1,6-hexanediyl-bis(2-(2-(1-ethylpentyl)-3-oxazolidinyl)ethyl)carbamate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item induced an increase of mutant frequency in mammalian cells in vitro in the presence of metabolic activation. However, no mutagenic activity was observed in two bacterial reverse mutation tests on Salmonella typhimurium. Additionally, there was no indication of a clastogenic effect of an intraperitoneal dose of 15 mg/kg bw test substance in the microncleus test on the mouse, i.e. in a somatic test system in vivo. Furthermore, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in liver or stomach cells in an in vivo Comet assay.

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:

1990-09-18 to 1990-09-27

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP and guideline study. No data were available on analytical inveatigations on the stability of the test substance in solution. However, this deviation did not limit the assessment of the results. Only four strains tested.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Directive 84/449/EEC

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 26 May 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-auxotrophic Salmonella typhimurium strains TA 1535, TA 100, TA 1537 and TA 98.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 fraction

Test concentrations with justification for top dose:

With S9 mix: 8, 40, 200, 1000, 5000 µg/plate
Without S9 mix: 8, 40, 200, 1000, 5000 µg/plate

Vehicle / solvent:

The solvent employed for the test substance was ethanol and for the positive controls DMSO.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

in strain TA1535, without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: nitrofurantoin

Remarks:

in strain TA100, without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene diamine

Remarks:

in strains TA1537 and TA 98, without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all strains, with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37 °C

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

The following criteria were used for the acceptance of an assay:
a) The negative controls have to be within the expected range, as defined by literature data and the laboratories' own historical data.
b) The positive controls have shown sufficient effects as defined by the laboratories' experience.
c) The titter determinations had to demonstrate sufficient bacterial density in the suspension.
An assay which was not in agreement with at least one of the above criteria was not used for assessment.
Furthermore the data generated in this assay have to be confirmed by two additional independent experiments. Even, if the criteria for points a, b and c were not met, an assay was accepted if it showed mutagenic activity of the test substance.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts for at least one strain is considered to be a positive result.
For TA 1535, TA 100 and TA 98 this increase should be about twice the amount of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Statistics:

not applicable

Species / strain:

E. coli WP2

Metabolic activation:

not specified

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

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:

valid

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

There was no indication of a bacteriotoxic effect for the test substance at doses up to and including 200 µg per plate. The total bacteria counts consistently produced results comparable to the negative control, or differed only insignificantly. Likewise an inhibition of growth was not detected.
Higher doses had a strong, strain-specific bacteriotoxic effect. Therefore they could only be used to a limited extent up to 5000 µg per plate.
None of the four strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the test with and without S9 mix and was confirmed by the results of the repeated tests.

Conclusions:

Under the experimental conditions, the test substance showed no mutagenic activity in this bacterial reverse mutation test on Salmonella typhimurium.

Executive summary:

The test substance was investigated in the Salmonella / microsome test for point-mutagenic effects in doses up to 5000 µg per plate on four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98. Doses up to and including 200 µg per plate did not cause any bateriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed.

At higher doses, the substance had a strain-specific bacteriotoxic effect, so that this range could only be used to a limited extend up to 5000 mg per plate for assessment purposes. Evidence of mutagenic activity for the test substance was not seen. No biologically-relevant increase of the mutant count, in comparison with the negative controls, was observed. Therefore, the test substance was considered to be non-mutagenic without and with S9 mix in the Salmonella/microsome test. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2 -phenylene-diamine and 2 -aminoanthracene had a marked mutagenic effect, as was seen by a biologically-relevant increase in mutant colonies compared to the corresponding negative controls.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2013-04-23 to 2013-05-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO K1

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F12 medium (F12-10) supplemented with 1 % of Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver.

Test concentrations with justification for top dose:

Experiment 1,5-hour treatment period without S9 mix: 200, 250, 300, 350, 400, 450 and 500 μg/mL
Experiment 1,5-hour treatment period with S9 mix: 200, 250, 300, 350, 400, 450 and 500 μg/mL
Experiment 2,20-hour treatment period without S9 mix: 200, 250, 300, 350, 400, 450, 500 and 550 μg/mL
Experiment 2,5-hour treatment period with S9 mix: 200, 250, 300, 350, 400, 450 and 500 μg/mL

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: The vehicle was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours (+/- S9 mix) and 20 hours (-S9 mix)
- Expression time: 8 days
- Selection time: 8 days

SELECTION AGENT: 6-thioguanine

NUMBER OF REPLICATIONS: one

NUMBER OF CELLS EVALUATED: 1E+06 cells: (5 plates at 2 x 1E+05 cells/plate)

DETERMINATION OF CYTOTOXICITY
- Method: colony forming efficiency, relative total growth

Evaluation criteria:

The test item is considered to be mutagenic in this assay if all the following criteria are met:
- The assay is valid.
- The mutant frequency at one or more doses is significantly greater than that of the relevant control.
- Increase of the mutant frequency is reproducible.
- There is a clear dose-response relationship.
The test item would have been considered to have shown no mutagenic activity if no increases were observed which met the criteria listed above.

Statistics:

Statistical analysis was done with SPSS PC+ software for the following data:
- mutant frequency between the negative (solvent) and the test item or positive control item treated groups.
The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO K1

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO K1

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Precipitation: Precipitation was observed from concentration of 500 μg/mL

RANGE-FINDING/SCREENING STUDIES:
Incozol EH was dissolved in DMSO. A clear solution was obtained up to a concentration of 50 mg/mL. For all test item concentrations examined, precipitation was observed from concentration of 500 μg/mL in the medium during the treatment period. The dose selection cytotoxicity assay was performed as part of this study to establish an appropriate concentration range for the Main Mutation Assays (Experiments 1 and 2), both in the absence and in the presence of a metabolic activation system (rodent liver S9 mix). Toxicity was determined by comparing the colony forming ability of the treated groups to the negative (solvent) control and results were noted as percentage of cells in relation to the negative control. The cytotoxicity results of the pre-experiments and solubility of test item were used for selection of concentrations for the Main Mutation Assays (Experiments 1 and 2). In the performed Main Mutation Assays the concentration levels were chosen mainly based on the solubility of test item.

COMPARISON WITH HISTORICAL CONTROL DATA:
The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures. The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory. The mutation frequency of EMS was 1446.27 and was slightly above the corresponding historical control data range. The slightly higher mutation frequencies were considered as acceptable and did not have any effect on the validity of the performed experiments.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Incozol EH did not induce an increase of cytotoxicity at any concentration used.

