Alcohols, C6-8, ethoxylated

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The evaluation of potential mutagenic properties of the submission substance was evaluated by performing in vitro mutagenicity studies (OECD TG 471, 473 and 476) with and without metabolic activation. In all available assays no indication of genetic toxicity was observed. Based hereupon, it can be reasonably concluded that the submission substance is not mutagenic in test systems for bacterial point mutation and/or mammalian cell systems regarding gen mutation or chromosome abberation neither with or without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 December 2021 To 25 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 476

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

To evaluate the mutagenic potential of the test item quantitation of forward mutations at the Hprt locus of CHO AA8 cells was performed.

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate

Type of assay:

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

Target gene:

Hprt locus of CHO AA8 cells.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

AA8 Celles

Details on mammalian cell type (if applicable):

CHO AA8 cells, Batch No.5000062 procured from American Type Culture Collection (ATCC) was used for the test.

Additional strain / cell type characteristics:

other:

Metabolic activation:

with and without

Metabolic activation system:

Sodium phenobarbitone and β-Naphthoflavone induced rat liver S9 homogenate was used as the metabolic activation system. The S9 homogenate was prepared from male wistar rats induced with intraperitoneal injection of sodium phenobarbitone and β-naphthoflavone at 16 mg/mL and 20 mg/mL respectively for 3 days prior to sacrifice. The S9 homogenate was prepared and stored in the test facility at -80±10ºC until use. Batch of S9 homogenate was assessed for sterility, protein content (Modified Lowry assay, Sword and Thomson, 1980) and for its ability to metabolize the promutagens 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 strain.
One mL of S9 homogenate was thawed immediately before use and mixed with 9 mL of co-factor solution containing 4 mM NADP, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in Phosphate Buffer Saline (PBS) of pH 7.37 and 7.34.
The details of S9 homogenate and activation mixture used in the study are given below:
Details of S9 homogenate:
Date of preparation:
09/12/2021
Batch No.:
S9-XI/21
Date of characterization
Sterility check:
09/12/2021
Activity check:
10/12/2021
Protein estimation:
13/12/2021
Expiry date:
08/12/2023

Test concentrations with justification for top dose:

Based on precipitation results, 0.125, 0.25, 0.5, 1, and 2 µL/mL were used for initial cytotoxicity testing.
Based in cytotoxicity results, 0.125, 025, 0.5, and 1 µL/mL were used for gene mutation testing with the survival rate being > 10 % at 1 µL/mL.

Vehicle / solvent:

- Vehicle used: DMSO

- Justification for choice of vehicle: Based in solubility testing

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: tetra plate cultures, each tetra plate treatments were pooled into pre-labeled tube and centrifuged at 800 rpm for 10 mintes. Supernatant was discarded, and cell pellet was retained and resuspended in culture media. Each treatment replicate was plated in a triplicate with cell concentration of 200 cells/5 mL media in 25 cm² flasks and incubated at 37 +/- 1 °C with 5 +/- 1 % CO2 for 9 days.
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
Approximately 2×106 (Initial cytotoxicity test and Gene mutation test) cells per culture flask were seeded using culture medium with 10% FBS with antibiotics (1% Penicillin and Streptomycin). Four additional flasks were seeded and kept for incubation along with flasks for treatment to determine cell count at the beginning of the treatment to determine the Adjusted Cloning Efficiency. The flasks were incubated at 37±1oC with 5±1% CO2 for 23 hours (Initial cytotoxicity test) and 23 hours and 15 minutes (Gene mutation test). Cells free of mycoplasma were used for the experiment.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Cells were exposed to the test item for 3 hours and 18 minutes at 37±1oC with 5±1% CO2.
- Harvest time after the end of treatment (sampling/recovery times): 8 days

FOR GENE MUTATION:
Post expression period of 8 days of mutant phenotype, each replicate treatment cultures were pooled and sub cultured in quintuplicates at a density of 4×105 cells per 25 cm2 flask with culture media containing 10 μM of 6-Thioguanine and 200 cells /25 cm2 flask in triplicates without 6-Thioguanine for determination of cloning efficiency. Flasks were incubated at 37±1°C with 5±1% CO2 for 9 days. Post incubation period, medium from each dish was aspirated and stained with 5% Giemsa stain, number of colonies formed were counted manually.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
For tests with exogenous metabolic activation, 1 mL of S9 mix was added to all the flasks. A volume of 100 μL of vehicle/different concentrations of test item was added to tetra plate cultures to get the required test concentration per mL of the test medium and volume of medium was made up to 10 mL. Cells were exposed to the test item for 3 hours and 18 minutes at 37±1oC with 5±1% CO2. For tests without exogenous metabolic activation, a volume of 100 μL of vehicle/different concentrations of test item was added to tetra plate culture to get the required test concentration per mL of the test medium and volume of medium was made up to 10 mL. Cells were exposed to the test item for 3 hours and 18 minutes at 37±1oC with 5±1% CO2. Post incubation period (Set 1 and 2), medium from each flask was aspirated and monolayer was washed with DPBS. Cells were trypsinized by adding trypsin-EDTA. Trypsinization was stopped by adding culture media followed by collecting the media with cells. Tetra plate treatments were pooled and collected in prelabelled tubes and centrifuged at 800 rpm for 10 minutes. Supernatant was discarded, and cell pellet was retained and resuspended in culture media.
Each treatment replicate was plated in triplicate with cell concentration of 200 cells / 5 mL media in 25 cm2 flasks and incubated at 37±1oC with 5±1% CO2 for 9 days. Post incubation period, medium from each culture flask was aspirated and stained with 5% Giemsa stain. Number of colonies formed was counted manually. Relative Survival Rate was used to determine cytotoxicity.

