AERO 5100 PROMOTER

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): positive with S. typhimurium TA 100 and TA 1535 with metabolic activation

HPRT (OECD 476): negative in L5178Y cells with and without metabolic activation

Micronucleus test (OECD 474), mouse: 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:

1 - 8 June 1998

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:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA 40 CFR 799 (1997) Toxic Substances Control Act Test Guidelines - Sub-section 799.9510, TSCA bacterial reverse mutation test. Federal Register, Vol. 62, No. 158

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (for S. typhimurium strains)
trp operon (for E. coli strain)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (single i.p. injection of 500 mg/kg bw)

Test concentrations with justification for top dose:

Experiment 1: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Experiment 2: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle/solvent used: ethanol
- Justification for choice of solvent/vehicle: the test substance was soluble in the vehicle at 50 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), 9-aminoacridine (9-AA), 2-nitrofluorene (2-NF), 2-aminoanthracene (2-AA), benzo[a]pyrene (BaP)

Remarks:

+S9: 2-AA (2 µg/plate, TA1535; 10 µg/plate, WP2uvrA/pKM101); BaP (5 µg/plate, TA1537, TA98, TA100) -S9: ENNG (5 µg/plate, TA1535; 3 µg/plate, TA100; 2 µg/plate, WP2uvrA/pKM101); 9-AA (80 µg/plate, TA1537); 2-NF (1 µg/plate, TA98)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: ca. 72 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduction in revertant colony numbers or thinning of the bacterial background lawn

Evaluation criteria:

The mutagenic activity of a test substance was assessed by applying the following criteria:
1) If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
2) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls in either mutation test it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
3) If the results obtained fail to satisfy the criteria for a clear positive or negative response given paragraphs 1) and 2), additional testing may be performed in order to resolve the issue of the test substance’s mutagenic activity in this test system. Should an increase in revertant colony numbers then be observed which satisfies paragraph 1) the substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.

Statistics:

Mean values and standard deviations were calculated.

Key result

Species / strain:

S. typhimurium, other: TA1535, TA 1537, TA98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

in TA1535 and TA100 with S9 mix

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains at 5000 µg/plate with and without S9 mix

Vehicle controls validity:

valid

Untreated 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

Remarks:

at 5000 µg/plate with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Table 1. Test results of Experiment 1.

With or without S9-Mix	Test substance concentration [μg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA1535	TA100	WP2 uvrA/ pKM101	TA1537	TA98
-	0 (EtOH)	19 ± 4	98 ± 15	101 ± 7	13 ± 3	40 ± 1
-	5	15 ± 2	89 ± 5	88 ± 15	10 ± 4	36 ± 7
-	15	15 ± 2	97 ± 9	102 ± 5	12 ± 2	44 ± 9
-	50	14 ± 2	96 ± 7	121 ± 3	12 ± 2	32 ± 5
-	150	14 ± 1	89 ± 7	106 ± 11	11 ± 1	41 ± 9
-	500	16 ± 3	89 ± 6	111 ± 9	12 ± 2	39 ± 6
-	1500	13 ± 1	94 ± 15	100 ± 6	11 ± 2	32 ± 5
-	5000	8 ± 4^T	62 ± 6^T	61 ± 15^T	3 ± 0^T	20 ± 2^T
Positive controls, –S9	Name	ENNG	ENNG	ENNG	9-AA	2-NF
	Concentration [μg/plate]	5	3	2	80	1
	Mean No. of colonies/plate (average of 3 ± SD)	148± 15	437± 20	2312± 48	925± 149	273± 48
+	0 (EtOH)	16± 3	97± 4	104± 10	14± 1	38±1
+	5	16± 3	100± 10	100± 9	14± 2	43± 7
+	15	25± 2	97± 3	96± 16	11± 1	40± 1
+	50	35± 4	116 ± 2	89± 3	9± 1	37± 1
+	150	76± 5	141± 14	90± 10	10± 2	38± 6
+	500	125± 6	165± 1	95± 2	9± 1	37± 3
+	1500	211± 26	171± 3	105± 9	10± 3	34 ± 5
+	5000	25± 1^T	56± 11^T	53± 11^T	6± 1^T	20± 2^T
Positive controls, +S9	Name	2-AA	BaP	2-AA	BaP	BaP
	Concentration [μg/plate]	2	5	10	5	5
	Mean No. of colonies/plate (average of 3 ± SD)	381± 45	492± 28	613± 13	215± 30	685± 33

EtOH: ethanol

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

2-NF: 2-nitrofluorene

BaP: benzo[a]pyrene

T: thinning of bacterial background lawn

 

Table 2. Test results of Experiment 2.

With or without S9-Mix	Test substance concentration [μg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA1535	TA100	WP2 uvrA/ pKM101	TA1537	TA98
-	0 (EtOH)	17 ± 2	107 ± 10	110 ± 4	14 ± 1	38 ± 5
-	50	17 ± 3	85 ± 19	95 ± 11	14 ± 1	40 ± 5
-	150	13 ± 4	90 ± 5	100 ± 14	11 ± 2	41 ± 4
-	500	13 ± 2	83 ± 8	96 ± 8	12 ± 2	36 ± 3
-	1500	12 ± 4	66 ± 7	90 ± 10	11 ± 2	34 ± 5
-	5000	5 ± 1^T	49 ± 7^T	37 ± 7^T	6 ± 1^T	9 ± 3^T
Positive controls, –S9	Name	ENNG	ENNG	ENNG	9-AA	2-NF
	Concentration [μg/plate]	5	3	2	80	1
	Mean No. of colonies/plate (average of 3 ± SD)	219 ± 18	437 ± 37	1787 ± 63	1324 ± 202	228 ± 5
+	0 (EtOH)	18± 4	101± 8	107± 12	13± 1	38± 1
+	50	26± 3	90± 11	103± 15	7± 2	35± 7
+	150	58± 5	138± 2	110± 12	7± 1	39± 6
+	500	93± 5	151± 4	113± 15	10± 2	36± 5
+	1500	143± 10	211± 21	107± 11	10± 2	33± 4
+	5000	14± 2^T	46± 5^T	72± 10^T	6± 0^T	11± 4^T
Positive controls, +S9	Name	2-AA	BaP	2-AA	BaP	BaP
	Concentration [μg/plate]	2	5	10	5	5
	Mean No. of colonies/plate (average of 3 ± SD)	495 ± 16	650 ± 30	813 ± 90	185 ± 22	497 ± 28

EtOH: ethanol

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

2-NF: 2-nitrofluorene

BaP: benzo[a]pyrene

T: thinning of bacterial background lawn

In Experiment 1 and 2, substantial increases in revertant colony numbers over control counts were observed in TA1535 following exposure to the test substance with metabolic activation. Small increases (< 2-fold) and substantial increased in revertant colony numbers over control counts were obtained in strain TA100 with metabolic activation in Experiment 1 and 2, respectively. In both strains increases were maximal at 1500 μg/plate and a clear dose-response relationship was observed.

Appropriate positive control substances induced substantial increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S9 mix.

Conclusions:

Under the conditions of the Ames test the substance was mutagenic in strains TA 1535 and TA 100 tested with metabolic activation and non mutagenic without meatbolic activation, while in strains TA 1537, TA 98 and WP2 uvr A pKM 101 it was non mutagenic with and without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Jun - 24 Aug 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted Jul 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

thymidin kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. J. Cole, Sussex University, UK

MEDIA USED
- Type and identity of media: R0p medium: RPMI 1640 supplemented with 0.1% Pluronic F68, 0.01% sodium pyruvate, 2mM L-glutamine, 45 IU/mL penicillin, 45 µg/mL streptomycin and 2.3 µg/mL Amphorericin B
R10p medium (general cell culture): R0p supplemented with 10% HiDHS
R20p medium (cloning): R0p medium without Pluronic F68 and incresed sodium pyruvate to 0.022% and supplemented with 20% HiDHS

- Properly maintained: not specified
- Periodically checked for Mycoplasma contamination: not specified
- Periodically checked for karyotype stability: not specified
- Periodically 'cleansed' against high spontaneous background: not specified

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats treated with Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

Pre-Experiment:
3 h and 24 h treatment: 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL without metabolic activation
24 h treatment: 0.01, 0.02, 0.05, 0.1, 0.1, 0.5, 1, 2, 5 and 10 µg/mL without metabolic activation
3 h treatment: 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL with metabolic activation

Main experiments:
3 h treatment: 5, 10*, 20*, 30*, 40* and 60* µg/mL without metabolic activation (test 1)
24 h treatment: 1, 2.5*, 5*, 7.5, 10*, 20*, 40* and 60 µg/mL without metabolic activation (test 2)

3 h treatment: 5*, 10*, 20*, 30*, 40 and 50 µg/mL with metabolic activation (test 1)
3 h treatment: 2.5, 5*, 10*, 15*, 20* and 30* µg/mL with metabolic activation (test 2)

*concentrations chosen for mutation rate analysis

Vehicle / solvent:

- Solvent used: ethanol
- Justification for choice of solvent: The test substance was found to be soluble in ethanol at a concentration of 625 mg/mL. On dosing a solution of 625 mg/mL at 0.8% (v/v) into aqueous tissue culture medium, giving a final conccntration of 5000 μg/mL, no precipitate was observed.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: 20-Methylcholanthrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: Test 1: 2 x 10E06; Test 2: 3 x 10E05

DURATION
- Exposure duration: 3 h with and without metabolic activation; 24 h without metabolic activation
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 11 - 12 days

SELECTION AGENT (mutation assays): 4 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicates in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER:
EVALUATION EQUATIONS:
- Growth in suspension was calculated as follows:
(cell count 24 h post treatment / 2 x 10E05) x (cell count 48 h post treatment /2 x 10E05)
* or previous day's cell count if less than 2 x 10E05/mL
- Relative total growth (RTG) over the experimental period was estimated from the following
equation:
RTG = (suspension growth (% control) x viability in agar (% control)) / 100
- The mutant frequency per 10E06 survivors (MF) was calculated as follows:
(600 / total no. of viable colonies ) x (total no. of mutant colonies / 3)

Evaluation criteria:

The criteria for a positive response were:
- An increase in mutant frequency in treated cultures of at least 100 relative to the concurrent solvent control.
- The demonstration of a statistically significant increase in mutant frequency following treatment with the test substance.
- Evidence of a dose relationship over at least two consecutive dose levels, in any increases in mutant frequency.
- Demonstration of reproducibility in any increase in mutant frequency.
- An increase in absolute colony numbers in the treated cultures.
- The relative total growth (RTG) of cultures showing an increase in mutant frequency should not be less than 10%.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-S9: at 30 and 40 µg/mL and above after 3 and 24 h treatment, respectively; +S9: 20 - 30 µg/mL and above

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 and osmolarity: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
- Precipitation: The test medium was checked for precipitation or phase separation at the end of each treatment period (4 h) prior to removal to the test item. Phase separation occurred at 522.5 µg/mL and above after 4 and 24 h treatment with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
A pre-experiment was performed in order to determine the concentration range for the mutagenicity experiments. The pre-experiment was performed in the presence (3 h treatment) and absence (3 h and 24 h treatment) of metabolic activation. Test item concentrations ranging 9.77 - 5000 µg/mL and 0.01 - 10 µg/mL (repeat test 24 h) were used. After 3 h exposure relevant toxic effects occurred at 19.53 µg/mL and above in the absence of metabolic activation and at 9.77 µg/mL and above in the presence of metabolic activation. After 24 h exposure toxic effects occured at 9.77 µg/mL and above and above in the absence of metabolic activation after 24 h exposure.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% occurred in both cultures of Experiment 1 at the highest analyzable concentration of 260 µg/mL without metabolic activation. Due to a very steep gradient of toxicity, no relevant cytotoxicity was noted up to the maximum analyzable concentrations in all of the other experimental parts. Exceedingly severe cytotoxicity precluded analysis at the next higher concentration even though the concentrations were spaced by less than a factor of 2.0 recommended by the OECD guideline 476.

Table 1: Test 1 - 3 h exposure - Without Metabolic Activation

Concentration [µg/mL]	Viability [% control]	Relative Total Growth [% control]	Mutants per 1E06 surviving cells	Mean mutants per 1E06 surviving cells
0 (ethanol)	100	100	55	50
			45
10	69.3	55	63	61
	88.3	64	58
20	96.5	56	59	64
	119.3	55	68
30	91.9	27	74	71**
	91.1	34	67
40	90.3	18	69	75**
	84.9	23	80
60	87.1	14	86	89***
	85.7	13	92
MMS (10 µg/mL)	64.9	20	351	330***
	65.1	18	308

MMS: Methyl methane sulfonate

*Significantly different from control p<0.001

** Significantly different from control p<0.01

 

Table 2: Test 1 - 3 h exposure - With Metabolic Activation

Concentration [µg/mL]	Viability [% control]	Relative Total Growth [% control]	Mutants per 1E06 surviving cells	Mean mutants per 1E06 surviving cells
0 (ethanol)	100	100	98	98
			98
5	105.5	94	168	137
	93.8	84	106
10	130.3	66	107	117
	98.6	57	127
20	91.6	17	148	156
	77.3	15	164
30	8.1	0.3	576	483
	9.5	0.4	390
20-MC (2.5 µg/mL)	75.9	84	250	205*
	78.8	86	159

20-MC: 20-Methylcholanthrene

*Significantly different from control p<0.05

 

Table 3: Test 2 - 24 h exposure - Without Metabolic Activation

Concentration [µg/mL]	Viability [% control]	Relative Total Growth [% control]	Mutants per 1E06 surviving cells	Mean mutants per 1E06 surviving cells
0 (ethanol)	100	100	101	117
			132
2.5	72.6	90	145	136
	81.9	108	127
5	58.6	75	182	155
	83.9	113	127
10	71.5	87	186	160
	88.5	107	133
20	101.7	81	121	110
	97.9	101	98
40	108.0	21	188	187
	115.0	21	185
MMS (5 µg/mL)	73.4	82	548	526**
	65.6	83	503

MMS: Methyl methane sulfonate

***Significantly different from control p<0.001

 

 Table 4: Test 2 - 3 h exposure - With Metabolic Activation

Concentration [µg/mL]	Viability [% control]	Relative Total Growth [% control]	Mutants per 1E06 surviving cells	Mean mutants per 1E06 surviving cells
0 (ethanol)	100	100	121	55
			122
5	82.2	82.2	126	72
	85.1	85.1	146
10	94.2	94.2	167	81**
	88.4	88.4	162
15	92.4	92.4	173	80**
	113.1	113.1	193
20	102.9	102.9	180	78**
	97.1	97.1	170
30	91.8	91.8	246	114***
	84.0	84.0	203
20-MC (2.5 µg/mL)	69.7	69.7	341	204***
	76.8	76.8	326

20-MC: 20-Methylcholanthrene

*Significantly different from control p<0.001

** Significantly different from control p<0.01

 

Conclusions:

Under the experimental conditions of the gene mutation assay the test item did not induce gene mutations at the HPRT locus in V79 cells with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Micronucleus test (OECD 474), mouse: negative

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

Study period:

13 Jan - 25 Feb 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 Dec 1992

Deviations:

no

GLP compliance:

yes

Type of assay:

other: mammalian erythrocyte micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ldt, Margate, UK
- Weight at study initiation: 28 - 30 g (males), 22 - 24 g (females)
- Housing: in groups of same sex in disposable cages
- Diet: pelleted expanded rat and mouse No. 1 diet SQC grade (Special Diets Services Ltd, Witham, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle used: corn oil
- Lot/batch no.: W 9088

Duration of treatment / exposure:

not applicable

Frequency of treatment:

single treatment

Post exposure period:

24 h after treatment (all groups), 48 h after treatment (negative control and high-dose group)

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

Range-finding study: 2 males and 2 females
Main study: 10 (negative control and high-dose group), 5 (positive control and low- and mid-dose group)

Control animals:

yes, concurrent vehicle

Positive control(s):

- mitomycin C
- Route of administration: gavage
- Concentration: 0.6 mg/mL in water
- Dose: 12 mg/kg bw

Tissues and cell types examined:

Tissue: bone marrow
Cell type: bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A range-finding study performed to find the maximum tolerated dose.

TREATMENT AND SAMPLING TIMES: Sampling was performed 24 h after treatment (all groups) and 48 h after treatment (negative control and high-dose group)

DETAILS OF SLIDE PREPARATION: Slides were fixed with methanol and stained with 10% Giemsa-solution for 10 minutes.

METHOD OF ANALYSIS: The proportion of immature erythrocytes is determined for each animal by counting of at least 1000 erythrocytes. 2000 polychromatic erythrocytes per animal were scored for the incidence of micronucleated erythrocytes.

Evaluation criteria:

A positive response is normally indicated by a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent control group (p < 0.01). Individual and/or group mean values should exceed the laboratory historical control range.
A negative result is indicated where individual and group mean incidences of micronucleated immature erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent control group (p > 0.01) and where these values fall within the historical control range.
An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.

Statistics:

The results for each treatment group were compared with the results for the concurrent control group using non-parametric statistics. Unless there is a substantial difference in response between sexes (which occurs only rarely) results for the two sexes are combined to facilitate interpretation and maximize the power of statistical analysis.
For incidences of micronucleated immature erythrocytes, exact one-sided p-values are calculated by permutation (StatXact, CYTEL Software Corporation). Comparison of several dose levels is made with the concurrent control using the Linear by Linear Association test for trend in a step-down fashion if significance is detected; for individual intergroup comparisons this procedure simplifies to a straightforward permutation test. For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 100 - 750 mg/kg bw
- Clinical signs of toxicity in test animals: Deaths (1/4, 2/4, 4/4, 3/4 and 4/4, respectively) occurred in all groups dosed with test substance at concentrations of 175, 250, 500, 600 and 750 mg/kg bw, respectively, and severe clinical signs were observed in the remaining animals. At 100 mg/kg bw, all animals showed signs of hunched posture, piloerection and irregular respiration but survived to terminal sacrifice. Results showed that a dose level of 100 mg/kg bw was approximately the maximum tolerated dose. This dose level was considered to be an appropriate maximum for use in the main study. Therefore, animals were treated with 25, 50 and 100 mg/kg bw test substance in the main study.

Table 1: Results of the in vivo micronucleus assay (males and females combined)

			Proportion of immature erythrocytes at sampling time		Mean number of micronucleated cells per 2000 immature erythrocytes at sampling time		Number of micronucleated cells per 2000 mature erythrocytes at sampling time
Exp. group	Number of animals per sampling time	Dose (mg/kg bw)	24 h	48 h	24 h	48 h	24 h	48 h
Vehicle control (corn oil)	5	-	41	42	0.8	0.4	1.0	2.0
Positive control (mitomycin C)		12	35	n.d	38.2***	n.d	1.8	n.d
Test substance		25	39	n.d	1.1	n.d	1.0	n.d
		50	34	n.d	1.2	n.d	1.5	n.d
		100	41	43	0.9	0.6	0.0	1.7

n.d. = not determined; *** statistically significant (p < 0.001)

CLINICAL SIGNS AND MORTALITY

No mortality was observed during the main study.

Clinical signs observed at 100 mg/kg bw were consistent with the maximum tolerated dose. No adverse clinical signs were observed for the vehicle or positive control group during the study period.

Conclusions:

Under the conditions of this mammalian erythrocyte micronucleus test in mice the test substances was non-clastogenic at any concentration tested up to 100 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

 Gene mutation in bacteria

A bacterial gene mutation assay with the test substance is available in accordance with OECD Guideline 471 and in compliance with GLP (May, 1998). In two independent experiments, the Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli strain WP2 uvrA/pKM101 were exposed to the test substance using the plate incorporation method. Based on the results of a pre-experiment, test substance concentrations of 5, 15, 50, 150, 500, 1500 and 5000 µg/plate were selected for the incubation with and without metabolic activation in Experiment 1 and concentrations of 50, 150, 500, 1500 and 5000 µg/plate for Experiment 2. In the Experiment 2 S9 mix concentrations were increased from 10% to 20%. The test substance showed bacterial toxicity at 5000 µg/plate in both experiments with and without metabolic activation. Biologically relevant increase in revertant numbers was observed in strain TA 1535 in the presence of metabolic activation in both experiments. In strain TA 100 revertant numbers were slightly increased (< 2 fold) in Experiment 1 with metabolic activation, but > 2 fold increased in Experiment 2 with metabolic activation (20% with S9 mix). The revertant frequencies of the vehicle control were within the expected range and the positive control chemicals induced marked increases in revertant colonies, demonstrating the effective performance of the experiments. Under the conditions of this experiment, the test substance is considered mutagenic in S. typhimurium strains TA 1535 and TA 100 in the presence of metabolic activation.

A second bacterial gene mutation assay with the test substance is available in according to a protocol of Ames et al. (1975) and in compliance with GLP (Finch, 1981). Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were exposed to the test substance using the plate incorporation method. Based on the results of a pre-experiment, test substance concentrations of 50, 100, 250, 500, 750, 1000 and 2500 µg/plate were selected for the incubation with and without metabolic activation. The test substance showed bacterial toxicity at 2500 µg/plate in all tester strains with and without metabolic activation. Additionally, cytotoxicity was observed at 1000 µg/plate in strains TA 100, TA 1535, TA 1537 and TA 1538 in the absence of metabolic activation and in TA 100 and TA 1535 in the presence of metabolic activation. In the presence of metabolic activation, incubation with the test substance at concentrations of 100, 250, 500 and 750 μg/plate significantly increased the number of revertant colonies in TA 100 and TA 1535. Additionally, a clear dose-response relationship was observed. Furthermore, significant increased numbers of revertant colonies were observed at 100 and 250 μg/plate in TA 1535 in the absence of metabolic activation. Since higher dose levels did not result in the induction of significantly greater numbers of revertant colonies nor a significant positive dose-response relationship was observed the results were considered to have no biological significance. The revertant frequencies of the vehicle control were within the expected range and the positive control chemicals induced marked increases in revertant colonies, demonstrating the effective performance of the experiments. Under the conditions of this experiment, the test substance is considered mutagenic in S. typhimurium strains TA 1535 and TA 100 in the presence of metabolic activation.

Gene mutation in mammalian cells

The mutagenic activity of the test substance was evaluated in an in vitro mammalian cell gene mutation test according to OECD Guideline 476 and in compliance with GLP (Lloyd, 1998). The test substance was assessed for its potential to induce gene mutations at the HPRT locus using L5178Y mouse lymphoma cells. The study was performed in four independent experiments, two in the presence and two in the absence of metabolic activation. Based on the results of a preliminary toxicity test, cells were exposed in the absence of metabolic activation to the test substance at concentrations of 10, 20, 30, 40 and 60 µg/mL for 3 h (test 1) and at 2.5, 5, 10, 20 and 40 µg/mL for 24 h (test 2). Further, cells were exposed in the presence of metabolic activation at 5, 10, 20 and 30 µg/mL (test 1)and 5, 10, 15, 20 and 30 µg/mL for 3 h (test 2). After expressions periods of 24 h and 48 h cells were assessed for viability and mutant frequency.