Mutagenesis assay results

 

Test group	Dose [µg/mL]	S9 mix	Treatment time [h]	Mutant frequency x 1E-06
				a	b
Experiment I
Solvent control	-	-	5	2.06	2.04
Positive control	EMS 1.0 µg/mL	-	5	1446.27 **	1282.35 **
Incozol EH	200	-	5	2.04	0.00
Incozol EH	250	-	5	1.01	3.06
Incozol EH	300	-	5	1.02	1.01
Incozol EH	350	-	5	4.08	5.10
Incozol EH	400	-	5	3.06	2.06
Incozol EH	450	-	5	4.21	3.16
Incozol EH	500	-	5	3.19	3.23
Solvent control	-	+	5	2.02	2.02
Positive control	DMBA 20 µg/mL	+	5	691.46 **	667.47 **
Incozol EH	200	+	5	2.02	3.03
Incozol EH	250	+	5	3.19	4.26
Incozol EH	300	+	5	6.12	4.04
Incozol EH	350	+	5	14.14 *	10.10 *
Incozol EH	400	+	5	15.15 **	15.15 *
Incozol EH	450	+	5	20.20 **	21.21 **
Incozol EH	500	+	5	25.51 **	26.53 **
Experiment II
Solvent control	-	-	20	2.02	2.00
Positive control	EMS 0.4 µg/mL	-	20	870.27 **	793.33 **
Incozol EH	200	-	20	3.03	1.01
Incozol EH	250	-	20	5.05	5.05
Incozol EH	300	-	20	4.12	4.08
Incozol EH	350	-	20	5.32	6.38
Incozol EH	400	-	20	4.12	3.09
Incozol EH	450	-	20	5.15	5.21
Incozol EH	500	-	20	4.08	4.08
Incozol EH	550	-	20	3.13	4.17
Solvent control	-	+	5	3.09	3.06
Positive control	DMBA 20 µg/mL	+	5	660.00 **	652.00 **
Incozol EH	200	+	5	4.12	6.19
Incozol EH	250	+	5	3.13	7.22
Incozol EH	300	+	5	6.38	8.51
Incozol EH	350	+	5	12.50	11.46
Incozol EH	400	+	5	18.09 *	18.95 **
Incozol EH	450	+	5	21.88 **	22.92 *
Incozol EH	500	+	5	26.88 **	31.58 **

 

a,b = parallel examination

*= p < 0.05

**= p < 0.01

EMS = Ethyl methanesulfonate

DMBA= 7,12-Dimethylbenzanthracene

Conclusions:

Incozol EH tested without metabolic activation (S9-mix) over a 5-hour and 20-hour period did not induce increases in mutant frequency over the background (negative solvent control) in this in vitro test in Chinese hamster ovary cells. Incozol EH tested with metabolic activation (S9-mix) over a 5-hour treatment period induced statistically and biologically significant increases in the mutant frequency. Incozol EH was mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.

Executive summary:

The test item, Incozol EH was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations and basis of solubility of test item made in a preliminary study (without and with metabolic activation using S9 mix). In the performed Main Mutation Assays the concentration levels were chosen mainly based on the solubility of test item.

Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:

Experiment 1,5-hour treatment period without S9 mix: 200, 250, 300, 350, 400, 450 and 500 μg/mL

Experiment 1,5-hour treatment period with S9 mix: 200, 250, 300, 350, 400, 450 and 500 μg/mL

Experiment 2,20-hour treatment period without S9 mix: 200, 250, 300, 350, 400, 450, 500 and 550 μg/mL

Experiment 2,5-hour treatment period with S9 mix: 200, 250, 300, 350, 400, 450 and 500 μg/mL

In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested without metabolic activation. There were no biologically significant differences between treatment and control groups and no dose-response relationships were noted.

In Experiment 1, the mutant frequency of the cells showed statistically and biologically significant increases in mutation frequency at the concentrations of 350, 400, 450 and 500 μg/mL, when the test item was tested with S9-mix.

In Experiment 2, the mutant frequency of the cells showed statistically significant increases in mutation frequency at the concentrations of 400, 450 and 500 μg/mL when the test item was tested with S9-mix. These increases were biologically significant and dose-response relationships were noted.

In Experiment 2, the mutant frequency of the cells did not show biologically or statistically significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours), further indicating that the findings in Experiment 1 were in the normal biological variation observed, when the test item was examined in the absence of metabolic activation.

The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures.

Incozol EH tested without metabolic activation (S9 mix) over a 5-hour and 20-hour period did not induce increases in mutant frequency over the background (negative solvent control) in this in vitro test in Chinese hamster ovary cells. Incozol EH tested with metabolic activation (S9 mix) over a 5-hour treatment period induced statistically and biologically significant increases in the mutant frequency. Incozol EH was mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1988-01-28 to 1988-02-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

Detailed description of experimental method available. Study is not conforming to OECD principles, because of the following deviations: No data concerning analytical investigations of compound and stability in solution were available. The same was true for storage stability. In addition, no inspections were performed by the quality assurance unit. The evaluation of the results was not limited by these deviations. Only four strains were tested.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-auxotrophic Salmonella typhimurium strains TA 1535, TA 100, TA 1537 and TA 98.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 fraction

Test concentrations with justification for top dose:

With S9 mix: 20, 100, 500, 2500, 12500 µg/plate
Without S9 mix: 20, 100, 500, 2500, 12500 µg/plate

Vehicle / solvent:

The solvent used for all substances was DMSO.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

in strains TA 1535, without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: nitrofurantoin

Remarks:

in strains TA 100, without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene

Remarks:

in strains TA 1537 and TA 98, without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

in strains TA 1535, TA1537, TA100 and T98, with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37 °C

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

The following criteria were used for the acceptance of an assay:
a) The negative controls have to be within the expected range, as defined by literature data and the laboratories' own historical data.
b) The positive controls have shown sufficient effects as defined by the laboratories' experience.
c) The titter determinations had to demonstrate sufficient bacterial density in the suspension.
An assay which was not in agreement with at least one of the above criteria was not used for assessment.
Furthermore the data generated in this assay have to be confirmed by two additional independent experiments. Even, if the criteria for points a, b and c were not met, an assay was accepted if it showed mutagenic activity of the test substance.

Evaluation criteria:

A reproducible and dose-related increase (i.e. >= twice the negative control count) in mutant counts for at least one strain is considered positive. Otherwise the result is evaluated as negative.
In case of questionable results, the investigations will continue, probably by the use of modifications, until a final evaluation is possible.

Statistics:

not applicable

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:

valid

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

There was no indication of a bacteriotoxic effect for the test substance at doses up to and including 100 µg per plate. The total bacteria counts consistently produced results comparable to the negative control, or differed only insignificantly. Likewise an inhibition of growth was not detected. Higher doses had a bacteriotoxic effect, specific to strain, and could therefore only partly be used for evaluation up to and including 12500 µg per plate.
One of the four strains used revealed without S-9 mix a dose-related increase in mutant counts to double those of the negative controls. This strain was TA 98.
This increase is to be regarded as a random result due to the low spontaneous rate of the strain. It must also be considered that the negative control with S-9 mix was within the range of values for treatment groups without S-9 mix, and therefore the increase without S-9 mix can be seen as biologically insignificant.
The doubling of Salmonella typhimurim TA 98 could not be confirmed, and is, therefore, regarded as random result due to the low spontaneous rate of the strain.

Conclusions:

Under the experimental conditions, the test substance showed no mutagenic activity in this bacterial reverse mutation test on Salmonella typhimurium.

Executive summary:

The test substance was investigated in the Salmonella / microsome test for point-mutagenic effects in doses up to 12500 µg per plate on four Salmonella typhimurium LT2 mutants. These were the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98.

Doses up to and including 100 µg per plate did not cause any bateriotoxic effects. The total bacteria counts remained unchanged. No inhibition of growth was observed. At higher doses, the substance had a strain-specific bacteriotoxic effect. Nevertheless, this range could be used for evaluation purposes. Evidence of mutagenic activity for the test substance was not found. Neither a dose-related doubling nor a biologically-relevant increase of the mutant count, in comparison with the negative controls, was observed. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2 -phenylene-diamine and 2 -aminoanthracene had a marked mutagenic effect, as was seen by a biologically-relevant increase in mutant colonies compared to the corresponding negative controls.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test item induced an increase of mutant frequency in mammalian cells in vitro in the presence of metabolic activation. However, no mutagenic activity was observed in two bacterial reverse mutation tests on Salmonella typhimurium. Additionally, there was no indication of a clastogenic effect of an intraperitoneal dose of 15 mg/kg bw test substance in the microncleus test on the mouse, i.e. in a somatic test system in vivo. Furthermore, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in liver or stomach cells in an in vivo Comet assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 1992-04-28 to 1992-05-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Directive 84/449/EEC

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

other: Bor: NMRI (SPF Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Bor: NMRI (SPF Han)
- Source: F. Winkelmann, Borchen
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 28 - 41 g
- Assigned to test groups randomly: yes
- Housing: The females were kept in groups of a maximum of three mice in Macrolon type I cages. Males were kept singly in type I cages. Bedding of soft wood granules, type S 8/15 was used. The animals were identified by cage and picric acid markers. The wood granules were spot-checked for contaminants at regular intervals.
- Diet: Altromin 1324 Standard diet was available ad libitum.
- Water: Drinking water. Tap water was available ad libitum.
- Acclimation period: at least one week
- Other: The breed's state of health was regularly spot-checked for the major specific pathogens. Only healthy animals without symptoms were used in the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22.5 - 23 °C
- Humidity: 43 - 48 %
- Air changes: ca. 10 times per hour
- Photoperiod: 12 hours of electrical light daily (6.00 to 18.00 hours), about 500 lux

Route of administration:

intraperitoneal

Vehicle:

The test substance was dissolved in polyethylene glycol 400 (PEG 400).

Details on exposure:

single treatment

Duration of treatment / exposure:

The substance was administered once.

Frequency of treatment:

once

Post exposure period:

48 hours

Dose / conc.:

15 mg/kg bw (total dose)

Remarks:

Basis: nominal conc.

No. of animals per sex per dose:

Each group comprised ten mice, five females and five males.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Cyclophosphamide
- Route of administration: intraperitoneal
- Dose: 20 mg/kg bw

Tissues and cell types examined:

Erythrocytes (evaluation of micronucleated polychromatic erythrocytes) in bone marrow of male and female mice

Details of tissue and slide preparation:

Preparation of specimens:
Schmid's method was used to produce the smears.
At least one intact femur was prepared from each sacrificed animal (not prepared with a spindle inhibitor). A suitable instrument was used to sever the pelvic bones and lower leg. The femur was separated from muscular tissue.
The lower-leg stump, including the knee and all attached soft parts, was separated in the distal epiphyseal cartilage by a gentle pull at the distal end. The proximal end of the femur was opened at its extreme end with a suitable instrument. A suitable tube was filled with sufficient fetal calf serum. A small amount of serum was drawn from the tube into a suitable syringe with a thin cannula. The cannula was pushed into the open end of the marrow cavity. The femur was then completely immersed in the calf serum and pressed against the wall of the tube, to prevent its slipping off. The contents were then flushed several times and the bone marrow was passed into the serum as a fine suspension. Finally the flushing might be repeated from the other end, after it had been opened. The tube containing the serum and bone marrow was centrifuged at ca. 1000 rpm for 5 minutes. The supernatant was removed with a pipette. The sediment was mixed to produce a homogeneous suspension. One drop of a viscous suspension was placed on a well-cleaned slide and spread with a suitable object, to allow proper evaluation of the smear. The labelled slides were dried overnight. If fresh smears needed to be stained, they needed to be dried with heat for a short period.

- Staining of smears: The smears were stained automatically with the Ames HemaTek Slide Stainer from the Miles Company.
- Covering of smears: The smears were transferred on a holder. A cuvette was filled with xylene, into which the holder was immersed of ca. 10 minutes. The slides were removed singly to be covered. A small amount of covering agent was taken from a bottle with a suitable object and applied to the coated side of the slide. A cover glass was then placed in position without trapping bubbles. The slides were not evaluated until the covering agent had dried.

Evaluation criteria:

Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as strained chromatin particles in the enucleated erythrocytes. They can be distinguished from artefacts be varying the focus.
Normally, 1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern. It is expedient to establish the ratio of polychromatic to normochromatic erythrocytes for two reasons:
1. Individual animals with pathological bone-marrow depressions may be identified and excluded from the evaluation.
2. An alternation of this ratio may show that the test substance actually reaches the target.
Therefore, the number of normochromatic erythrocytes per 1000 polychromatic ones was notes. If the ratio for a single animal amounts to distinctly more than 3000 normochromatic erythrocytes per 1000 polychromatic ones, or if such a ratio seems likely without other animals in the group showing similar effects, then the case may be regarded as pathological and unrelated to treatment, and the animal may be omitted from the evaluation. A relevant treatment-related alteration of the ratio polychromatic to normochromatic erythrocytes can only be concluded if it is clearly lower for a majority of the animals in the treated group than in the negative control.
Reference to historical data, or other considerations of biological relevance was also taken into account in the evaluation of data obtained.

Statistics:

The test substance with the highest mean and the positive control were checked by Wilcoxon's non-parametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error probability was below 5 % and the treatment group figure was higher than that of the negative control.
The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevant increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chi2-test. A variation was considered statistically significant if the error probability was below 5 % and the treatment group figure was higher than that of the negative control.
In addition, standard deviations (1s ranges) were calculated for all the means.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

The animals treated with the test substance showed symptoms of toxicity after administration. One of forty animals died before the end of the test due to the acute intraperitoneal toxicity of 15 mg/kg test substance.

Vehicle controls validity:

valid

Negative controls validity:

other: Yes, based on literature data and laboratories' own experience

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
The selection of the test substance dose was based on the pilot test, in which groups of five animals, including both females and males, were intraperitoneally administered 10 mg/kg, 17.5 mg/kg, 25 mg/kg and 100 mg/kg test substance.
The following symptoms were recorded for up to 48 hours: apathy, roughened fur, staggering gait, spasm and difficulty in breathing. In addition, 1 of 5 animals died in the 25 mg/kg group and all animals died in the 100 mg/kg group. Based on these results, 15 mg/kg test substance was chosen as MTD for this test.