Evaluation criteria:

Acceptability Criteria
Acceptance of a test is based on the following criteria:
1 The concurrent vehicle control is considered acceptable for addition to the laboratory historical vehicle control database as described in OECD guidelines for testing of chemicals, No. 476.
2 Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative/vehicle control.
3 Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
4 Adequate number of cells and concentrations are analysable (according to OECD guidelines for testing of chemicals, No. 476).
5 The criteria for the selection of top concentration are consistent with those described in OECD guidelines for testing of chemicals, No. 476.

Interpretation of Results
A test chemical is clearly positive if, in any of the experimental conditions examined:
1 At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2 The increase is concentration-related when evaluated with an appropriate trend test.
3 Any of the results are outside the distribution of the historical negative/vehicle control data.
When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
A test chemical is considered clearly negative if, in all experimental conditions examined:
4 None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
5 There is no concentration-related increase when evaluated with an appropriate trend test.
6 All results are inside the distribution of the historical negative/vehicle control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells

Statistics:

Data of mutant frequencies were analyzed for differences among vehicle control, treatment and positive control groups by performing power transformation procedure by Snee and Irr (1981) with which, the observed mutant frequency was transformed using the formula:
Y=(X+A)B
Where,
BIO-GNT 1805 Study Report Page 18 of 35
Y = transformed mutant frequency, X = observed mutant frequency
[Where X=No. of mutant colonies per replicateACE value×100
and A, B = constants (viz. A = 1 and B = 0.15)]
Statistical analysis of the experimental data was carried out using SPSS Statistical package version 22.0. If the analysis of variance is significant at p < 0.05, Dunnett’s test will be conducted, comparing each treatment group and the positive control to the vehicle control.
The statistical significances are designated by the superscripts as given below:
* Statistically significant (p<0.05) change than the vehicle control group.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

AA8

Metabolic activation:

with and 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 specified

Positive controls validity:

valid

Additional information on results:

Based on the results obtained, the test item is considered as non-mutagenic at and up to the concentration of 1 μL/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.

Conclusions:

Based on the results obtained, the test item is considered as non-mutagenic at and up to the concentration of 1 μL/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.

Executive summary:

The test item was evaluated for gene mutation test in CHO AA8 cells.
The test item was found miscible in DMSO at 200 μL/mL. Precipitation test was conducted at 0.125, 0.25, 0.50, 1 and 2 μL/mL concentrations. Post 3 hours and 45 minutes of incubation, no precipitation observed at any of the test concentrations. No change in pH was observed at the tested concentrations up to 2 μL/mL.
Based on precipitation results, 2 μL/mL was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 0.125, 0.25, 0.5, 1 and 2 μL/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (3 hours and 18 minutes). Cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival in the test.
The results of the initial cytotoxicity test indicated that the Relative Survival was greater than 10 % at 1 μL/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on these results, 1 μL/mL was selected as highest concentration for gene mutation test.
The gene mutation test was conducted at the concentrations of 0.125, 0.25, 0.5 and 1 μL/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (4 hours and 15 minutes).
Benzo(a) pyrene and 4 Nitroquinoline N-oxide were used as Positive controls for the gene mutation test.
Cytotoxicity as Relative Survival was 17.05 to 18.39% in presence of metabolic activation and absence of metabolic activation at the highest tested concentration of 1 μL/mL.
There was no statistically significant increase in mutant frequencies at any of the concentrations tested when compared with the vehicle control. Moreover, treatment with the test item resulted in mutant frequencies which fell within acceptable ranges with regard to historical controls.
There was statistically significant increase in mutant frequencies for positive controls when compared with the vehicle control in both metabolic activation and absence of metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 December 2021 to 20 April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2016

Deviations:

yes

Remarks:

SPSS software version 27 was used for statistical analysis instead of version 22. This deviation does not have any impact on outcome of the study as upgraded version of software was used for analysis.

