Reaction products of alcohols, C11-14-iso, C13 rich and phosphorus pentoxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in-vitro genetic toxicity study:

- Ames: Negative

- Chromosomal Aberration Test: Negative

- Mammalian Cell Gene Mutation (Hprt): Negative

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:

13 December 2017 to 09 February 2018

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:

In accordance with the OECD guideline for testing of chemicals No. 471 “Bacterial Reverse Mutation
Test”, adopted on 21st July 1997.

Deviations:

no

Principles of method if other than guideline:

- Principle of test:
- Short description of test conditions:
- Parameters analysed / observed:

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:Clariant Produkte (Deutschland) GmbH,
- Expiration date of the batch: 2019-02-22
- Purity : 98.1%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility a test substance in the vehicle: Dimethly sulphoxide


OTHER SPECIFICS:

Target gene:

Histidine Locus

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

1 mL of S9 homogenate was mixed with 9 mL co factor

Test concentrations with justification for top dose:

On the basis of test item solubility, precipitation and initial cytotoxicity test 2 µL/plate was considered as the highest test concentration for mutation assay and other concentrations tested are 0.02, 0.06, 0.20, 0.63 µL/plate

Vehicle / solvent:

The test item was miscible in dimethyl sulphoxide at 50 µL/mL- Vehicle(s)/solvent(s) used:Dimethyl sulphoxide
- Justification for choice of solvent/vehicle:The test item was miscible in dimethyl sulphoxide at 50 µL/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in

DURATION
- Preincubation period: 30 Minutes

NUMBER OF REPLICATIONS: Triplicates

CYTOTOXICITY :cytotoxicity by lawn evaluation & mutation assay by counting revertant colonies.

Rationale for test conditions:

Not applicable

Evaluation criteria:

The test will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and TA102 or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537.

Statistics:

Not applicable

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 applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Insoluble in water
- Precipitation: The test item resulted in mild precipitation at 5 and 4 µL/plate, minimal precipitation at 3 µL/plate and no precipitation up to 2 µL/plate


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Plate incorporation method (WITH S9)
Tester Strain TA98 TA100 TA1535 TA 537 TA102
Mean 412. 0 385.5 143.2 118.2 606.2
±SD 21.7 20.9 9.6 8.7 27.6
Min 280 306 117 100 510
Max 440 419 292 149 670

Plate incorporation method (WITHOUT S9)
Tester Strain TA98 TA100 TA1535 TA 537 TA102
Mean 399.7 378.3 145.0 120. 2 613.4
±SD 36.2 23.9 12.6 8.5 22.6
Min 310 311 112 103 589
Max 428 446 190 158 694

Pre incubation method (WITH S9)
Tester Strain TA98 TA100 TA1535 TA1537 TA102
Mean 410.1 386.0 144.2 117.1 606.5
±SD 18.2 22.0 11.3 6.5 26.7
Min 290 298 103 102 508
Max 428 425 187 128 670

Pre incubation method (WITHout S9)
Tester Strain TA98 TA100 TA1535 TA1537 TA102
Mean 404.0 379.1 146.5 118.7 616.0
±SD 29. 0 23.7 14.5 5.5 25.9
Min 314 317 110 105 590
Max 428 450 200 134 699

Conclusions:

Based on the results obtained from the study, it is concluded that the test item, is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration 2 µL/plate under the test conditions

Executive summary:

The test item was evaluated for mutagenicity in Bacterial Reverse Mutation Test as per the OECD guidelinefor testing of chemicalsNo. 471, “Bacterial Reverse Mutation Test”, adopted on21^stJuly 1997.

On the basis of test item solubility and precipitation tests, the initial cytotoxicity test was performed at 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5µL/plate. Initial cytotoxicity test was performed withSalmonella typhimuriumTA100 both in the presence and absence of metabolic activation system.

The tester strain,Salmonella typhimuriumTA100 treated with test item in the presence and absence of metabolic activation system resulted in cytotoxicity and it was observed by a thinning of the bacterial background lawn. Those concentrations are graded as 1+(extremely reduced lawn) for 4 and5µL/plate,2+(moderately reduced lawn) for3µL/plate and 3 + (Slightly reduced lawn)for 2 µL/platewhen compared to vehicle control.