Cytotoxicity was observed after treatment with the test substance in all the tests, both in the absence and the presence of metabolic activation. In the absence of metabolic activation, increases in mutation frequency (relative to the control value) were observed in treated cultures in both tests and statistically significant increases were observed at 30, 40 and 60 μg/mL in the first test (p<0.01, or less), but an increase of 100 over the control value was not achieved in either test, and all mutation frequency values were within the normal control ranges. In the presence of metabolic activation, increases in mutation frequency (relative to the control value) were observed in treated cultures in both tests and statistically significant increases were observed at 10, 15, 20 and 30 μg/mL in the second test (p<0.01, or less), but an increase of 100 over the control value was not achieved in either test, and all mutation frequency values were within the normal control ranges with one exception. In test 1, there was evidence of mutagenic activity in cultures treated with the test substance at 30 μg/mL in the presence of metabolic activation, but the RTG value of 0.4% was considerably below the required value of 10-20%, therefore the observation was of little biological significance. Appropriate reference mutagens, 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, the test substance did not induce gene mutations at the HPRT locus in L5178Y mouse lymphoma cells under the experimental conditions reported. Therefore, the test substance is not considered to be mutagenic in this HPRT assay.

 

In a supporting study, the mutagenic activity of the test substance was evaluated in an in vitro mammalian cell gene mutation test similar to a protocol of OECD Guideline 476 (Finch, 1981). The test substance was assessed for its potential to induce gene mutations at the HPRT locus using chinse hamster ovary (CHO) cells. The study was performed in four independent experiments, two in the presence and two in the absence of metabolic activation. A dose-range-finding study showed cytotoxicity for 100% of the cells at test substance concentrations of 80-150 µg/mL

in the presence of metabolic activation and at 70-150μg/mL in the absence of metabolic activation.Based on these results, cells were exposed to test substance at concentrations of 80, 90, 100, 110 and 120 µg/mL in the presence of metabolic activation and at 30, 40, 50, 60 and 70 µg/mL in the absence of metabolic activation for 5 h. After expression periods of 8 days cells were assessed for viability and mutant frequency.

Exposure of the cells to the test substance did not result in the induction of significantly greater (p > 0.05) mutation frequencies compared to the corresponding solvent controls at any dose level with and without metabolic activation. Appropriate reference mutagens, 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, the test substance did not induce gene mutations at the HPRT locus in CHO cells under the experimental conditions reported. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

In vivo

An in vivo micro nucleus test in CD-1 mice is available with the test substance in accordance with OECD Guideline 474 and in compliance with GLP (Mason, 2000). In a preliminary dose range finding study, death occurred in all groups dosed with test substance at concentrations of 175, 250, 500, 600 and 750 mg/kg bw, respectively, and severe clinical signs were observed in the remaining animals. At 100 mg/kg bw, all animals showed signs of hunched posture, piloerection and irregular respiration but survived to terminal sacrifice. Based on this result, in the main study, 5 animals per sex and dose were once treated intraperitoneally with 25 or 50 mg/kg bw test substance and 10 animals per sex and dose received 100 mg/kg bw test substance or vehicle (water) only. Mytomycin C was used as positive control and was administered via gavage at a dose level of 0.6 mg/mL in water. Bone marrow smears were prepared on microscope slides 24 h (all animals) and 48 h (negative and high dose group) after treatment, allowed to air dry, fixed in methanol, and stained in 10% Giemsa. One smear of each animal was examined for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes/animal.

The test item did not significantly increase the incidence of micronucleated immature erythrocytes and no substantial decrease in proportion of immature erythrocytes was observed at any of the dose levels tested. The positive control substance significantly increased the number of micronucleated erythrocytes in males and females.

Based on the results of this micronucleus test in erythrocytes, the test substance was considered to be non-clastogenic or cytotoxic to bone marrow cells in male and female CD-1 mice under the conditions of this experiment.

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.