Principles of method if other than guideline:

The purpose of this study was to determine the genotoxic potential of the test item to cause structural chromosomal aberrations in cultured Human Peripheral Blood Lymphocytes.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

Justification for Selection of Test System:
The in vitro chromosomal aberration test employs primary cell cultures derived from healthy human donor. The primary cell cultures of human whole blood were selected on the basis of growth ability in culture, stability of the karyotype. This provides the opportunity to test using the same test system which the in vitro test is predictive of in vivo genotoxic events. Further as per the regulatory requirements the human peripheral blood lymphocytes is one of the recommended test system.

Source of the Test System:
Blood Procured from Sanjeevini blood bank (Vidya Nagar, Tumkur) from male healthy adult young 24 year and 22 years (male) of age with no known illness or recent exposure to genotoxic chemicals or radiation was used.

Culture Media:
RPMI Media supplemented with 10% FBS and antibiotics (1% Penicillin-Streptomycin) was used. The pH of the culture medium used was 7.34 to 7.35. The media was stored at 2 to 8ºC till use and was thawed to room temperature before use.

Metabolic activation:

with and without

Metabolic activation system:

Sodium phenobarbitone and β-Naphthoflavone induced rat liver S9 homogenate was used as the metabolic activation system. The S9 homogenate was prepared from liver of male Wistar Rats induced with intraperitoneal injection of sodium phenobarbtone and β-naphthoflavone at 16 mg/mL and 20 mg/mL respectively for 3 days prior to sacrifice. The S9 homogenate was prepared and stored in the test facility at -80±10ºC until use. Batch of S9 homogenate was assessed for sterility, protein content and for its ability to metabolize the promutagens 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 tester strain.
1 mL of S9 homogenate was thawed immediately before use and mixed with the 9 mL of co-factor solution containing 4 mM NADP, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in Phosphate Buffer Saline (PBS) of pH 7.35 for initial cytotoxicity test and 7.38 for chromosomal aberration test.

Test concentrations with justification for top dose:

Initial cytotoxicity was conducted at concentrations of 0.125, 0.25, 0.5, 1 and 2 µL/mL. The percentage reduction in Mitotic Index was in the range of 46.42 to 48.50 at 1 µL/mL. As the percentage reduction in MI was not more than 45±5% at 1 µL/mL, same was selected as the highest concentration for the chromosomal aberration test. Other concentrations tested were 0.25 and 0.5 µL/mL.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Human Peripheral Blood Lymphocytes were used.

Evaluation criteria:

The concurrent vehicle control is considered acceptable for addition to the laboratory historical negative control database.
Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
• All three experimental conditions should be tested unless one resulted in positive results.
• Adequate number of cells (at least 300 well spread metaphases per concentration) and concentrations (at least three analyzable concentrations) should be analyzed.
The criteria for the selection of concentrations for chromosomal aberration test should be fulfilled .
The test is acceptable as all the criteria mentioned above have been met.

Statistics:

Data (Percentage of cells with aberrations) was analyzed using SPSS Software version 27 for differences among solvent/vehicle control, positive control and test item groups using ANOVA following Dunnett’s test at a 95% level of confidence (p<0.05) and the statistical significance was designated by the superscripts through out the report as stated below:
* Statistically significant (p<0.05) change than the vehicle control group.

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Solubility, Precipitation and pH Test
The test item found miscible in DMSO at 200 µL/mL. Precipitation test was conducted at 0.125, 0.25, 0.5, 1 and 2 µL/mL. After 24 hours of incubation, no precipitation was observed at 0.125 and 2 µL/mL, No change in pH was observed in any of the concentrations tested upto 2 µL/mL.
Initial Cytotoxicity Test
The percentage reduction in Mitotic Index at 0.125, 0.25, 0.5, 1 and 2 µL/mL was 11.88, 19.97, 33.33, 46.42 and 72.47% in presence of metabolic activation (short term), 11.29, 19.95, 37.53, 47.24 and 75.85 in absence of metabolic activation (short term) and 10.30, 20.73, 38.07, 48.50 and 77.05 in absence of metabolic activation (long term) respectively.

Chromosomal Aberration Test
The reduction in MI observed at 1 µL/mL was 45.57% in the presence of metabolic activation and 47.55% in the absence of metabolic activation for short term treatments. Similarly, 48.27% in the absence of metabolic activation system for long term treatment.
The observed mean percent aberrated cells at 0.25, 0.5 and 1 µL/mL were 1.00, 1.00 and 1.33 in the presence of metabolic activation (short term treatment 3 to 6 hours), 1.00, 1.00 and 1.33 in the absence of metabolic activation (short term treatment 3 to 6 hours) and 1.00, 1.33 and 1.33 in the absence of metabolic activation (long term 20 to 24 hours) respectively.
There was no statistically significant increase in the number of aberrated cells when compared with vehicle control at any of the concentration levels tested.
Positive control, 10 µg/mL of Cyclophosphamide Monohydrate, in the presence of metabolic activation (3 to 6 hours), induced 10.00% of aberrated cells which was statistically significant compared to the vehicle control (1.00%). The reduction in mitotic index was 8.32% when compared with the vehicle control for short term treatment.
Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (3 to 6 hours), induced 10.34% of aberrated cells which was statistically significant to the vehicle control (1.33%). The reduction in mitotic index observed was 8.34% when compared with the vehicle control for short term treatment.
Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (20 to 24 hours), induced 10.34% of aberrated cells which was statistically significant to the vehicle control (1.33%). The reduction in mitotic index observed was 8.38% when compared with the vehicle control for long term treatment.

Remarks on result:

other: non-clastogenic

 

 SUMMARY OF PERCENTAGE MITOTIC INDEX FOR INITIAL CYTOTOXICITY TEST

           -1

Set No.		Treatment		Concentrations (µL/mL )		Average % Mitotic Index		% Reduction of Mitotic Index
Set 1 (+S9)                       (3 to 6 hours)		Vehicle Control		0		7.41		-
		test item		0.125		6.53		11.88
			0.25		5.93		19.97
			0.5		4.94		33.33
			1		3.97		46.42
			2		2.04		72.47
Set 2  (-S9)  (3 to 6 hours)		Vehicle Control		0		7.62		-
		test item		0.125		6.76		11.29
			0.25		6.10		19.95
			0.5		4.76		37.53
			1		4.02		47.24
			2		1.84		75.85
Set 3      (-S9)                      (20 to 24 hours)		Vehicle Control		0		7.67		-
		test item		0.125		6.88		10.30
			0.25		6.08		20.73
			0.5		4.75		38.07
			1		3.95		48.50
			2		1.76		77.05

    +S9: With metabolic activation, -S9: Without metabolic activation.                                   

 

 

SUMMARY OFCHROMOSOMAL ABERRATIONS AND MITOTIC INDEX

                                                                                                                                                                                   

Set No.		Treatment		Concentrations (µL/mL)		Mean % MI		Mean % Reduction in MI		Mean of Total Aberrations with Gaps		Mean of Total Aberrations without Gaps		Mean of Total Aberrant cells without Gaps		Mean of Percentage Aberrated Cells
Set 1  (+S9) (3 to 6 hours)		Vehicle Control		0		7.33		NA		1.5		1.5		1.5		1.00
		Positive Control (Cyclophosphamide monohydrate)		10 (µg/mL)		6.72		8.32		16.0		16.0		15.0		10.00*
		test item		0.25		5.97		18.55		1.5		1.5		1.5		1.00
			0.5		4.96		32.33		1.5		1.5		1.5		1.00
			1		3.99		45.57		2.0		2.0		2.0		1.33

 MI: Mitotic Index; *: Statistically significant; +S9: With metabolic activation.

 

 

 

  SUMMARY OF CHROMOSOMAL ABERRATIONS AND MITOTIC INDEX

                                                                                         x 2

Set No.	Treatment	Concentrations (µL/mL)	Mean % MI	Mean % Reduction in MI	Mean of Total Aberrations with Gaps	Mean of Total Aberrations without Gaps	Mean of Total Aberrant cells without Gaps	Mean of Percentage Aberrated Cells
Set 2 (-S9) (3 to 6 hours)	Vehicle Control	0	7.55	NA	2.0	2.0	2.0	1.33
	Positive Control (Mitomycin-C)	0.05 (µg/mL)	6.92	8.34	17.0	16.5	15.5	10.34*
	test item	0.25	6.11	19.07	1.5	1.5	1.5	1.00
		0.5	5.01	33.64	1.5	1.5	1.5	1.00
		1	3.96	47.55	2.0	2.0	2.0	1.33

 MI: Mitotic Index; *: Statistically significant; -S9: Without metabolic activation.

 

 

SUMMARY OF CHROMOSOMAL ABERRATIONS AND MITOTIC INDEX

Set No.	Treatment	Concentrations  ( µL/mL )	Mean % MI	Mean % Reduction in MI	Mean of Total Aberrations with Gaps	Mean of Total Aberrations without Gaps	Mean of Total Aberrant cells without Gaps	Mean of Percentage Aberrated Cells
Set 3  (-S9)  (20 to 24 hours)	Vehicle Control	0	7.52	NA	2.00	2.00	2.00	1.33
	Positive Control (Mitomycin-C)	0.05 (µg/mL)	6.89	8.38	16.0	15.5	15.5	10.34*
	test item	0.25	5.87	21.94	1.5	1.5	1.5	1.00
		0.5	4.67	37.90	2.0	2.0	2.0	1.33
		1	3.89	48.27	2.0	2.0	2.0	1.33

 

    x 2

MI: Mitotic Index; *: Statistically significant; -S9: Without metabolic activation.

 

Conclusions:

Based on the results obtained, the test item is considered as non-clastogenic up to the concentration of 1 µL/mL both in the presence and absence of metabolic activation under the presented test conditions.

Executive summary:

The test item was evaluated for chromosomal aberrations in human lymphocytes.The test item was found miscible in DMSO at 200 µL/mL. Precipitation and pH test was conducted at 0.125, 0.25, 0.5, 1 and 2 µL/mL. Post 24 hours of incubation, no precipitation was observed at 0.125, 0.25, 0.5, 1 and 2 µL/mL. No change in pH was observed in any of the concentrations tested upto 2 µL/mL. Hence, 2 µL/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.125, 0.25, 0.5, and 1 µL/mL of test item.

In initial cytotoxicity test, the percentage reduction in Mitotic Index was in the range of 46.42 to 48.50% at 1µL/mL in presence of metabolic activation (short term), in absence of metabolic activation (short term), and in absence of metabolic activation (long term), respectively. The percentage reduction in mitotic index was in the range of 10.30 to 38.07 at 0.125 to 0.5 µL/mL. As the percentage reduction in mitotic index was not more than 45±5% at 1 µL/mL, same was selected as the highest concentration for the chromosomal aberration test. Other concentrations tested were 0.5 and 0.25 µL/mL.

In the chromosomal aberration test, the cells were treated with test item at the concentrations of 0.25, 0.5 and 1 µL/mL using DMSO as the vehicle. The treatment was carried out in duplicates for the short term period (3 to 6 hours) both in the presence and absence of metabolic activation and for the long term period (20 to 24 hours) in the absence of metabolic activation. Cyclophosphamide Monohydrate (+S9 for short term) at the concentration of 10 µg/mL and Mitomycin-C at the concentration of 0.05 µg/mL (-S9 both for short term and long term) were used as positive controls.

The treated cells were harvested at about 1.5 normal cell cycle length after treatment. During harvesting of cultures, thecells were treated with a metaphase-arresting substance (colchicine), stained and metaphase cells were analysed microscopically for the structural chromosomal aberrations.

There was no statistically significant increase in the number of aberrated cells when compared with vehicle control at any of the test item concentration levels tested.Positive controls induced statistically significant aberrant cells when compared to vehicle control.

The concurrent vehicle control values were within the 95% control limits of the distribution of the laboratory’s historical vehicle control database.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June 2022

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:

adopted on 26th June 2020

Deviations:

no

Principles of method if other than guideline:

The objective of Bacterial Reverse Mutation Test was to evaluate the test item for its ability to induce reverse mutations either in the presence or absence of a metabolic activation at the histidine locus of Salmonella typhimurium and at the tryptophan locus of Escherichia coli tester strains.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine locus of Salmonella typhimurium and at the tryptophan locus of Escherichia coli tester strains

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate

Test concentrations with justification for top dose:

Based on the results of cytotoxicity 5 µL/plate was considered as the highest testing concentration with other concentrations 0.05, 0.16, 0.5, 1.6 µL/plate for mutation assay.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item found miscible in DMSO at a concentration of 50 µL/mL.

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

Remarks:

2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation and preincubation
- Cell density : 18×108 cells/mL

DURATION
- Preincubation period: 21 to 25 minutes
- Exposure duration: 64 hours and 20 minutes

NUMBER OF REPLICATIONS: 03

DETERMINATION OF CYTOTOXICITY
- Method: Lawn intensity and number of revertant colonies

Rationale for test conditions:

The Bacterial reverse mutation test was considered acceptable as it meets the following criteria:
• The positive control substances produced a significant increase in mutant colony frequencies.
• The spontaneous reversion rates in the solvent and negative control (if used) are in the range of in house historical control data and preferably within the range reported in the literature or if it’s a new batch as per certificate of analysis received by the vendor.
• The tester strains must meet all required genetic characterization.
• Tester Strains used should be in the approximate range of 1×109 to 2×109 cells/mL.
• Minimum of five analyzable test item dose concentrations should be used to evaluate the mutation assay and none of these must show any signs of contamination.
• Cytotoxicity must be defined as greater than 50% reduction in mean revertants per concentration relative to the mean solvent/vehicle control value or by a thinning of the bacterial background lawn

Evaluation criteria:

The conditions necessary for determining a positive result are, there should be a dose related increase in the mean revertants per plate of at least that one tester strain over a minimum of two increasing doses of the test item either in the presence or absence of the metabolic activation system.
The test will be judged positive if the increase in mean revertants at the limit dose tested is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and Escherichia coli WP2 uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal

Statistics:

not applicable

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Insoluble, hence the test item was dissolved in DMSO.
- Precipitation: The test item resulted in no precipitation from 0.00625 to 5 µL/plate at the tested concentrations.

RANGE-FINDING/SCREENING STUDIES: Initial cytotoxicity test

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Plate Incorporation Method:
Positive Control Mean 384.5 400.7 144.3 125.4 395.3 370.1 391.8 135.4 117.9 388.3
±SD 9.6 9.8 7.8 8.8 11.1 11.1 9.3 9.4 7.7 11.1
Min 360 374 125 106 359 340 358 108 91 354
Max 412 423 159 156 416 405 412 153 148 412
Preincubation Method:
Positive Control Mean 384.5 401.0 143.5 125.1 394.2 369.6 392.3 133.7 117.1 387.1
±SD 9.6 9.3 8.5 9.0 10.7 10.9 9.2 10.5 8.2 10.8
Min 358 372 111 97 364 340 368 106 95 352
Max 411 424 159 150 414 407 416 156 139 412

- Solvent historical control data:
Plate Incorporation Method:
Vehicle Control (DMSO) Mean 29.9 107.4 19.6 9.8 75.5 26.9 102.4 17.8 8.6 71.4
±SD 3.1 4.3 2.5 1.8 4.3 2.9 4.4 2.1 1.5 4.0
Min 22 95 14 6 63 17 90 13 6 59
Max 42 123 27 14 85 38 114 24 12 82
Preincubation Method:
Vehicle Control (DMSO) Mean 30.0 107.8 19.4 9.8 76.0 27.0 102.2 18.1 8.8 71.6
±SD 2.7 4.9 2.5 1.9 4.2 2.8 4.8 2.2 1.5 3.6
Min 23 91 13 6 62 20 90 13 6 61
Max 39 125 28 14 86 35 118 24 13 81


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Lawn intensity, number of revertant colonies

 

TABLE 1.  SUMMARY OF INITIAL CYTOTOXICITY TEST-SALMONELLA TYPHIMURIUMTA100TESTER STRAIN

Test Item Concentration (µL/plate)	No. of Revertants/plate
	With S9						Without S9
	R1	R2	R3	Average	±SD	Bacterial Lawn Intensity	R1	R2	R3	Average	±SD	Bacterial Lawn Intensity
Vehicle Control	111	103	105	106	4.2	*4+,4+,4+	102	100	97	100	2.5	*4+,4+,4+
0.00625	110	104	101	105	4.6	*4+,4+,4+	93	98	101	97	4.0	*4+,4+,4+
0.0125	106	112	105	108	3.8	*4+,4+,4+	107	102	106	105	2.6	*4+,4+,4+
0.025	114	103	102	106	6.7	*4+,4+,4+	101	97	93	97	4.0	*4+,4+,4+
0.05	103	108	112	108	4.5	*4+,4+,4+	103	101	95	100	4.2	*4+,4+,4+
0.1	99	107	110	105	5.7	*4+,4+,4+	106	97	91	98	7.5	*4+,4+,4+
0.2	98	105	113	105	7.5	*4+,4+,4+	102	93	99	98	4.6	*4+,4+,4+
0.4	107	114	110	110	3.5	*4+,4+,4+	100	105	97	101	4.0	*4+,4+,4+
0.8	102	108	111	107	4.6	*4+,4+,4+	106	99	91	99	7.5	*4+,4+,4+
1.6	113	104	109	109	4.5	*4+,4+,4+	95	93	90	93	2.5	*4+,4+,4+
3.2	110	103	102	105	4.4	*4+,4+,4+	91	99	100	97	4.9	*4+,4+,4+
5	105	114	103	107	5.9	*4+,4+,4+	90	97	104	97	7.0	*4+,4+,4+

     Values of Revertants are in Mean±SD

     Lawn intensity: 4+= Thick lawns: Distinguished by a healthy (Normal) background lawn compared tovehiclecontrol

plates.

     *Lawn intensity of replicates 1, 2, 3 respectively.

 

 