On the basis of cytotoxicity results 2 µL/plate was considered as the highest test concentration for mutation assay.

The test concentrations tested in the mutation assay were selected based on the results of solubility, precipitation and initial cytotoxicity test. The two independent trials (trial 1 and 2) were conducted by plate incorporation method and pre incubation method in the presence and absence of metabolic activation system. In mutation assay the test item was tested at the concentrations of 0.02, 0.06, 0.20, 0.63 and 2µL/plate. Vehicle control (dimethyl sulphoxide) and appropriate positive controls (2-nitrofluorene, sodium azide and 9-Aminoacridine, Mitomycin C for trials “without metabolic activation” and 2-Aminoanthracene for trials “with metabolic activation”) were tested simultaneously.

The tester strains used in the mutation assay wereSalmonella typhimuriumTA98, TA100, TA102, TA1535 and TA1537.

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 both the trials, 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 2.2 to 14.7 fold increase under identical conditions.

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 2017 to 08 February 2018

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:

adopted on 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Mammalian Chromosomal Aberration Test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: DEG4410913
- Expiration date of the batch: 2019-02-22
- Purity test date: 98.1%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility of the test substance in the solvent/vehicle: dimethyl sulfoxide

Species / strain / cell type:

lymphocytes: primary culture

Details on mammalian cell type (if applicable):

CELLS USED
- Sex, age and number of blood donors if applicable: Male, 23 and 25 years of age and 2.
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Modal number of chromosomes: 46


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: 37±1ºC with 5±1% CO2
- Properly maintained: yes

Cytokinesis block (if used):

Not Applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Based on the results of solubility, precipitation and pH tests, an initial cytotoxicity test was conducted
for the selection of test concentrations for the chromosomal aberration test. The concentrations sele
cted for initial cytotoxicity test were 0.03125, 0.0625, 0.125 and 0.25 mg/mL.
Based on the results of initial cytotoxicity test, the concentrations selected for the chromosome
aberration test are .015625, 0.03125 and 0.0625 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide
- Justification for choice of solvent/vehicle: Test item was checked for solubility in DMSO and found soluble in DMSO at 200 mg/mL

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:

DURATION
- Preincubation period: 48 hours
- Exposure duration: 3-6 hours and 20-24 hours


SPINDLE INHIBITOR (cytogenetic assays):: Colchicine
STAIN (for cytogenetic assays): 5% Giemsa
NUMBER OF REPLICATIONS: Two
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: few drops of the cells
suspension was aspirated and dropped onto the chilled slides pre labeled . The slides were air dried.
Three slides were prepared for each treatment. Slides were stained using 5% Giemsa stain for 20
minutes.
NUMBER OF CELLS EVALUATED: 500 cells were scored per each replicate for determination of
mitotic index.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 metaphase

Rationale for test conditions:

The primary cell cultures of lymphocytes from the human whole blood are 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.

Evaluation criteria:

Cytotoxicity was determined by calculating percentage reduction in mitotic index (%).
The cells were evaluated for structural aberrations in 150 metaphase plates for each replicate and the
metaphases with aberrations were recorded in raw data and presented in study report. Gaps were recorded separately and reported but generally not included in the total aberration frequency a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
a) At least one of the test concentrations exhibits a statistically significant increase compared with the
concurrent negative/vehicle control.
b) The increase is dose-related when evaluated with an appropriate trend test.
When all of these criteria are met, the test chemical is then considered able to induce chromosomal
aberrations in cultured mammalian cells in this test system.
• Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
a) None of the test concentrations exhibits a statistically significant increase compared with the
concurrent negative/vehicle control
b) There is no concentration-related increase when evaluated with an appropriate trend test

Statistics:

Data (Percentage of cells with aberrations) was analyzed using SPSS Software version 22 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).

