ± 0.60	0.0	0.0
	5000 mg/kg bw	1	-0.20 ± 0.20	1.3	0.0
		2	-1.18 ± 0.39	0.0	0.0
		3	-0.92 ± 0.75	1.3	0.0

UDS: Average of net nuclear grain counts on triplicate coverslips (150 total cells), ± standard deviation between coverslips

*: Average values for triplicate coverslips

Conclusions:

The substance was tested for genotoxicity following OECD 486. The tets substance was inactive in the in-vitro/in-vivo Rat Epatocyte UDS Assay over a dosing range of about 500 to 4000 mg/kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

For further details refer to the attached document on genotoxicity assessment. 

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), a mutation means a permanent change in the amount or structure of the genetic material in a cell. The term ‘mutation’ applies both to heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known.

The more general terms ‘genotoxic’ and ‘genotoxicity’ apply to agents or processes which alter the structure, information content, or segregation of DNA, including those which cause DNA damage by interfering with normal replication processes, or which in a non- physiological manner (temporarily) alter its replication.

For the purpose of classification for germ cell mutagenicity, substances are allocated to one of two categories:

 

Category 1: substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans.

Category 1A: based on positive evidence from human epidemiological studies.

Category 1B: based on:

- Positive result(s) from in vivo heritable germ cell mutagenicity tests in mammals; or

- Positive result(s) from in vivo somatic cell mutagenicity tests in mammals, in combination with some evidence that the substance has potential to cause mutations to germ cells.

- Positive results from tests showing mutagenic effects in the germ cells of humans, without demonstration of transmission to progeny; for example, an increase in the frequency of aneuploidy in sperm cells of exposed people.

 

Category 2: substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans, based on positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

- Somatic cell mutagenicity tests in vivo, in mammals; or

- Other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays

 

Based on the negative results of the in vivo tests on the similar substance, OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) and OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)  no classification for mutagenicity is applied following Regulation 1272/2008.
This classification will be confirmed after the results of the OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay).