2,4-di-tert-butylphenyl 3,5-di-tert-butyl-4-hydroxybenzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was not mutagenic in two Ames tests and in an HPRT test with and without microsomal activation.

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:

1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

only four strains tested

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of liver from rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

5 µg/0.1 mL, 15 µg/0.1 mL, 45 µg/0.1 mL, 135 µg/0.1 mL, 405 µg/0.1 mL

Vehicle / solvent:

- Vehicle/solvent used: Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Remarks:

for details see table below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar

DURATION
- Exposure duration: The plates were incubated for about 48 hours at 37 °C in darkness.

NUMBER OF REPLICATIONS: Three Petri dishes were prepared per strain and per group.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth, number of revertants

POSITIVE CONTROLS
WITHOUT S9
TA 1535: N-methyl N'-nitro-N-nitrosoguanidine; 3 and 5 µg/0.1 mL phosphate buffer
TA 1537: 9(5)aminoacridine hydrochloride monohydrate; 25, 50 and 100 µg/0.1 mL DMSO
TA 98: daunoblastin; 2,5, 5 and 10 µg/0.1 mL phosphate buffer
TA 100: 4-nitroquinoline-N-oxide; 0.0625, 0.125 and 0.25 µg/0,1 mL phosphate buffer
WITH S9
TA 1535: cyclophosphamide; 100 and 250 µg/0.1 mL phosphate buffer

Evaluation criteria:

The test substance was considered to be non-mutagenic if the colony count in relation to the negative control was not doubled at any concentration.

Statistics:

When the colonies had been counted, the arithmetic mean was calculated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the highest concentration the substance precipitated in soft agar.

Conclusions:

No evidence of the induction of point mutations by the test article or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 November 2014 - 28 November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Principles of method if other than guideline:

only E. coli WP2 uvr A tested

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-experimet/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II without S9 mix: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II with S9 mix: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: 2-aminoanthracene, 10 µg/plate (+S9)

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours at 37 °C in the dark.

NUMBER OF REPLICATIONS: three plates/concentration

DETERMINATION OF CYTOTOXICITY
- Method: reduction of revertants, clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar in the test tubes at 5000 µg/plate in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 µg/plate in experiment I without S9 mix and from 333 to 5000 µg/plate with S9 mix and in experiment II from 2500 to 5000 µg/plate with and without S9 mix.

RANGE-FINDING/SCREENING STUDIES:
To evaluate the toxicity of the test item a pre-experiment was performed with WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. Since no toxic effects were observed 5000 µg/plate was chosen as maximal concentration. The pre-experiment is reported as main experiment I, since the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in both experiments with and without S9 mix.

Summary of Experiment I - Plate Incorporation

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				WP2 uvrA
Without Activation	Acetone			37 ± 3
	Untreated			41 ± 3
	Tinuvin 120	3 µg		44 ± 6
		10 µg		42 ± 9
		33 µg		37 ± 2
		100 µg		43 ± 6
		333 µg		45 ± 2
		1000 µg		38 ± 4
		2500 µg		39 ± 5
		5000 µg		32 ± 2^P M
	MMS	2.0 µL		892 ± 16
With Activation	Acetone			51 ± 5
	Untreated			44 ± 3
	Tinuvin 120	3 µg		46 ± 8
		10 µg		52 ± 2
		33 µg		51 ± 4
		100 µg		52 ± 7
		333 µg		57 ± 1^P
		1000 µg		50 ± 5^P M
		2500 µg		39 ± 2^P M
		5000 µg		43 ± 3^P M
	2-AA	10.0 µg		225 ± 119

Summary of Experiment II - Pre-Incubation

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				WP2 uvrA
Without Activation	Acetone			46 ± 6
	Untreated			43 ± 1
	Tinuvin 120	33 µg		49 ± 4
		100 µg		52 ± 6
		333 µg		48 ± 16
		1000 µg		45 ± 4
		2500 µg		48 ± 11^P
		5000 µg		45 ± 2^M P
	MMS	2.0 µL		870 ± 114
With Activation	Acetone			59 ± 6
	Untreated			61 ± 3
	Tinuvin 120	3 µg		56 ± 11
		10 µg		62 ± 2
		33 µg		62 ± 6
		100 µg		60 ± 7
		333 µg		67 ± 6
		1000 µg		61 ± 8
		2500 µg		64 ± 4^P
		5000 µg		65 ± 7^M P
	2-AA	10.0 µg		357 ± 46

Key to Positive Controls: MMS methyl methane sulfonate; 2-AA 2-aminoanthracene

Key to Plate Postfix Codes: P Precipitate; M Manual count

Conclusions:

The test item did not induce gene mutations by base pair changes in the genome of the strain used and is therefore considered to be non-mutagenic in this Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test article suspended in Acetone to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the the Escherichia coli strain WP2 uvrA. The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-experimet/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II without S9 mix: 33; 100; 333; 1000; 2500; and 5000 µg/plate; with S9 mix 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes at 5000 µg/plate in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 µg/plate in experiment I without S9 mix and from 333 to 5000 µg/plate with S9 mix and in experiment II from 2500 to 5000 µg/plate with and without S9 mix. The undissolved particles had no influence on the data recording. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in both experiments with and without S9 mix. No substantial increase in revertant colony numbers was observed following treatment with the test article in strain WP2uvrA at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Appropriate reference mutagens were used as positive controls and showed a distinct in­crease of induced revertant colonies. In conclusion, it can be stated that during the described mutage­nicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes in the genome of the strain used. Therefore, the test article is considered to be non-mutagenic in this Escherichia coli reverse mutation assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 November 2014 - 08 January 2015

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)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Target gene:

hprt (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1%).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for spontaneus mutant frequency: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I, with and without S9: (0.37); 1.1; 3.3; 10; 30; 90 µg/mL
Experiment II, with and without S9: (1.1); 3.3; 10; 30; 90, 270 µg/mL
Numbers in parantheses: these cultures were discontinued.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

with S9: DMBA, 2.2 µg/mL; without S9: EMS, 150 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without S9)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15-16 days

SELECTION AGENT (mutation assays): 11 μg/mL 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: two independent cultures were used

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, cell density

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation fre¬quency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance was considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item was observed at 90.0 µg/mL and above with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
No relevant toxic effect occurred up to the maximum concentration tested with and without metabolic activation following 4 hours of treatment. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) prior to removal to the test item. Precipitation occurred at 31.3 µg/mL and above after 4 hours treatment with and without metabolic activation. There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. The dose range of the first experiment was set according precipitation observed in the pre-experiment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effect indicated by a relative cloning efficiency I and cell density below 50% in both parallel cultures was observed up to the highest concentration of both experiments with and without metabolic activation.

Summary of Results

	conc. (µg/ml)	P	S9 Mix	relative cloning efficiency I (%)	relative cell density (%)	relative cloning efficiency II (%)	mutant colonies / 10^6cells	induction factor	relative cloning efficiency I (%)	relative cell density (%)	relative cloning efficiency II (%)	mutant colonies / 10^6cells	induction factor
Experiment I / 4h treatment				culture I					culture II
solvent control (DMSO)			-	100	100	100	7.1	1	100	100	100	14.9	1
positive control (EMS)	150		-	95.1	122.3	81.4	236.1	33.2	106.4	73.4	103.4	149.5	10
test item	0.37		-	103	culture was not continued#				104.9	culture was not continued#
test item	1.1		-	98.5	90.3	86.5	48.2	6.8	104.7	71.1	109.1	21.9	1.5
test item	3.3		-	94.2	79.4	78.5	19.4	2.7	109	116.9	105.2	17.4	1.2
test item	10		-	95.6	68.8	145	9.1	1.3	106.7	70.5	111.9	17.1	1.1
test item	30		-	95.6	42.9	121.3	11.8	1.7	109.2	28.6	109.1	10.5	0.7
test item	90	P	-	87.5	39	132.3	3.1	0.4	106.7	80.8	116.2	4.8	0.3
solvent control (DMSO)			+	100	100	100	9.6	1	100	100	100	11.1	1
positive control (DMBA)	2.2		+	79.4	77.3	104.5	294.9	30.8	86.9	89.8	102.6	416.1	37.6
test item	0.37		+	90.9	culture was not continued#				98.9	culture was not continued#
test item	1.1		+	94.9	58.9	101.4	11.4	1.2	87.1	133.3	99.7	13	1.2
test item	3.3		+	91.2	55.5	101.9	12.7	1.3	94.3	118.1	98.1	16.4	1.5
test item	10		+	90.6	89.4	102.6	17.2	1.8	112.6	114.3	100.7	19.2	1.7
test item	30		+	93.2	76	102.1	12.5	1.3	85.6	118	100.1	13.7	1.2
test item	90	P	+	89	89.4	102.1	20.3	2.1	85.1	97.9	99.8	21.8	2
Experiment II / 4h treatment				culture I					culture II
solvent control (DMSO)			-	100	100	100	7.3	1	100	100	100	6.6	1
positive control (EMS)	150		-	101.5	108.3	101.3	130.4	17.9	100.7	119.6	91.8	90	13.6
test item	1.1		-	101.4	culture was not continued#				98	culture was not continued#
test item	3.3		-	98.8	98.6	103.7	5.1	0.7	97.6	125.9	89.8	16	2.4
test item	10		-	101.5	120.5	103.7	15.9	2.2	94.6	110.4	84.7	3.3	0.5
test item	30		-	101.1	85.5	100.6	17.4	2.4	98.9	134.3	78.4	11.1	1.7
test item	90	P	-	99.2	109.2	101.3	22.1	3	97.8	107.1	76.8	21.6	3.3
test item	270	P	-	100.4	111.5	101.7	19	2.6	99.9	115	79.9	13.6	2
solvent control (DMSO)			+	100	100	100	28.5	1	100	100	100	5.8	1
positive control (DMBA)	2.2		+	97.6	97.9	101.2	277.4	9.7	99.2	94.3	39.5	381.4	65.5
test item	1.1		+	100.3	culture was not continued#				95.6	culture was not continued#
test item	3.3		+	99.3	109	99.5	19.3	0.7	97.6	105.7	86	7.1	1.2
test item	10		+	95.9	97.9	93.3	29.6	1	98.3	107.5	83.8	8.4	1.4
test item	30		+	95	100.5	111.6	37.6	1.3	98.3	105.6	90.2	9.5	1.6
test item	90	P	+	98.3	87.1	103.4	22.8	0.8	100.1	97.3	110.2	11	1.9
test item	270	P	+	95.6	95.8	101.6	19.5	0.7	96.3	92.4	78.7	16.7	2.9

P = Precipitation visible at the end of treatment

# Culture was not continued since a minimum of only four concentrations is required by the guidelines

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.

Executive summary:

In a GLP-compliant genotoxicity study according to OECD guideline 476 the test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The main experiments were performed with and without liver microsomal activation and a treatment period of 4 hours. The highest concentration of 4000 µg/mL in the pre-experiment was limited by the solubility of the test item in organic solvents. The concentration range of the main experiments was limited by the precipitation of the test item. The test item was dissolved in DMSO. The tested concentrations in the main experiments ranged from 0.37 to 270 µg/ml. Precipitation of the test item was observed at 90.0 µg/mL and above with and without metabolic activation. No relevant cytotoxic effect indicated by a relative cloning efficiency I and cell density below 50% in both parallel cultures was observed up to the highest concentration of both experiments with and without metabolic activation. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In an in vivo Micronucleus Test in somatic interphase nuclei the test item showed no evidence of mutagenic effects in Chinese hamsters. In an in vivo Dominant Lethal Study the test substance showed no dominant lethal effect in the progeny of male mice.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

no

Type of assay:

other: micronucleus assay

Species:

hamster, Chinese

Strain:

other: Cricetulus griseus

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Housing: Kept in an air-conditioned room
- Diet (e.g. ad libitum): NAFAG No. 196
- Water: Tap water ad libitum.
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature: 23 °C +/- 1 °C
- Humidity: 55 % +/- 5 %
- Photoperiod: The room was illuminated for 12 hours

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The preparation was administered orally to groups of 6 female and 6 male animals each.
- Test substance: 500, 1000 and 2000 mg/kg in 20 mL/kg sesame oil.
- Cyclophosphamide (ENDOXAN ): 128 mg/kg in 20 mL/kg sesame oil (positive control).
- 20 mL sesame oil/kg (negative control).

DIET PREPARATION
- Rate of preparation of diet (frequency): daily one application on 2 consecutive days

Duration of treatment / exposure:

48 hours

Frequency of treatment:

daily one application on 2 consecutive days

Post exposure period:

none

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

3

Control animals:

yes

Positive control(s):

- Cyclophosphomamide
- Route of administration: orally
- Doses / concentrations: 128 mg/kg

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES:
Treatment consisted of daily one application on 2 consecutive days. 24 h after the second application the animals were sacrificed.

DETAILS OF SLIDE PREPARATION:
Bone marrow was harvested from the shafts of both femurs. In a siliconized pipette filled with 0.5 yl rat serum the bone marrow was drawn up. In order to receive a homogeneous suspension the content of pipette was aspirated gently about three times. Small drops of the mixture were transferred on the end of a slide, spread out by pulling it behind a polished cover glass and the preparations were air-dried. At the next day the slides were stained in undiluted May-Grunwald solution for 2 min then in May-Griinwald solution/ water 1/1 for 2 min and then in Giemsa's, 40% for 20 min. After being rinsed in methanol 55% for 5-8 sec. and washed off twice in water, they are left immersed in water for approx. 2 min. After rinsing with distilled water and airdrying the slides were cleared in Xylol and mounted in Eukitt.

METHOD OF ANALYSIS:
The slides of three female and three male animals per group were examined. 1000 bone marrow cells each were scored per animal and the following anomalies were registered: Single Jolly bodies, fragments of nuclei in erythrocytes, micronuclei in erythroblasts, micronuclei in leucopoietic cells, polyploid cells.

Statistics:

The significance of difference was assessed by X^2 test.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control. By contrast, the positive control (cyclophosphamide, 128 mg/kg) yielded a marked increase of -the percentage of cells with anomalies. Here the mean percentage of anomalies was 5.17, whereas the negative control yielded a percentage of 0.083. The difference is highly significant (p<0.05).

	Number of animals	Sex of animals	Single Jolly bodies	Fragments of nuclei in erythrocytes	Micronuclei in erythroblasts	Micronuclei in leucopoietic cells	Polyploid cells	Total
Control	1	f						0.0
(Sesame oil)	2	f	0.2					0.2
	3	f						0.0
	4	m	0.1					0.1
	5	m					0.1	0.1
	6	m					0.1	0.1
Cyclophosphamide	1	f	4.8	1.1	0.1	0.1	0.3	6.4
(128 mg/kg)	2	f	3.7	0.8	0.2		0.3	5.0
	3	f	2.9	0.4	0.3	0.1	0.2	3.9
	4	m	3.3	0.6	0.2	0.1	0.4	4.6
	5	m	1.8	0.1	0.1		1.7	3.7
	6	m	4.3	2.2	0.3	0.2	0.4	7.4
Test item	1	f	0.2					0.2
(500 mg/kg)	2	f					0.1	0.1
	3	f					0.1	0.1
	4	m						0.0
	5	m	0.1					0.1
Test item	6	m						0.0
(1000 mg/kg)	1	f					0.2	0.2
	2	f				0.1	0.1	0.2
	3	f	0.3				0.1	0.4
	4	m					0.1	0.1
	5	m						0.0
	6	m						0.0
Test item	1	f	0.3					0.3
(2000 mg/kg)	2	f	0.1					0.1
	3	f	0.1				0.1	0.2
	4	m					0.2	0.2
	5	m	0.1					0.1
	6	m						0.0

Conclusions:

Under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with preparations of the test article.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro - Ames Test

The test substance was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The study satisfies the OECD Guideline 471 with one deviation: 4 strains had been tested instead of 5. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 5, 15, 45, 135, and 405 µg/0.1 ml. These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the numbers of bacteria in the treated and control cultures that have undergone back-mutation to histidine-protototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors) In the experiments performed with and without microsomal activation, no mutagenic effects were observed.

To investigate the mutagenic potential of the test substance in an E. coli strain WP2 uvrA, a GLP-compliant study was performed with the test article suspended in Acetone. The plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested up to 5000 µg/plate. The test item precipitated in the overlay agar in the test tubes at 5000 µg/plate in both experiments and in the overlay agar on the incubated agar plates from 333 µg/plate. No toxic effects, evident as a reduction in the number of revertants occurred in both experiments with and without S9 mix. No substantial increase in revertant colony numbers was observed in strain WP2uvrA at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Appropriate reference mutagens were used as positive controls and showed a distinct in­crease of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes in the genome of the strain used.

In vitro - HPRT Test

A GLP-compliant study following OECD guideline 476 was performed to assess the test item's potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The study was performed in two independent experiments, using identical experimental procedures. In both experiments the treatment period was 4 hours with and without metabolic activation. The main experiments were evaluated at concentration range of 0.37 -270 µg/ml. Precipitation of the test item was observed at 90.0 µg/ml and above with and without metabolic activation. No relevant cytotoxic effect indicated by a relative cloning efficiency I and cell density below 50% in both parallel cultures was observed up to the highest concentration of both experiments with and without metabolic activation. No relevant and reproducible increase in mutant colony numbers/10⁶ cells was observed in the main experiments up to the maximum concentration. The induction factor exceeded the threshold of three times the corresponding solvent control in both cultures of the second experiment at 90.0 µg/mL without metabolic activation. This effect however, was based upon a rather low solvent control of 7.3 and 6.6 mutant colonies/10⁶ cells respectively, and thus, biologically irrelevant. Another increase exceeding the threshold of three times the corresponding solvent control and the range of the historical solvent control data occurred in culture I of the first experiment at 1.1 µg/mL without metabolic activation. However, this increase was neither reproduced at any higher concentration nor in the parallel culture performed under identical experimental conditions. Therefore, it was again judged as biologically irrelevant. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the second culture of the first experiment without metabolic activation. This trend however, was inverse going down versus increasing concentrations and consequently, irrelevant. In the second experiment a significant trend was noted in the second culture with metabolic activation. Since the mutation frequency neither exceeded the historical range of solvent controls nor the threshold as indicated above, the statistical result is considered as biologically irrelevant fluctuation. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.8 up to 28.5 mutants per 10⁶ cells; the range of the groups treated with the test item was from 3.1 up to 48.2 mutants per 10⁶ cells. EMS (150 µg/mL) and DMBA (2.2 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.

In vivo

The in-vivo Micronucleus test is used for the detection of damage induced by the test substance to the chromosomes or the mitotic apparatus as sampled in bone marrow and/or peripheral blood cells of animals, usually rodents. The test item was administered by gavage. Treatment consisted of one daily application on 2 consecutive days. The animals were sacrificed 24 h after the second application. From the bone marrow smears were made. The experiment was performed to evaluate any mutagenic effect on somatic interphase cells in vivo. Mutagenic effects present themselves in interphase cells in form of nucleus anomalies of bone marrow cells. These anomalies occur in interphase cells as a consequence of damage during the mitotic process. The increase in anomalies shows a clear dose dependency, comparable to the occurrence of chromosome aberrations in metaphase preparations. The bone marrow smears from animals treated with various doses of preparation of the test item showed no significant difference from the control. The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group. By contrast, a positive control experiment with cyclophosphamide (128 mg/kg) yielded 5.17% cells with anomalies of nuclei. This is significantly different from the controls treated with the vehicle (sesame oil) alone. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with preparation of the test substance.

In an in-vivo dominant lethal study, mice were exposed to the test substance and mated to untreated virgin females. The calculation of the dominant lethal effect is based on comparison of the live implants per female in the treated group to the live implants per female in the control group. The test item was administered orally in single doses to male albino mice (NMRI-derived) which were then mated to untreated females over a period of six weeks. At the end of each week the females were replaced by new ones. Doses of 1000 and 3000 mg/kg were given. The experiment was done to evaluate any cytotoxic or mutagenic effects on the male germinal cells as expressed by the loss of pre-implantation zygotes as well as by the rate of deaths of post-implantation stages of embryonic development. The females mated to males which had been treated with the compound did not differ significantly from the females mated to controls, neither in mating ratio nor in the number of implantations and embryonic deaths (resorptions). No evidence of dominant lethal effect was observed in the progeny of male mice treated with the test item.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is considered not to be classified under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218.