Reaction mass of 3-phenoxybenzyl (1R,3R)-2,2-dimethyl-3-(2-methylprop-1-en-1-yl)cyclopropanecarboxylate and 3-phenoxybenzyl (1R,3S)-2,2-dimethyl-3-(2-methylprop-1-en-1-yl)cyclopropanecarboxylate (4:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

d-Phenotrhin was tested for mutagenicity in three different in vitro assays, namely an Ames test (OECD 471), a mouse lymphoma test (OECD 476) and a chromosomal aberration test (OECD 473), all with and without metabolic activation.

In all three studies, no signs of mutagenicity were seen and thus, an in vivo study was not required to conclude upon mutagnicity. As a result, the substance d-Phenothrin is considered non-mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Mar - Apr 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP / guideline study with no deficiencies

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Purity according to analytical certificate: 97.9%
Isomers: 16.7% d-cis isomer, 79.7% d-trans isomer, 1.0% l-trans isomer, 2.6% l-cis isomer
In study report falsely reported as CAS 26002-80-2.

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: not reported

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 fraction from phenobarbital/ß-naphoflavone treated male rats

Test concentrations with justification for top dose:

Assay 1 (-S9, 3 h): 5, 10, 20, 40, 60, 80 µg/mL
(+S9, 3 h): 5, 10, 20, 40, 80 µg/mL
Assay 2 (-S9, 24 h): 2.5, 5, 10, 20, 30, 40 µg/mL
(+S9, 3 h): 11.9, 17.8, 26.7, 40, 60 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: preferred vehicle for non-water-soluble substances

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h or 24 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 - 14 days

SELECTION AGENT (mutation assays): 5-trifluorothymidine

NUMBER OF REPLICATIONS: 2

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

OTHER EXAMINATIONS:
- Colony sizing

Evaluation criteria:

A test is considered positive if
- the mutant frequency at one or more doses is statistically significantly greater than for the negative controls
- there is a significant dose-relationship as indicated by the linear trend analysis

Statistics:

According to UKEMS guidelines
- Test for consistency between plates (chi² distribution)
- Heterogeneity factor for replicate cultures (H)
- Test for overall consistency
- Updated heterogeneity factors
- Comparison of each treatment with the control
- Test for linear trend

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at >= 40 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no change due to test substance
- Effects of osmolality: no change due to test substance

COMPARISON WITH HISTORICAL CONTROL DATA:
Mutant frequencies for solvent and positive controls were in the range of repective historical controls.

Table 1: 3 h exposure - Without Metabolic Activation

Concentration [µg/ mL]	Relative survival [%]	Relative Total Growth [%]	Mutants per 1E+06 cells	P-Value	Quotient large / small colonies
0 (DMSO)	100	100	60.42	–	0.28
5.00	97	109	58.89	NS	–
10.0	84	100	61.14	NS	–
20.0	77	69	64.34	NS	–
40.0	10	7	80.10	NS	–
60.0	3	1	139.2	tox	–
80.0	2	1	118.9	tox	–
MMS, 10.0	73	31	427.6	–	0.33
MMS: methylmethanesulphonate, NS: not significant, tox: excessive toxicity

 

 

Table 2: 24 h exposure - Without Metabolic Activation

Concentration [µg/ mL]	Relative survival [%]	Relative Total Growth [%]	Mutants per 1E+06 cells	P-Value	Quotient large / small colonies
0 (DMSO)	100	100	53.43	–	0.24
2.50	110	103	39.67	NS	–
5.00	99	90	42.85	NS	–
10.0	90	88	36.73	NS	–
20.0	91	89	32.55	NS	–
30.0	63	62	35.69	NS	–
40.0	33	36	37.34	NS	–
MMS, 5.00	63	73	381.7	–	0.41
MMS: methylmethanesulphonate, NS: not significant

 

Table 3: 3 h exposure - With Metabolic Activation (1^st assay)

Concentration [µg/ mL]	Relative survival [%]	Relative Total Growth [%]	Mutants per 1E+06 cells	P-Value	Quotient large / small colonies
0 (DMSO)	100	100	76.46	–	0.22
5.00	100	113	67.90	NS	–
10.0	100	103	57.72	NS	–
20.0	85	98	74.99	NS	–
40.0	71	72	70.11	NS	–
80.0	4	4	91.09	tox	–
B[a]P, 2.00	50	35	623.8	–	0.34
B[a]P: benzo[a]pyrene, NS: not significant, tox: excessive toxicity