TABLE 1.  SUMMARY OF COLONY COUNTS OF REVERTANTS - TRIAL-I

          Plate Incorporation Method

                                                                                                                                                         Refer: Appendix - 1

Treatment	Test Concentration (µL/plate)		No. of Revertants (Mean of 3 Plates)
			With S9						Without S9
			Salmonella typhimurium				E. coliWP2 uvrA (pKM101)		Salmonella typhimurium				E. coliWP2 uvrA (pKM101)
			TA            98	TA100	TA 1535	TA 1537			TA             98	TA100	TA 1535	TA 1537
VehicleControl	100 µL of DMSO	Mean	30.0	105.3	17.7	9.3	75.0		21.7	98.0	18.3	10.7	72.0
		±SD	3.6	2.1	1.2	1.5	3.6		2.5	4.0	3.2	1.5	2.6
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,  4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+
test item	0.05	Mean	30.0	101.0	18.0	9.7	73.7		22.7	100.0	17.7	9.7	71.7
		±SD	4.0	4.0	2.0	2.5	2.5		1.5	4.6	2.1	1.2	3.1
	Fold Increase		1.0	1.0	1.0	1.0	1.0		1.0	1.0	1.0	0.9	1.0
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+
	0.16	Mean	31.0	99.0	17.3	8.3	74.7		24.3	99.0	16.3	9.7	69.7
		±SD	2.0	4.6	1.5	2.3	2.9		4.0	2.0	0.6	0.6	2.5
	Fold Increase		1.0	0.9	1.0	0.9	1.0		1.1	1.0	0.9	0.9	1.0
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+
	0.5	Mean	31.7	101.0	17.7	11.0	73.7		24.0	99.0	17.3	10.3	71.0
		±SD	3.2	4.0	1.5	1.7	3.2		1.7	2.6	2.3	1.5	2.6
	Fold Increase		1.1	1.0	1.0	1.2	1.0		1.1	1.0	0.9	1.0	1.0
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+
	1.6	Mean	29.3	102.3	17.7	10.0	72.3		25.3	99.7	17.0	9.7	72.7
		±SD	2.1	3.5	2.1	1.7	3.1		3.1	2.1	2.0	1.5	3.2
	Fold Increase		1.0	1.0	1.0	1.1	1.0		1.2	1.0	0.9	0.9	1.0
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+
	5	Mean	31.0	101.0	18.3	9.7	71.3		24.0	97.3	18.7	10.0	70.3
		±SD	3.6	2.0	0.6	1.5	3.5		2.6	2.5	3.2	1.0	2.5
	Fold Increase		1.0	1.0	1.0	1.0	1.0		1.1	1.0	1.0	0.9	1.0
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+
PositiveControl	100 µL of respective Positive Control	Mean	383.0	395.3	145.7	126.7	384.7		368.7	401.0	136.0	110.7	389.0
		±SD	8.5	6.1	8.6	8.6	12.3		6.5	9.0	6.0	11.0	10.8
	Fold Increase		12.8	3.8	8.2	13.6	5.1		17.0	4.1	7.4	10.4	5.4
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+

Lawn intensity: 4+= Thick lawn: Distinguished by a healthy (Normal) background lawn compared tovehiclecontrol plates.

Values of Revertants are in Mean±SD

Positive controls:

With S9:

ForSalmonella typhimuriumTA98, TA100, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene

ForE.coliuvrA pKM 101 =30 µg/plate of 2-Aminoanthracene

Without S9:

For TA98: 2 µg/plate of 2-Nitrofluorene

For TA100 and TA1535: 1 µg/plate of Sodium azide

For TA1537: 50 µg/plate of 9-Aminoacridine

For E.coliuvrA pKM 101:5µg/plate of 4 Nitroquinoline N-oxide

 

 

TABLE 1.  SUMMARY OF COLONY COUNTS OF REVERTANTS - TRIAL-II

Preincubation Method

                                                                                                                                             Refer: Appendix - 2

Treatment	Test Concentration (µL/plate)		No. of Revertants (Mean of 3 Plates)
			With S9						Without S9
			Salmonella typhimurium				E. coliWP2 uvrA (pKM101)		Salmonella typhimurium				E. coliWP2 uvrA (pKM101)
			TA            98	TA100	TA 1535	TA 1537			TA             98	TA100	TA 1535	TA 1537
VehicleControl	100 µL of Dimethyl Sulphoxide	Mean	28.3	102.7	22.3	9.0	77.3		27.0	101.0	21.0	8.3	72.3
		±SD	2.5	3.1	2.1	2.0	3.1		2.0	2.6	2.6	1.5	3.5
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,  4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+
test item	0.05	Mean	29.0	104.0	22.0	8.7	76.7		28.3	98.7	18.7	8.7	71.3
		±SD	2.6	3.6	2.0	2.1	3.5		2.1	3.5	2.5	1.5	3.2
	Fold Increase		1.0	1.0	1.0	1.0	1.0		1.0	1.0	0.9	1.0	1.0
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+
	0.16	Mean	28.0	102.3	21.0	8.0	75.3		27.7	94.3	20.0	7.7	71.7
		±SD	2.6	5.7	2.0	1.0	3.5		3.5	5.1	2.0	0.6	3.2
	Fold Increase		1.0	1.0	0.9	0.9	1.0		1.0	0.9	1.0	0.9	1.0
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+
	0.5	Mean	29.3	101.3	20.7	9.0	74.7		27.3	100.0	19.7	8.3	71.0
		±SD	1.5	3.5	1.5	1.0	2.5		2.5	3.6	2.5	2.3	3.6
	Fold Increase		1.0	1.0	0.9	1.0	1.0		1.0	1.0	0.9	1.0	1.0
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+
	1.6	Mean	27.3	99.0	18.3	7.3	74.3		25.0	93.7	17.7	7.0	72.3
		±SD	2.5	4.6	1.5	0.6	4.2		1.0	3.1	1.5	1.0	3.1
	Fold Increase		1.0	1.0	0.8	0.8	1.0		0.9	0.9	0.8	0.8	1.0
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+
	5	Mean	21.0	71.3	13.7	3.7	55.0		18.0	63.3	13.3	3.3	50.3
		±SD	3.0	3.5	2.1	0.6	3.0		1.0	1.5	2.1	0.6	2.5
	Fold Increase		0.7	0.7	0.6	0.4	0.7		0.7	0.6	0.6	0.4	0.7
	Lawn Intensity		3+, 3+,3+	3+, 3+,3+	3+, 3+,3+	3+, 3+,3+	3+, 3+,3+		3+, 3+,3+	3+, 3+,3+	3+, 3+,3+	3+, 3+,3+	3+, 3+,3+
PositiveControl	100 µL of respective Positive Control	Mean	382.3	400.7	143.0	132.3	392.0		364.0	388.3	140.0	119.0	386.3
		±SD	11.2	10.7	5.6	7.5	4.0		9.5	7.8	7.5	9.0	4.0
	Fold Increase		13.5	3.9	6.4	14.7	5.1		13.5	3.8	6.7	14.3	5.3
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+