RESULTS OF DEFINITIVE STUDY
Toleration by the animals:
The following compound-related symptoms until sacrifice were observed after single intraperitoneal administration of 15 mg/kg: apathy, staggering gait, spasm, sternal recumbency, twitching and difficulty in breathing. Their feeding behaviour was normal. One of the 40 animals died during the test period, due to the acute toxicity of 15 mg/kg test substance.
Animals of the negative control showed apathy and spasm for up to four hours after treatment. Thereafter, their external appearance and physical activity remained unaffected. Their feeding behaviour was normal. No symptoms were recorded for the positive control. No animals died in the control groups.

Microscopic evaluation:
There were no relevant variations between males and females. Therefore, these were evaluated jointly.
The ratio of polychromatic to normochromatic erythrocytes was not altered by the treatment with the test substance, being 1000:1096 (1s = 223) in the negative control, 1000:1692 (1s = 1119) in the 16 hours group, 1000:1449 (s = 457) in the 24 hours group and 1000:884 (1s = 292) in the 48 hours group. Weak but relevant variations were thus noted. However, statistically significant differences were not found.
No biologically important or statistically significant variations existed between the negative control and the group treated with test substance, with respect to the incidence of micronucleated polychromatic erythrocytes.
Further, no biologically significant variations between the negative control and the test substance groups in the number of micronucleated normochromatic erythrocytes were found, since normochromatiic erythrocytes originated from polychromatic ones.
- The positive control caused a clear increase in the number of polychromatic erythrocytes with micronuclei. The incidence of micronucleated cells was 18.4/1000 (1s = 9.4), which represents a biologically relevant increase in comparison with the negative control.

Conclusions:

There was no indication of a clastogenic effect of an intraperitoneal dose of 15 mg/kg test substance in the micronucleus test on the mouse, i.e. in a somatic test system in vivo.

Executive summary:

The test substance was tested in a mouse micronucleus assay according OECD guideline no. 474 and EU method B.12. Possible clastogenic effect to the chromosomes of bone-marrow erythroblasts of the test substance were investigated in male and female mice.

The treated animals received a single intraperitoneal administration of either test substance or cyclophosphamide (positive control). The femoral marrow of groups treated with test substance was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours. The doses of test substance and positive control were 15 and 20 mg/kg bw, respectively. The animals treated with test substance showed symptoms of toxicity after administration. One of the forty animals died before the end of the test due to the acute inraperitoneal toxicity of 15 mg/kg test substance. There was weekly altered ratio between polychromatic and normochromatic erythrocytes.

The results with the test substance gave no relevant indications of clastogenic effects after single intraperitoneal treatment with 15 mg/kg. The known mutagen and clastogen, cyclophosphamide, had a clear clastogenic effect at an intraperitoneal dose of 20 mg/kg bw. The number of micronucleated polychromatic erythrocytes increased to a biologically relevant degree.

The number of microncleated normochromatic erythrocytes did not increase relevantly in any of the groups. The test substance was judged to be not clastogenic in vivo.

Reference 2

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2016-02-17 and 2016-06-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

OECD Guidelines for Testing of Chemicals, Section 4, No. 489. In vivo Mammalian Alkaline Comet Assay, adopted 26th September, 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT., Cserkesz u. 90., H-1103 Budapest, Hungary
- Age at study initiation: 50 - 55 days
- Weight at study initiation: 247 - 276 g
- Assigned to test groups randomly: yes, under following basis: All animals were sorted according to body weight by computer and grouped according to weight ranges.
- Housing: 3 animals / cage; at the positive control groups 2 animals / cages
- Diet: ad libitum (ssniff® SM R/M-Z+H complete diet)
- Water: ad libitum (tap water)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8 - 22.7
- Humidity (%): 32 - 54
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: propylene glycol 400 (PEG 400)
- Justification for choice of solvent/vehicle: The was chosen as vehicle for the formulation of test item solutions, based on the earlier formulation experiences originated from acute oral toxicity and from previous repeated dose oral gavage toxicity studies performed with this test item. The suitability of PEG 400 vehicle in the in vivo Comet assay was checked, controlled in a separate validation study (Study code: 392-489-1283) performed in the testing laboratory under the same conditions as the present study.
- Concentration of test material in vehicle: 200, 100 and 50 mg/mL
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations were prepared before each treatment. The test item was formulated in the vehicle in nominal concentrations of 200, 100 and 50 mg/mL.

Duration of treatment / exposure:

2 days

Frequency of treatment:

- Test animals: twice (once on the day 0 and 24 hours thereafter)
- Positive controls animals: once during the experiment on the day 1

Post exposure period:

3 - 4 hours after the second treatment

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Numbers of treated animals: 6 animals in the dose groups and negative control group; 4 animals in the positive control groups
Numbers of analysed animals: 5 animals in the dose groups and negative control group; 3 animals in the positive control groups

Control animals:

yes, concurrent vehicle

Positive control(s):

1,2-Dimethylhydrazine dihydrochloride (DMH); Ethyl methanesulfonate (EMS)
- Justification for choice of positive control(s): The known mutagen DMH was used in the testing laboratory as positive control item (for liver and duodenum samples) in several earlier studies (performed under comparable test conditions with the conditions of the present study) therefore its use as an additional group was considered. The EMS is widely used and proposed (OECD 489 guideline) know positive mutagen for any tissue.
- Route of administration: oral (gavage)
- Doses / concentrations: 20 mg/kg bw/d (DMH); 200 mg/kg bw/d (EMS)

Tissues and cell types examined:

liver and glandular stomach

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
For dose selection the guideline proposal and the information obtained from an acute oral toxicity study and from a previous repeated dose oral gavage toxicity study with Incozol EH in rats (results of this study were provided by the Sponsor) was taken into consideration. Based on this information the maximum dose were 2000 mg/kg bw/day, and in addition to the maximum dose two additional doses 1000 and 500 mg/kg bw/day were selected. For the correct identification of the maximum tolerated dose (MTD) where no death, evidence of pain, suffering or distress occurs, a range finding test was performed in the testing laboratory, using the same species, strain, sex, and treatment regimen to be used in the main study.
In the range-finding test (based on the available information) two animals (male rats, CRL (WI) BR of Wistar origin) were treated with the test item by oral administration at 10 mL/kg body weight treatment volume at the dose level of 2000 mg/kg bw/day. The treatment was performed on two consecutive days with 24h interval. At the chosen concentration level neither mortality nor any clinical sign, any suffering of animals were observed; therefore 2000 mg/kg body weight/day was chosen as the highest dose level in the present study.

SAMPLING TIMES:
The sampling was performed 3 - 4 hours after the second treatment.

DETAILS OF SLIDE PREPARATION:
- Liver Single Cell Preparation:
A portion of the left lateral lobe of the liver was removed and washed in the cold mincing buffer until as much blood as possible was removed; thereafter placed in mincing buffer (ice cold Hank’s Balanced Salt Solution (HBSS) containing 20 mM EDTA and 10 % DMSO), minced with a pair of fine scissors to release the cells. The cell suspension was kept on ice for about 30 seconds to allow the large clumps to settle. The supernatant was pipetted into an Eppendorf tube and used for comet slides.