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

TABLE 1.           SUMMARY OF PERCENTAGE MITOTIC INDEX FOR INITIAL CYTOTOXICITY TEST

   Refer Appendix-1

Set No.			Treatment		Concentration (mg/mL)			Mitotic Index				Mean Mitotic Index		Mean Percentage Mitotic Index			Percentage Reduction in Mitotic Index
				R1				R2
Set 1 (+S9)                  (3-6 hours)			Vehicle control		-			0.0579		0.0534			0.0556		5.56			-
			Test item [Hostacor ITD]		0.03125			0.0457		0.0490			0.0474		4.74			14.83
				0.0625			0.0336		0.0279			0.0307		3.07			44.73
				0.125			0.0197		0.0179			0.0188		1.88			66.19
				0.25			0.0039		0.0020			0.0030		0.30			94.68
Set 2 (-S9)                     (3-6 hours)	Vehicle control			-	0.0489	0.0556	0.0522			5.22		-
	Test item [Hostacor ITD]			0.03125	0.0415	0.0397	0.0406			4.06		22.30
				0.0625	0.0274	0.0314	0.0294			2.94		43.75
				0.125	0.0159	0.0217	0.0188			1.88		64.00
				0.25	0.0059	0.0020	0.0039			0.39		92.44
Set 3 (-S9)                   (20-24 hours)		Vehicle control	-		0.0452	0.0552		0.0502	5.02		-
		Test item [Hostacor ITD]	0.03125		0.0398	0.0356		0.0377	3.77		24.98
			0.0625		0.0218	0.0313		0.0266	2.66		47.08
			0.125		0.0098	0.0196		0.0147	1.47		70.67
			0.25		0.0020	0.0000		0.0010	0.10		98.04

+S9: With metabolic activation; -S9: Without metabolic activation  

 

TABLE 2.     SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX

                                                                                              Refer Appendix2

Set No.	Treatment	Concentration (mg/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) (3-6 hours)	Vehicle control	-	5.26	-	1.00	1.00	1.00	0.67
	Positive Control (Cyclophosphamide)	10 µg/mL	4.28	18.71	21.50	20.50	18.00	12.00*
	Test item  [Hostacor ITD]	0.015625	4.91	6.74	3.00	2.50	1.50	1.00
		0.03125	4.08	22.54	2.00	1.00	1.00	0.67
		0.0625	3.27	37.94	4.50	3.50	2.50	1.67

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

 

TABLE 2 (Contd..,). SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX

                                                                                         Refer Appendix 2

Set No.	Treatment	Concentration (mg/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) (3-6 hours)	Vehicle control	-	5.23	-	2.50	1.50	1.00	1.00
	Positive Control (Mitomycin-C)	0.05 µg/mL	4.04	22.73	23.50	22.00	17.50	11.67*
	Test item  [Hostacor ITD]	0.015625	4.85	7.29	3.00	3.00	2.00	1.33
		0.03125	3.68	29.70	2.00	2.00	2.00	1.33
		0.0625	3.17	39.41	3.50	3.50	3.00	2.00

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

 

 

TABLE 2 (Contd..,). SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX

Refer Appendix 2

Set No.	Treatment	Concentration (mg/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) (20-24 hours)	Vehicle control	-	4.85	-	1.50	1.00	1.00	0.67
	Positive Control (Mitomycin-C)	0.05 µg/mL	3.67	24.30	24.50	23.50	18.50	12.33*
	Test item  [Hostacor ITD]	0.015625	4.36	10.14	2.50	2.00	2.00	1.33
		0.03125	3.66	24.50	3.00	2.00	2.00	1.34
		0.0625	2.86	41.15	3.50	2.00	2.00	1.33

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

 

Conclusions:

Based on the results obtained, the test item is considered as non-clastogenic at and up to the concentration of 0.0625 mg/mL, both in the presence and absence of metabolic activation under the test conditions.

Executive summary:

The test item was evaluated for chromosomal aberration in human lymphocytes, as per the OECD guideline for the testing of chemicals,No. 473  “In vitroMammalian Chromosomal AberrationTest” adopted on 29 July 2016.

Test item was found soluble in dimethyl sulfoxide (DMSO) at 200 mg/mL. Precipitation test was conducted at concentrations of 0.0625, 0.125, 0.25, 0.5, 1.0 and 2.0 mg/mL of test item. After 23 hours of incubation no precipitation and no significant change in pH was observed at any of the concentrations tested at and up to 0.125 mg/mL. As there was slight precipitation and no significant change in pH observed at 0.25 mg/mL, the same was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.03125, 0.0625 and 0.125 mg/mL.

In the initial cytotoxicity test, the test item resulted in not greater than 47.08% reduction of Mitotic Index (MI) up to the concentration of 0.0625 mg/mL both at short term (3 to 6 hours) and long term treatment (20 to 24 hours). Hence, 0.0625 mg/mL was selected as highest concentration. The other concentrations selected as low and mid concentration were 0.015625 and 0.03125 mg/mL respectively.

In the chromosomal aberration test, the cells were treated withthe test itemat the concentrations of0.0625,0.03125and0.015625mg/mLconcentrationsusing DMSO as vehicle. The treatment was carried out in duplicates for short term (3 to 6 hours) both in the presence and absence of metabolic activation and long term (20 to 24 hours) in the absence of metabolic activation.

Concentration of 10 µg/mL of Cyclophosphamide Monohydrate (+S9) and           0.05µg/mLof Mitomycin-C (-S9 both for short term and long term) were used as positive controls. Cells were arrested at metaphase using 0.3 µg/mL of colchicine. The cells were harvested between 2 to 3 hours of cell cycle arrest and slides were prepared and stained using 5% Giemsa stain.

The results indicatedno statistically significant increase in the number of aberrant cells when compared with vehicle control at any of the concentrations tested.The mean percentage reduction in mitotic index observed at 0.0625 mg/mL, the highest concentration tested was 37.94 in the presence of metabolic activation and 39.41 and 41.15 in the absence of metabolic activation for short and long term treatments, respectively.

The respective positive controls tested induced 11.67% to 12.33% of mean aberrant cells which was statistically significant. The mean percentage reduction in mitotic index was in the range of 18.71 to 24.30 when compared with the respective vehicle controls.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 December 2017 TO 05 May 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

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


CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29th July 2016

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:98.1%
- Expiration date of the batch:2019-02-22

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:Ambient (21 to 29°C)
- Solubility and stability of the test substance in the solvent/vehicle:Dimethly sulphoxide

Target gene:

Hprt locus of CHO AA8 cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:American Type Culture Collection (ATCC)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:MEM Alpha media and 5±1% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

1 mL of S9 homogenate was mixed with 9 mL co factor

Test concentrations with justification for top dose:

Based on the results of solubility, pH, precipitation test and initial cytotoxicity test concentrations of 0.0078, 0.156, 0.0312 and 0.0625 mg/mL using DMSO as a vehicle were tested.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:Dimethyl sulphoxide
- Justification for choice of solvent/vehicle: test item was found soluble in Dimethyl sulphoxide at 200 mg/mL.

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:

METHOD OF APPLICATION: in culture media
- Cell density at seeding (if applicable): Approximately 2×106

DURATION
- Exposure duration: 3 to 6 hours
- Expression time (cells in growth medium): 7 to 9 days

NUMBER OF REPLICATIONS:04

DETERMINATION OF CYTOTOXICITY
- Method: Relative Survival (RS)

Rationale for test conditions:

Not applicable

Evaluation criteria:

There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in mutant frequency. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results.

Statistics:

Yes

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:The pH tested was comparable to the vehicle control.
- Water solubility: No
- Precipitation:Slight precipitation was observed at the concentration of 0.50 mg/mL

RANGE-FINDING/SCREENING STUDIES: Initial cytotoxicity test : Relative Survival

The Cytotoxicity level was determined using the following formulae:

Adjusted Cloning
Efficiency (ACE) = CE × No. of cells at the end of treatment
No. of Cells at the beginning of the treatment

Relative Survival (RS) = ACE (Treated) × 100
ACE (Vehicle Control)
Cloning efficiency (CE) is the percentage of cells plated at a low density that are able to grow into a colony that can be counted

Conclusions:

Based on the results obtained, the test item is considered as non-mutagenic at and up to the concentration of 0.0625 mg/mL, both in the presence and absence of metabolic activation under the laboratory conditions tested.

Executive summary:

The test item was evaluated for gene mutation test in CHO AA8 cells, as per the OECD guideline for the testing of chemicals,No. 476 “In vitroMammalian Cell Gene Mutation Tests using theHprtandxprtgenes” adopted on 29^thJuly 2016.The test item found soluble in DMSO at 200 mg/mL. The test item precipitates at and up to 1 and 2 mg/mL in culture medium and slight precipitate at 0.50 mg/mL. The pH tested at concentrations up to 0.50 mg/mL was comparable to the vehicle control.Based on the results of solubility, pH and precipitation test, an initial cytotoxicity test was conducted at the concentrations of 0.0312, 0.0625, 0.125, 0.25and0.50mg/mLusing DMSO as a vehicle in tetra plates/group in the presence and absence of metabolic activation (3 to 6 hours).The results of the initial cytotoxicity test indicated that the test item was excessive cytotoxic to CHO AA8 cells as the Relative Survival of the treated CHO AA8 cells at the tested concentrations up to 0.0625 mg/mL was more than 20%, when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Above this level, survival rate decrease to below 5%.The gene mutation test was conducted at the concentrations of 0.0078, 0.156, 0.0312 and 0.0625 mg/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (3 to 6 hours). Positive controlsBenzo(a)pyrene and4 Nitroquinoline N-oxidewere used for the gene mutation test. Cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival in the test. There was no statistically significant increase in the number of mutant colonies at any of the concentrations tested when compared with vehicle control.

Positive controls resulted in mutant frequencies,which were statistically significant when compared with the vehicle control.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The submission substance was evaluated for mutagenicity in Bacterial Reverse Mutation Test as per the OECD guidelinefor testing of chemicals No. 471, “Bacterial Reverse Mutation Test”, adopted on21^stJuly 1997. The tester strains used in the mutation assay were Salmonella typhimuriumTA98, TA100, TA102, TA1535 and TA1537. In mutation assay the test item was tested at the concentrations of0.02, 0.06, 0.20, 0.63 and 2µL/plate.

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 both the trials, 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. Based on the results obtained from the study, it is concluded that the test item, is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration 2 µL/plate under the test conditions.

The submission substance was evaluated for chromosomal aberration in human lymphocytes, as per the OECD guideline for the testing of chemicals,No. 473  “In vitro Mammalian Chromosomal Aberration Test” adopted on 29 July 2016. In the chromosomal aberration test, the cells were treated withthe test itemat the concentrations of 0.0625,0.03125 and 0.015625 mg/mLconcentrationsusing DMSO as vehicle.

The results indicated no statistically significant increase in the number of aberrant cells when compared with vehicle control at any of the concentrations tested. The mean percentage reduction in mitotic index observed at 0.0625 mg/mL, the highest concentration tested was 37.94 in the presence of metabolic activation and 39.41 and 41.15 in the absence of metabolic activation for short and long term treatments, respectively. Based on the results obtained, the test item is considered as non-clastogenic at and up to the concentration of 0.0625 mg/mL, both in the presence and absence of metabolic activation under the test conditions.

The submission substance was evaluated for gene mutation test in CHO AA8 cells, as per the OECD guideline for the testing of chemicals,No. 476 “In vitroMammalian Cell Gene Mutation Tests using theHprtandxprtgenes” adopted on 29^thJuly 2016. The gene mutation test was conducted at the concentrations of 0.0078, 0.156, 0.0312 and 0.0625 mg/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (3 to 6 hours). There was no statistically significant increase in the number of mutant colonies at any of the concentrations tested when compared with vehicle control. Based on the results obtained, the submission substance is considered as non-mutagenic at and up to the concentration of 0.0625 mg/mL, both in the presence and absence of metabolic activation under the laboratory conditions tested.

Justification for classification or non-classification

In reliable in-vitro gene mutation studies in bacteria and mammalian cells, as well as chromosome aberration tests no mutagenic potential was found for the submission substance. Based on the available data no classification according to Regulation (EC) No. 1272/2008 and Council Directive 67/548/EEC on mutagenicity is warranted.