 

Table 4: 3 h exposure - With Metabolic Activation (2^nd assay)

Concentration [µg/ mL]	Relative survival [%]	Relative Total Growth [%]	Mutants per 1E+06 cells	P-Value	Quotient large / small colonies
0 (DMSO)	100	100	44.88	–	0.15
11.9	81	96	42.40	NS	–
17.8	81	85	38.52	NS	–
26.7	90	81	48.52	NS	–
40.0	56	50	48.76	NS	–
60.0	24	20	58.89	NS	–
B[a]P, 2.00	42	47	345.8	–	0.44
B[a]P: benzo[a]pyrene, NS: not significant

 

 

Conclusions:

The test substance is negative in the mouse lymphoma assay, with and without metabolic activation.

Executive summary:

The test item D-Phenothrin was examined for mutagenic activity by assaying for the induction of 5-trifluorothyrnidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment (in the absence and presence of S9 metabolic activation) using a fluctuation method. Test item solutions were prepared using dimethylsulphoxide.

A preliminary cytotoxicity assay was performed. The test item was assayed at a maximum dose-level of 313 µg/mL and a wide range of lower dose-levels: 156, 78.1, 39.1, 19.5, 9.77, 4.88, 2.44 and 1.22 µg/mL. Upon addition of the test item to the cultures precipitation was observed at the two higher dose-levels (313 and 156 µg/mL) both in the absence and presence of S9 mix. At the end of the treatment incubation period, precipitation was present at the same dose-levels. Using the short treatment time in the presence of S9 metabolic activation, severe toxicity, reducing survival to 1% of the negative control value, was observed at the two higher concentrations, while at the next lower dose-level (78.1 µg/mL) survival was reduced to 24%. No relevant toxicity was observed at the remaining concentrations. In the absence of S9 metabolic activation, using a short treatment time (3 hours), severe toxicity was observed at the three higher concentrations reducing survival to less than 10% of the concurrent negative control value. At the next lower concentration (39.1 µg/mL) moderate toxicity, reducing survival to 55% of the negative control value, was observed. Using a long treatment time (24 hours) no cells survived at the three higher concentrations. Marked toxicity reducing survival to 23% of the control was observed at 39.1 µg/mL, while no toxicity was observed at the remaining concentrations.

Two independent assays for mutation at the TK locus were performed using dose-levels described in the following table:

Assay 1: 3 hours at dose levels 80.0, 60.0, 40.0, 20.0, 10.0, and 5.0 µg/mL without S9 and 80.0, 40.0, 20.0, 10.0, and 5.0 µg/mL with S9

Assay 2: 24 hours at dose levels 40.0, 30.0, 20.0, 10.0, 5.0, and 2.5 µg/mL without S9, and 3 hours at 60.0, 40.0, 26.7, 17.8, and 11.9 µg/mL with S9

Both in the absence and presence of S9 metabolic activation no statistically significant increases in mutant frequency were observed following treatment with the test item at any dose-level and treatment time.

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the negative control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

It is concluded that D-Phenothrin does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb - Aug 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP / guideline study without relevant deficiencies

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no justification for duplicate plating was provided

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

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:

S9 fraction (Aroclor 1254 induced rat liver homogenate) supplemented with cofactors

Test concentrations with justification for top dose:

Trial I: 0.156, 0.312, 0.625, 1.25, 2.5, and 5.0 µL/plate
Trial II: 0.051, 0.128, 0.32, 0.8, 2.0, and 5.0 µL/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: preferred vehicle for non-water-soluble compounds

Untreated negative controls:

yes

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-aminofluorene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: inhibition of background lawn

Evaluation criteria:

A positive result is defined as a dose-dependent and reproducible increase at one or more concentrations in the number of histidine-independent revertant colonies per plate in at least one strain (in the presence or absence of metabolic activation). A test substance for which the results do not meet the above criteria is considered non-mutagenic in this test. If the test substance does not induce a statistically significant dose-dependent increase in revertant frequency but does induce revertant frequency at one dose level that is at least two-fold the spontaneous control value, the result is considered equivocal.
Negative and equivocal results in the first trial are confirmed by a second trial with an alteration in concentration spacing and/or metabolic activation.

Statistics:

Simple linear regression analysis was performed for TA1537, TA1535, TA98, TA100 and TA102, separately, to assess the dose-dependent nature of any increase in revertant colonies.

Key result

Species / strain:

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

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:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Cytotoxicity was not observed at any of the concentrations tested (up to 5 µL/plate), either in the absence or in the presence of metabolic activation. Hence, 5.0 µL of d-phenothrin/plate was selected as the highest test concentration in all trials.

Remarks on result:

other: all strains/cell types tested

Table 1: Test Results of Trial I (plate incorporation)

With or without S9-Mix	Test substance concentration (μL/plate)	Mean number of revertant colonies per plate (average of 2 plates)
		Base-pair substitution type			Frameshift type
		TA100	TA1535	TA102	TA98	TA1537
-	Neg. control	114.0	14.5	203.0	17.0	6.0
-	Veh. control	96.5	10.0	214.5	17.0	5.0
-	0.156	97.5	8.5	196.5	12.0	5.5
-	0.312	103.5	10.5	233.0	16.5	7.0
-	0.625	114.5	9.0	167.5	11.5	6.5
-	1.25	96.0	12.5	196.0	9.0	3.5
-	2.5	103.5	11.0	192.0	13.5	10.5
-	5.0	115.0	9.0	184.0	8.0	10.0
-	Pos. control	1620.0	107.5	944.0	1030.0	167.5
+	Neg. control	105.5	9.0	231.0	31.5	5.0
+	Veh. control	118.0	13.5	264.0	32.0	5.0
+	0.156	112.0	6.0	258.0	25.5	3.5
+	0.312	101.0	9.5	210.0	27.0	6.5
+	0.625	98.0	12.0	230.5	29.5	7.5
+	1.25	127.5	12.5	208.0	27.5	5.5
+	2.5	121.0	12.5	204.0	21.0	7.0
+	5.0	128.5	8.0	240.5	17.0	5.0
+	Pos. control	1085.0	422.0	1075.5	926.0	70.0
Positive controls used: -S9: TA1537: 9-Aminoacridine (75 µg/plate), TA1535: sodium azide(0.5 µg/plate), TA98 = 2-nitrofluorene (7.5 µg/plate), TA100: sodium azide (5 µg/plate), TA102: mitomycin-C (0.5 µg/plate) +S9, all strains: 2-aminofluorene (20 µg/plate)

  

Table 2: Test Results of Trial II (plate incorporation)

With or without S9-Mix	Test substance concentration (μL/plate)	Mean number of revertant colonies per plate (average of 2 plates)
		Base-pair substitution type			Frameshift type
		TA100	TA1535	TA102	TA98	TA1537
-	Neg. control	129.0	8.5	252.0	25.5	6.0
-	Veh. control	139.0	11.0	272.0	26.5	8.5
-	0.051	116.5	10.5	257.0	17.0	8.5
-	0.128	107.5	14.0	262.5	19.0	6.5
-	0.32	107.0	10.0	224.0	23.5	1.5
-	0.8	121.5	9.5	231.5	23.0	7.0
-	2.0	127.0	11.5	272.5	14.0	7.5
-	5.0	135.5	6.5	250.5	19.0	9.5
-	Pos. control	960.0	151.5	2615.0	1032.5	189.0
+	Neg. control	129.5	13.5	256.5	31.0	11.5
+	Veh. control	130.0	16.0	267.5	33.0	8.0
+	0.051	131.0	9.0	255.5	25.0	4.0
+	0.128	118.0	7.0	247.5	23.5	2.5
+	0.32	110.0	14.5	258.0	21.5	5.5
+	0.8	118.5	11.0	248.5	23.5	9.0
+	2.0	113.5	13.5	238.5	29.0	7.0
+	5.0	119.0	13.0	235.0	24.0	9.0
+	Pos. control	1081.0	409.5	1113.5	1105.5	87.5
Positive controls used: -S9: TA1537: 9-Aminoacridine (75 µg/plate), TA1535: sodium azide(0.5 µg/plate), TA98 = 2-nitrofluorene (7.5 µg/plate), TA100: sodium azide (5 µg/plate), TA102: mitomycin-C (0.5 µg/plate) +S9, all strains: 2-aminofluorene (20 µg/plate)

 

Conclusions:

The test substance is negative in the Ames test (strains TA 1535, TA 1537, TA 98, TA100, and TA 102), with and without metabolic activation.