Lawn intensity: 4+= Thick lawn: Distinguished by a healthy (Normal) background lawn compared tovehiclecontrol plates.

3+=Slightly thin lawn: Distinguished by a noticeable thinning of the background lawn compared tovehiclecontrol plates.

Values of Revertants are in Mean±SD

Positive controls:

With S9:

ForSalmonella typhimuriumTA98, TA100, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene

ForE.coliuvrA pKM 101 = 30 µg/plate of 2-Aminoanthracene

Without S9:

For TA98: 2 µg/plate of 2-Nitrofluorene

For TA100 and TA1535: 1 µg/plate of Sodium azide

For TA1537: 50 µg/plate of 9-Aminoacridine

ForE.coliuvrA pKM 101: 5 µg/plate of 4 Nitroquinoline N-oxide

 

 

APPENDIX 1.INDIVIDUAL VIABLE COUNT OF TESTER STRAINS

Name of the Strain		Required CFU/mL		No. of colonies of 10^-7dilutions in triplicates				Average		ObtainedCFU/mL(1 to 2×10^9cells/mL)
			R1		R2		R3
SalmonellatyphimuriumTA98		1-2×10^9		174		170		168		171		1.71×10^9
SalmonellatyphimuriumTA100		1-2×10^9		167		172		175		171		1.71×10^9
SalmonellatyphimuriumTA1535		1-2×10^9		162		169		173		168		1.68×10^9
SalmonellatyphimuriumTA1537		1-2×10^9		171		165		167		168		1.68×10^9
E.coliWp2 uvrA pKM 101		1-2×10^9		175		166		168		170		1.70×10^9

 R: Replicate

Conclusions:

Based on the results of the study, it is concluded that the test item is non-mutagenic in the Bacterial Reverse Mutation Test up to the highest tested concentration of 5 µL/plate under the test conditions, when tested on Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA (pKM101) tester strains.

Executive summary:

 

The test item was evaluated for mutagenicity in Bacterial Reverse Mutation Test.

The test item foundmisciblein DMSO at a concentration of 50 µL/mL and resulted in no precipitation from 0.00625 to 5 µL/plate tested concentrations.

On the basis of precipitation test, 5µL/plate was selected as the highest test concentration for initial cytotoxicity test.

Salmonella typhimuriumTA100 was exposed to vehicle control0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 and 5 µL/plate of test item concentrations. The test item resulted no cytotoxicity from0.00625 to5µL/platein the tester strain Salmonella typhimuriumTA100 with lawn intensity 4+ (Thick lawn) in the presence and absence of metabolic activation system when compared to vehicle control 4+ (Thick lawn).

Based on the results of initial cytotoxicity test, concentrations of0.05, 0.16, 0.5,            1.6  and 5µL/plate were selected for testing in the mutation test.

The test item was assessed for its mutagenic effects usingSalmonella typhimuriumtester strains: TA98, TA100, TA1535, TA1537 and  EscherichiacoliWP2 uvrA (pKM101). The test item was tested for plate incorporation method (Trial I) and for preincubation method (Trial II) in presence and absence of metabolic activation system using DMSOasthe vehicle and appropriate positive controls (2-nitrofluorene, sodium azide, 9-Aminoacridine and 4-nitroquinoline N-oxide for trials “without metabolic activation” and 2-Aminoanthracene for trials “with metabolic activation”) were tested simultaneously. Two trials were carried out for this study in triplicate.

Based on the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in trial I in the presence and absence of metabolic activation. Whereas in trial II the mean numbers of revertant colonies at the tested concentrations (except 5µL/plate) were comparable to those of the vehicle control in the presence and absence of metabolic activation. There was no appreciable increase in number of revertant colonies at any of the tested concentrations in both the trials. The number of revertant colonies in the positive controls resulted in 3.8 to 17.0 fold increase under identical conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data from three independent mutagenicity assays on the submission substance, a respective mutagenic potential of the submission substance can most probably be excluded. Thus, the submission substance is considered to be devoid of any mutagenic potential and does not have to be classified for mutagenicity in accordance with the criteria laid down in the EU Classification, Labellling and Packaging Regulation (EC) 1272/2008 (CLP).