- Glandular Stomach Single Cell Preparation:
The stomach was open and washed free shortly from food using cold phosphate buffered saline. The forestomach was removed and discarded. The glandular stomach was then placed into cold mincing buffer and incubated on ice for about 15 minutes. After the incubation the surface epithelia was gently scraped about two times using a scalpel blade. This layer was discarded and the gastric mucosa was rinsed with cold mincing buffer. Thereafter, the stomach epithelium was carefully scraped 4 - 5 times with scalpel blade. The obtained cell suspension was kept on ice for about 30 seconds to allow the large clumps to settle. The supernatant was pipetted into an Eppendorf tube and used for comet slides.

- Preparation of the Comet Assay Slides:
The slide preparation was done within one hour after single cell preparation. Four slides were prepared for each animal for each tissue sample, with six animals per dose group and vehicle control and four animals for the positive control groups. In summary, 24 slides per treatment per tissue for the treatment groups and vehicle control and 16 slides per tissue for the positive controls were prepared in total. The slides were adequately coded (with number: animal number, parallel number and M letter for liver and G letter for stomach).

Pre-treatment of slides: Conventional (superfrost) slides were dipped in hot 0.5 % normal melting point agarose in water. After gently remove the underside of the slides were wiped in order to remove the excess of agarose. The slides were then laid on a flat surface and were let allow drying.

Embedding the cells: Before the use a volume of 130 μL of 0.5 % normal melting point agarose (NMA) was added on a microscope slide pre-layered with 0.5 % NMA (see above) and covered with a glass coverslip. The slides were placed on a tray until the agarose hardens (~ 5 minutes). After the cell isolations the cell suspension was mixed with 0.5 % 1.0 % Low Melting Point Agarose (LMPA); thereafter 85 μL (~1 – 3 x 1E+04 cells) of this mixture was added on the microscope slide after gentle slide off the coverslip.
The microscope slides were covered with a new coverslip. After the LMPA-cell mixture hardens an additional 70 μL of NMA was dropped on the microscope slide after a gentle slide off the (second) coverslip and an additional new coverslip was laid on the slide. After the repeated NMA layer hardens the coverslip was removed.

- Lysis:
After the top layer of agarose solidifies and the last glass coverslip was removed the slides were immersed in chilled lysing solution. The slides were kept overnight in lysing solution at 2 – 8 °C (in refrigerator) in the dark.
After the incubation period, the slides were rinsed to remove residual detergents and salts prior to the alkali unwinding step. This rinsing procedure was performed in electrophoresis buffer.

METHOD OF ANALYSIS:
- Unwinding and Electrophoresis:
The slides were removed from the lysing solution and randomly placed on a horizontal gel electrophoresis unit. The unit was filled up with freshly prepared electrophoresis solution until the surfaces of the slides are completely covered with the solution (to about 1 - 2 mm above the slides).
During the unwinding and electrophoresis a balanced design was used to place slides in the electrophoresis tank to mitigate the effects of any trends or edge effect within the tank and to minimize batch to batch variability.
The slides were left for 30 min. for the DNA to unwind. Thereafter the electrophoresis was conducted for 30 min. by applying a constant voltage of 25 V and an electric current of about 300 mA (260 - 299 mA). The same volume of the electrophoresis solution was used at every run, therefore at constant voltage slight change in the electric current was noticed. All of these steps were sheltered from the daylight to prevent the occurrence of additional DNA damage.
The temperature of the electrophoresis solution (before the unwinding the electrophoresis solution was kept in refrigerator, its temperature was noticed: 5.2 - 5.8 °C) through unwinding and electrophoresis was maintained at a low temperature, at 5 °C using a special cooler designed for Comet electrophoresis tank.
The temperature of the electrophoresis solution before the unwinding, during the unwinding and electrophoresis was recorded.

- Neutralization and Preservation of Slides:
After electrophoresis, the slides were removed from the electrophoresis unit, covered with neutralization solution left stand for about 5 minutes, thereafter blotted and covered again with neutralization solution. This procedure was repeated for three times. Subsequently the slides were exposed for additional 5 minutes to absolute ethanol in order to preserve all of the slides. The slides were stored at room temperature until scored.

- Staining:
The slides were air dried and then stored at room temperature until they were scored for comets. Just prior the scoring the DNA, the slides were stained using 50 μL of 20 μg/mL Ethidium bromide.

- Evaluation of slides:
Every animal were euthanized and their cells were isolated. For each animal, each tissue 4 slides were prepared (8 slides per animal, 48 slides per test item doses and negative control and 32 slides per positive controls).
Three slides of five animals per vehicle control and test item treatments were stained and analysed (6 slides per animal, 30 slides per dose) and three slides of three animals per positive controls were stained and analysed (6 slides per animal, 18 slides per dose).
Coded slides were stained and blind scored. The slides were examined with an appropriate magnification (200x) using fluorescent microscope (Olympus BH-2) equipped with an appropriate excitation filter (TRITC) and with an Alpha DCM 510B CMOS camera.
For image analysis the Andor Kinetic Imaging Komet 6.0 (Andor Technology) was used.
For each tissue sample fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analysed cells per test item treatment, per vehicle control and 450 per positive controls).
DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, tail moment and tail length.
Olive Tail Moment (OTM): is expressed in arbitrary units, is calculated by multiplying the percentage of DNA (fluorescence) in the tail by the length of the tail in μm. The tail length is measured between the centre of the comet head and the end of the comet tail.
The tail % DNA (also known as tail intensity) was applied for the evaluation and interpretation of the results and determined by the DNA fragment intensity in the tail expressed as a percentage of the cell’s total intensity.
In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis). The ghost cells were recorded for each slide per animal, per type of the treatment and per tissue. Ghost cells were excluded from the image analysis data collection.

Evaluation criteria:

The test chemical is clearly negative if:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
- there is no concentration-related increase when evaluated with an appropriate trend test;
- all results are inside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administration;
- direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) is demonstrated.

The test chemical is clearly positive if:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
- the increase is dose-related when evaluated with an appropriate trend test;
- any of the results are outside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administration;

Statistics:

The heterogeneity of the obtained data was tested. The statistical significance of % tail DNA values, OTM tail length values; furthermore the number of ghost cells was carried out using the appropriate statistical method, using SPSS PC+ software.
The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. In case of a positive analysis, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data were not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. In case of a positive analysis result, the inter-group comparisons were performed using Mann-Whitney U-test.

Key result

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: 2000 mg/kg bw/day
- Clinical signs of toxicity in test animals: neither mortality nor any clinical signs were observed

RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: The highest dose (2000 mg/kg bw/day) is selected according to the criteria required by the OECD 489 guideline. Exposure via oral route was chosen as this route was considered to be the most relevant exposure route.

Results of Analytical Measurements

Based on the results of the available validation study the test item proved to be stable in PEG 400 formulations at 20 and ~200 mg/mL concentration levels at least for 6 hours at room temperature and for 1 day in refrigerator (5 ± 3 °C), (Study No.: 644-100-4845). The homogeneity of the samples was good, the test item in PEG 400 formulations was considered to be homogeneous. The measured concentration values remained within the ± 5 % of nominal range at 200 and 100 mg/mL concentrations (99 and 100 % for the 200 mg/mL solution; 95 and 96 % for the 100 mg/mL solution); but slightly lower values 87 % of the nominal were obtained at the nominal concentration of 50 mg/mL. Based on the analytical results the real measured test item concentrations were (when taking the average of the measured values into consideration): 199, 95.6 and 43.6 mg/mL that corresponded to 1990, 956 and 436 mg/kg bw/d treatment concentrations. The measured lower values at lower test item concentration (50 mg/mL) were considered as acceptable and caused rather on the test item instability (facilitated by some inevitable, suspected water content of PEG 400) than as a technical error. In this particular unequivocally negative test the somewhat lower measured concentration levels (especially the measured: 43.6 mg/mL instead of 50 mg/mL (436 mg/kg bw/d instead of 500 mg/kg bw/d) have not any influential effect on the results and final conclusion of the study; therefore the nominal concentration values 200, 100 and 50 mg/mL (2000, 1000 and 500 mg/kg bw/d) were applied and referred throughout the study.

 

Mortality and Clinical Observations

No mortality was observed during the treatments and expression period in any dose group and in the controls. Toxic symptoms any clinical signs were not observed during the treatments: 1, 2 and 4 hours after the first treatment (day 0), just before the second treatment on day 1; furthermore 1 hour after the second treatment and shortly before sampling time (on day 1). At the tissue isolation after the opening of the stomach a characteristic strong chemical smell was noticed at 500, 1000 and 2000 mg/kg bw/d; the smell intensity increased dose-dependently. At 1000 and 2000 mg/kg bw/d doses attenuated gastric mucosa layer was noticed. The animal weights increased in average of 3.85 % in the negative control group, when compared the weight values just before the first treatment and just before the sacrifice. Animal weight increases were obtained in all treated concentrations and also at the positive controls. The weight increases remained in the same range 3.56 - 4.07 % at the doses of 500 and 1000 mg/kg bw/d and at the positive controls and the increase was lower 2.28 % at the highest dose group of 2000 mg/kg bw/d.

 

Cytotoxicity, Ghost Cells

In this study as the first indication of the possible cytotoxicity was estimated by Trypan blue dye exclusion technique. This screening technique as an indicator provided preliminary information from the effectiveness and success of the single cell preparation. The cell concentrations of the isolated cell suspensions were in the same (1E+06) order of magnitude. The viability values of both the liver and the stomach cell suspensions remained in the same, vehicle control range at all test item treatment doses and positive controls. The screened average viability values varied between 82 - 90 % at the liver cell preparations and 81 - 85 % at the stomach cell preparations. The decrease of viability was not more than 30 % when compared to the concurrent control in any case. In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis). Ghost cells results from a total migration of the DNA from the nucleus into the comet tail, reducing the size of the head to a minimum. The number of ghost cells did not differ statistically significantly from that of the vehicle control at the examined doses and at the positive control treatments in the stomach samples. The numbers of ghost cells did not differ statistically significantly from that of the vehicle control at the examined doses and at the EMS positive control treatments in the liver samples, and a statistically significant increase of ghost cells was noticed at the DMH treatments in the case of liver samples.

 

% Tail DNA, Olive Tail Moment and Tail Length Comparisons

The tail % DNA (also known as tail intensity) was applied for the evaluation and interpretation of the results and determined by the DNA fragment intensity in the tail expressed as a percentage of the cell’s total intensity. The mean % tail DNA values of each treatment were slightly lower than the vehicle control value at both, at the stomach and liver samples. The mean % tail DNA values in the stomach samples did not differ statistically significantly from that of the vehicle control at the 500 and 1000 mg/kg bw/d doses and statistical significant difference was noticed at the 2000 mg/kg bw/d dose. The statistical significance was considered as not relevant in mutagenicity point of view since the significance linked with a lower (but acceptable) value than the corresponding vehicle control value. The mean % tail DNA values in the liver samples did not differ statistically significantly from that of the vehicle control at the whole examined dose range. Additionally the % tail DNA mean median values were calculated and investigated. In the case of stomach samples the same tendencies were obtained as it was noticed at the mean values. The slightly lower (in comparison with the negative control value) mean median values did not show statistical significant difference in the whole examined dose range. The % tail DNA mean median values in the liver samples remained nearly in the same range (varied between 3.95 - 4.51 %). The slightly higher value 4.51 (vehicle control: 4.23) was obtained at the highest test item dose of 2000 mg/kg bw/d. The % tail DNA values in the liver samples did not differ statistically significantly from that of the vehicle control at the whole examined dose range. The mean median value calculations were performed in the case of the % tail DNA parameter, only. The analysis of these values confirmed the results obtained at the mean value calculations. Additionally the tail length and olive tail moment values of the vehicle control and each treatment were compared. Statistical significances at the OTM values of the stomach samples was not noticed at the 500 and 1000 mg/kg bw/d doses and statistical significant difference was noticed at the 2000 mg/kg bw/d dose. Similarly to the % tail DNA evaluations, the statistical significance was considered as not relevant in mutagenicity point of view. Statistical significances were not noticed at the OTM values of the liver samples in the whole dose range. In the case of the tail length comparisons statistical significances were not noticed at the in the stomach samples, not relevant significance was noticed in the liver samples at the dose of 1000 mg/kg bw/d and statistical significance was noticed at the dose of 2000 mg/kg bw/d. In this latter case the tail length values were higher than the tail length values of the negative control; however the higher value did not accompanied with dose relationship and taking the other parameters (% tail DNA and OTM) the statistical significance was not considered as biologically relevant.

Conclusions:

Under the experimental conditions, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in liver or in stomach cells. Thus, the test item is negative as it did not show genotoxic activity in the examined tissues.

Executive summary:

The purpose of the comet assay (single cell gel electrophoresis assay) was to evaluate the mutagenic potential of the test item by measuring its ability to induce DNA damage in the target organs, tissues. The genotoxicity potential of the test item was investigated on isolated liver and stomach cells under alkaline conditions in the male WISTAR rats administered orally twice with 2000, 1000 and 500 mg/kg body weight/day on day 0 and 24 hours thereafter. The negative control (propylene glycol 400) and positive controls (1,2-dimethylhydrazine dihydrochloride and ethyl methanesulfonate) were tested in parallel. Each dose group and the negative control group consisted of 6 animals; the positive control groups consisted of 4 animals. 

 

Formulations were prepared before each treatment. The test item was formulated in the vehicle in nominal concentrations of 200, 100 and 50 mg/mL. The measured concentration values remained within the ± 5 % of nominal range at 200 and 100 mg/mL concentrations (99 and 100 % for the 200 mg/mL solution; 95 and 96 % for the 100 mg/mL solution); but slightly lower values 87 % of the nominal were obtained at the nominal concentration of 50 mg/mL. In this particular unequivocally negative test the somewhat lower measured concentration levels (especially the measured: 43.5 and 43.7 mg/mL instead of 50 mg/mL (435 and 437 mg/kg bw/d instead of 500 mg/kg bw/d) have not any influential effect on the results and final conclusion of the study; therefore the nominal concentration values 200, 100 and 50 mg/mL (2000, 1000 and 500 mg/kg bw/d) were applied and referred throughout the study. Analysis of formulations (for checking of each concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility according to the validated analytical method (Study code: 644-100-4845).

3-4 hours after the second treatment (doses and vehicle control) and 3 - 4 hours after the treatment (positive controls) the animals were euthanized and the cells of the target tissues were isolated. Cytotoxicity was determined on a small sample of each isolated cell suspension following the Trypan blue dye exclusion technique, directly after sampling. The slide preparation was done within one hour after single cell preparation. Therefore the following steps were performed: embedding the cells, lysis (pH > 10), alkaline unwinding (pH > 13) followed by electrophoresis (pH > 13). After electrophoresis slides were neutralized and preserved in absolute ethanol. Just prior scoring the DNA, the slides were stained using Ethidium bromide.

For each animal, each tissue 4 slides were prepared (8 slides per animal, 48 slides per test item doses and negative control and 32 slides per positive controls).

Three slides of five animals per vehicle control and test item treatments were stained and analysed (6 slides per animal, 30 slides per dose) and three slides of three animals per positive controls were stained and analysed (6 slides per animal, 18 slides per dose).

The comets were measured via a digital camera linked to an image analyser system using a fluorescence microscope equipped with an appropriate excitation filter at a magnification of 200X. For image analysis the Komet 6.0 F (Andor Technology) was used.

For each tissue sample fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analysed cells per test item treatment, per vehicle control and 450 per positive controls).

In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis). Ghost cells were excluded from the image analysis data collection.

 

All of the validity criteria regarding the negative and positive control treatments as well as the number of analysed cells, and the investigated dose levels were met (See: Validity of the Study).

No mortality was observed during the treatments and expression period in any dose group and in the controls. Toxic symptoms any clinical signs were not observed during the treatments. At the tissue isolation after the opening of the stomach a characteristic strong chemical smell was noticed at the test item treatments and the smell intensity increased dose-dependently.

The animal weights increased in all treatments, test item, negative and positive control treatments. The weight increases remained in the same range 3.56 - 4.07 % at the negative and positive controls and at the doses of 500 and 1000 mg/kg bw/d and the increase was lower 2.28 % at the highest dose group of 2000 mg/kg bw/d.

At the screening cytotoxicity measurements (using Trypan blue dye exclusion method) significant cytotoxicity was not noticed in any test item and control item treatments.

The number of ghost cells did not differ statistically significantly from that of the vehicle control at the examined doses and at the positive control treatments in the stomach samples.

The numbers of ghost cells did not differ statistically significantly from that of the vehicle control at the examined doses and at the EMS positive control treatments in the liver samples, and a statistically significant increase of ghost cells was noticed at the DMH treatments in the case of liver samples.

DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, Olive Tail Moment (OTM) and tail length.

The % tail DNA mean values of each treatment were slightly lower than the vehicle control value at both, at the stomach and liver samples.

The % tail DNA mean values in the stomach samples did not differ statistically significantly from that of the vehicle control at the 500 and 1000 mg/kg bw/d doses and statistical significant difference was noticed at the 2000 mg/kg bw/d dose. The statistical significance was considered as not relevant in mutagenicity point of view since the significance linked with a lower (but acceptable) value than the corresponding vehicle control value.

The % tail DNA values in the liver samples did not differ statistically significantly from that of the vehicle control at the whole examined dose range.

Similarly to the % tail DNA values in the stomach and liver samples sporadic statistical significances occurred at the tail moment and tail length comparisons as well; however the statistical significances were not considered as biologically relevant.

Additionally the % tail DNA mean median values were investigated and found the same tendencies as at the mean values in the case of stomach samples and the mean median values of the liver samples were nearly in the same range in the vehicle control and treated doses. Any tendency and/or statistical significance at the test item treated doses was not noticed.

 

In conclusion, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in liver or in stomach cells. Thus, the test item is negative, did not show genotoxic activity in the examined tissues.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vivo

 

Micronucleus test

The test substance was tested in a mouse micronucleus assay according OECD guideline no. 474 and EU method B.12. Possible clastogenic effect to the chromosomes of bone-marrow erythroblasts of the test substance were investigated in male and female mice.

The treated animals received a single intraperitoneal administration of either test substance or cyclophosphamide (positive control). The femoral marrow of groups treated with test substance was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours. The doses of test substance and positive control were 15 and 20 mg/kg bw, respectively. The animals treated with test substance showed symptoms of toxicity after administration. One of the forty animals died before the end of the test due to the acute inraperitoneal toxicity of 15 mg/kg test substance. There was weekly altered ratio between polychromatic and normochromatic erythrocytes.

The results with the test substance gave no relevant indications of clastogenic effects after single intraperitoneal treatment with 15 mg/kg. The known mutagen and clastogen, cyclophosphamide, had a clear clastogenic effect at an intraperitoneal dose of 20 mg/kg bw. The number of micronucleated polychromatic erythrocytes increased to a biologically relevant degree.

The number of microncleated normochromatic erythrocytes did not increase relevantly in any of the groups. The test substance was judged to be not clastogenic in vivo.

 

In vivo alkaline comet assay

The purpose of the comet assay (single cell gel electrophoresis assay) was to evaluate the mutagenic potential of the test item by measuring its ability to induce DNA damage in the target organs, tissues. The genotoxicity potential of the test item was investigated on isolated liver and stomach cells under alkaline conditions in the male WISTAR rats administered orally twice with 2000, 1000 and 500 mg/kg body weight/day on day 0 and 24 hours thereafter. The negative control (propylene glycol 400) and positive controls (1,2-dimethylhydrazine dihydrochloride and ethyl methanesulfonate) were tested in parallel. Each dose group and the negative control group consisted of 6 animals; the positive control groups consisted of 4 animals.

Formulations were prepared before each treatment. The test item was formulated in the vehicle in nominal concentrations of 200, 100 and 50 mg/mL. The measured concentration values remained within the ± 5 % of nominal range at 200 and 100 mg/mL concentrations (99 and 100 % for the 200 mg/mL solution; 95 and 96 % for the 100 mg/mL solution); but slightly lower values 87 % of the nominal were obtained at the nominal concentration of 50 mg/mL. In this particular unequivocally negative test the somewhat lower measured concentration levels (especially the measured: 43.5 and 43.7 mg/mL instead of 50 mg/mL (435 and 437 mg/kg bw/d instead of 500 mg/kg bw/d) have not any influential effect on the results and final conclusion of the study; therefore the nominal concentration values 200, 100 and 50 mg/mL (2000, 1000 and 500 mg/kg bw/d) were applied and referred throughout the study. Analysis of formulations (for checking of each concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility according to the validated analytical method (Study code: 644-100-4845).

3 - 4 hours after the second treatment (doses and vehicle control) and 3 - 4 hours after the treatment (positive controls) the animals were euthanized and the cells of the target tissues were isolated.

Cytotoxicity was determined on a small sample of each isolated cell suspension following the Trypan blue dye exclusion technique, directly after sampling.

The slide preparation was done within one hour after single cell preparation. Therefore the following steps were performed: embedding the cells, lysis (pH > 10), alkaline unwinding (pH > 13) followed by electrophoresis (pH > 13). After electrophoresis slides were neutralized and preserved in absolute ethanol. Just prior scoring the DNA, the slides were stained using Ethidium bromide.

For each animal, each tissue 4 slides were prepared (8 slides per animal, 48 slides per test item doses and negative control and 32 slides per positive controls).

Three slides of five animals per vehicle control and test item treatments were stained and analysed (6 slides per animal, 30 slides per dose) and three slides of three animals per positive controls were stained and analysed (6 slides per animal, 18 slides per dose).

The comets were measured via a digital camera linked to an image analyser system using a fluorescence microscope equipped with an appropriate excitation filter at a magnification of 200x. For image analysis the Komet 6.0 F (Andor Technology) was used.

For each tissue sample fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analysed cells per test item treatment, per vehicle control and 450 per positive controls).

In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis). Ghost cells were excluded from the image analysis data collection.

All of the validity criteria regarding the negative and positive control treatments as well as the number of analysed cells, and the investigated dose levels were met (See: Validity of the Study).

No mortality was observed during the treatments and expression period in any dose group and in the controls. Toxic symptoms any clinical signs were not observed during the treatments. At the tissue isolation after the opening of the stomach a characteristic strong chemical smell was noticed at the test item treatments and the smell intensity increased dose-dependently.

The animal weights increased in all treatments, test item, negative and positive control treatments. The weight increases remained in the same range 3.56 - 4.07 % at the negative and positive controls and at the doses of 500 and 1000 mg/kg bw/d and the increase was lower 2.28 % at the highest dose group of 2000 mg/kg bw/d.

At the screening cytotoxicity measurements (using Trypan blue dye exclusion method) significant cytotoxicity was not noticed in any test item and control item treatments.

The number of ghost cells did not differ statistically significantly from that of the vehicle control at the examined doses and at the positive control treatments in the stomach samples.

The numbers of ghost cells did not differ statistically significantly from that of the vehicle control at the examined doses and at the EMS positive control treatments in the liver samples, and a statistically significant increase of ghost cells was noticed at the DMH treatments in the case of liver samples.

DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, Olive Tail Moment (OTM) and tail length.

The % tail DNA mean values of each treatment were slightly lower than the vehicle control value at both, at the stomach and liver samples.

The % tail DNA mean values in the stomach samples did not differ statistically significantly from that of the vehicle control at the 500 and 1000 mg/kg bw/d doses and statistical significant difference was noticed at the 2000 mg/kg bw/d dose. The statistical significance was considered as not relevant in mutagenicity point of view since the significance linked with a lower (but acceptable) value than the corresponding vehicle control value.

The % tail DNA values in the liver samples did not differ statistically significantly from that of the vehicle control at the whole examined dose range.

Similarly to the % tail DNA values in the stomach and liver samples sporadic statistical significances occurred at the tail moment and tail length comparisons as well; however the statistical significances were not considered as biologically relevant.

Additionally the % tail DNA mean median values were investigated and found the same tendencies as at the mean values in the case of stomach samples and the mean median values of the liver samples were nearly in the same range in the vehicle control and treated doses. Any tendency and/or statistical significance at the test item treated doses was not noticed.

In conclusion, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in liver or stomach cells. The test item is negative, did not show genotoxic activity in the examined tissues.

 

 

 

In vitro

 

Ames test

The test substance was investigated in the Salmonella / microsome test for point-mutagenic effects in doses up to 5000 µg per plate on four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98. Doses up to and including 200 µg per plate did not cause any bateriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed.

At higher doses, the substance had a strain-specific bacteriotoxic effect, so that this range could only be used to a limited extend up to 5000 mg per plate for assessment purposes. Evidence of mutagenic activity for the test substance was not seen. No biologically-relevant increase of the mutant count, in comparison with the negative controls, was observed. Therefore, the test substance was considered to be non-mutagenic without and with S9 mix in the Salmonella/microsome test. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2 -phenylene-diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically-relevant increase in mutant colonies compared to the corresponding negative controls. This result is supported by a second Ames test available. No evidence of mutagenic activity was found for the test substance. Neither a dose related doubling nor a biologically relevant increase of the mutant count in comparison with the negative controls, were observed.

 

HPRT test

The test item, Incozol EH was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations and basis of solubility of test item made in a preliminary study (without and with metabolic activation using S9 mix). In the performed Main Mutation Assays the concentration levels were chosen mainly based on the solubility of test item.

Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:

Experiment 1, 5-hour treatment period without S9 mix:

200, 250, 300, 350, 400, 450 and 500 μg/mL

Experiment 1, 5-hour treatment period with S9 mix:

200, 250, 300, 350, 400, 450 and 500 μg/mL

Experiment 2, 20-hour treatment period without S9 mix:

200, 250, 300, 350, 400, 450, 500 and 550 μg/mL

Experiment 2, 5-hour treatment period with S9 mix:

200, 250, 300, 350, 400, 450 and 500 μg/mL

In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested without metabolic activation. There were no biologically significant differences between treatment and control groups and no dose-response relationships were noted.

In Experiment 1, the mutant frequency of the cells showed statistically and biologically significant increases in mutation frequency at the concentrations of 350, 400, 450 and 500 μg/mL, when the test item was tested with S9-mix.

In Experiment 2, the mutant frequency of the cells showed statistically significant increases in mutation frequency at the concentrations of 400, 450 and 500 μg/mL when the test item was tested with S9-mix. These increases were biologically significant and dose-response relationships were noted.

In Experiment 2, the mutant frequency of the cells did not show biologically or statistically significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours), further indicating that the findings in Experiment 1 were in the normal biological variation observed, when the test item was examined in the absence of metabolic activation.

The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures.

Incozol EH tested without metabolic activation (S9 mix) over a 5-hour and 20-hour period did not induce increases in mutant frequency over the background (negative solvent control) in this in vitro test in Chinese hamster ovary cells. Incozol EH tested with metabolic activation (S9 mix) over a 5-hour treatment period induced statistically and biologically significant increases in the mutant frequency.

Incozol EH was mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.

 

 

 

Conclusion

There are two bacterial reverse mutation assays (Ames test), one mammalian cell gene mutation test in vitro, one in vivo micronucleus test and one in vivo comet assay. Although the test item induced an increase of mutant frequency in mammalian cells in vitro in the presence of metabolic activation, no mutagenic activity was observed in two bacterial reverse mutation tests on Salmonella typhimurium. Additionally, there was no indication of a clastogenic effect of an intraperitoneal dose of 15 mg/kg bw test substance in the micronucleus test on the mouse, i.e. in a somatic test system in vivo. Furthermore, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in liver or stomach cells in an in vivo Comet assay. Thus, the test substance is considered to be not mutagenic.

Justification for classification or non-classification

Based on the available in vitro and in vivo the test item is considered to be not mutagenic according to Regulation (EC) No 1272/2008 (CLP), as amended for the 17th time in Regulation (EU) 2021/849.