Executive summary:

This study was performed to evaluate d-Phenothrin for its possible mutagenic activity, by the bacterial reverse mutation test, using five histidine deficient(his) mutant tester strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and TA102.The methods followed were as per the OECD guideline No 471 (July 1997).

The treatments were performed by a plate incorporation technique both in the absence and in the presence of metabolic activation (S9 mix). The S9 mix of 5% and 10% v/v consisted of an S9 fraction (Aroclor 1254 induced rat liver homogenate) supplemented with cofactors.

Before conducting the mutagenicity test, d-Phenothrin was evaluated for its possible cytotoxicity in strain TA100, both in the absence and in the presence of S9 mix (5% v/v). The test concentration for cytotoxicity was selected based on the solubility and precipitation test. Cytotoxicity to the tester strain was tested at 0.019, 0.039, 0.078, 0.156, 0.312, 0.625, 1.25, 2.5 and 5.0µL/plate both in the presence (S9 mix 5% v/v) and in the absence of the metabolic system. Cytotoxicity is characterised by inhibition of the background bacterial lawn and reduction in the number of colonies. Cytotoxicity was not observed at any of the concentrations tested, either in the presence or the absence of the metabolic activation system. Hence, 5.0µL of was selected as the highest concentration to be tested in the mutagenicity study, both in the presence and in the absence of the metabolic activation system, for all the tester strains.

Trial 1

Based on the results of the cytotoxicity study, d-Phenothrin was evaluated for its possible mutagenic effect in five strains of typhimurium at dose levels of 0.156, 0.312, 0.625, 1.25, 2.5 and 5.0µL/plate both in the presence (5% v/v S9 mix) and in the absence of metabolic activation for Trial I (spacing between dose levels being factor 2). The stock solutions of d-Phenothrin were prepared in dimethyl sulfoxide (DMSO). Duplicate plates were maintained for each test concentration of d-Phenothrin, negative vehicle and positive controls.

The results of exposure at the dose levels of 0.156, 0.312, 0.625, 1.25, 2.5 and 5.0µL/plate both in the presence (5% v/v S9 mix) and in the absence of metabolic activation did not reveal any positive mutagenic effect when compared with the concurrent vehicle controls in the tester strains TA1537, TA1535, TA98, TA100 and TA102. The analysis of the results of Trial I did not reveal any statistically significant increase in number of revertant colonies, either in the presence or in the absence of metabolic activation.

 

Trial 2

A second trial was conducted to confirm the negative results obtained in Trial 1. In Trial 2, the concentration spacing was modified using a factor of 2.5 and the conversion of S9 mix was increased to 10% v/v. The highest concentration being 5.0µL/plate, five lower concentrations viz., 2.0, 0.8, 0.32, 0.128 and 0.051µL/plate were tested both in the presence and in the absence of metabolic activation. Duplicate plates were maintained for each test concentration of d-Phenothrin, negative, vehicle and positive control.

No statistically significant increase in the number of revertant colonies were observed in any of the tester strains viz., TA 1537, TA1535, TA98, TA100 and TA102, at any of the concentrations of d-Phenothrin tested, either in the presence (10% v/v S9 mix) or absence of metabolic activation.

The values of negative and vehicle controls in all tester strains during both the trials were within limits. The positive controls exhibited a clear increase in the number of revertants both in the absence and in the presence of metabolic activation with known mutagens when compared with the respective negative and vehicle controls. This demonstrated the efficiency of the test system and suitability of the procedures employed in the study.

From the results of this study, it is concluded that d-Phenothrin, up to the concentration of 5.0µL/plate, both in the presence and absence of metabolic activation, is non-mutagenic to all the five tester strains of Salmonella typhimurium TA1537, TA1535, TA98, TA100 and TA102, when tested under the specified conditions.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 2006 - Feb 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP / guideline study with minor deficiencies

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

The highest concentration did not elicit 50% cytotoxicity or reach 5 µg/mL. The frequency of gaps was not reported.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F-12 with 10% FCS
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 liver fraction from Aroclor 1254 treated rats supplemented with co-factors

Test concentrations with justification for top dose:

Trial I (4-h exposure): 0.025, 0.05, and 0.1 µL/mL (-S9); 0.01875, 0.0375, 0.075 µl/mL (+ 1% S9)
Trial II (18- to 21-h exposure): 0.025, 0.05, and 0.1 µL/mL (-S9)
Trial III (4-h exposure): 0.01875, 0.0375, 0.075 µl/mL (+ 5% S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: preferred vehicle for non-water-soluble compounds

Untreated negative controls:

yes

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:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h or 18 - 21 h
- Fixation time (start of exposure up to fixation or harvest of cells): 18 - 21 h (1.5 cell cycles)

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per slide = 200 per concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: not reported

Evaluation criteria:

A positive result is defined as a dose-dependent and reproducible increase at one or more concentrations in the number of histidine-independent revertant colonies per plate in at least one strain (in the presence or absence of metabolic activation). A test substance for which the results do not meet the above criteria is considered non-mutagenic in this test. If the test substance does not induce a statistically significant dose-dependent increase in revertant frequency but does induce revertant frequency at one dose level that is at least two-fold the spontaneous control value, the result is considered equivocal.
Negative and equivocal results in the first trial are confirmed by a second trial with an alteration in concentration spacing and/or metabolic activation.

Statistics:

Data were evaluated by Student's t-test

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reduction of mitotic index to 7% of controls with 5% S9, ca 70% without S9, no cytotoxicity with 1% S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1: Phase I - 4 h exposure, without S9 mix
Concentration [µg/mL]	Replicate	Mitotic index [%]	Mitotic index [% DMSO control]	No. of aberrant cells		Others	Total No. Of aberrations	% aberrant cells
				Chromatid type	Chromosome type
0 (water)	1	1.89	100%	0	0	0	0	0
	2	2.66	141%	0	0	0	0	0
0 (DMSO)	1	1.69	89%	0	0	0	0	0
	2	2.09	111%	1	0	1E	2	1
0.025	1	2.21	117%	0	0	0	0	0
	2	2.16	114%	0	0	0	0	0
0.05	1	1.98	105%	0	0	1DL	1	1
	2	2.18	115%	2	0	1DL	3	3
0.1	1	1.47	78%	3	0	0	3	3
	2	1.10	58%	0	2	0	2	2
MMC, 0.5	1	1.08	57%	15	17	1DL, 3E, 10F, 1R	48	11
	2	1.40	74%	13	0	4E, 4F	21	12
MMC = mitomycin C, F= Fragment, E=Exchange, DL=Deletion, D=Dicentric, R=Ring
Table 2: Phase I - 4 h exposure, with 1% S9 mix
Concentration [µg/mL]	Replicate	Mitotic index [%]	Mitotic index [% DMSO control]	No. of aberrant cells		Others	Total No. Of aberrations	% aberrant cells
				Chromatid type	Chromosome type
0 (water)	1	2.46	115%	0	0	0	0	0
	2	2.55	120%	0	0	0	0	0
0 (DMSO)	1	2.21	104%	0	0	0	0	0
	2	2.05	96%	0	1	2E	3	2
0.01875	1	2.13	100%	1	0	0	1	1
	2	2.14	100%	1	0	0	1	1
0.0375	1	1.83	86%	0	0	0	0	0
	2	2.11	99%	2	2	0	4	3
0.075	1	2.36	111%	1	1	0	2	2
	2	1.76	83%	3	3	0	6	4
CP, 25	1	1.28	60%	3	4	3DL, 2D, 4E, 9F, 1Cb	26	19
	2	1.39	65%	2	4	3DL, 3D, 3E, 15F, 3R	33	20
CP = cyclophosphamide, F= Fragment, E=Exchange, DL=Deletion, D=Dicentric, Cb=Centromeric break, R=Ring
Table 3: Phase II - continuous exposure, without S9 mix
Concentration [µg/mL]	Replicate	Mitotic index [%]	Mitotic index [% DMSO control]	No. of aberrant cells		Others	Total No. Of aberrations	% aberrant cells
				Chromatid type	Chromosome type
0 (water)	1	1.09	94%	0	0	0	0	0
	2	1.94	167%	0	0	0	0	0
0 (DMSO)	1	1.06	91%	1	0	0	1	1
	2	1.26	109%	1	1	0	2	2
0.025	1	1.16	100%	1	0	1E	2	2
	2	0.98	84%	0	0	0	0	0
0.05	1	0.99	85%	1	2	1E	4	4
	2	1.19	103%	1	0	0	1	1
0.1	1	0.78	67%	2	1	0	3	3
	2	0.88	76%	0	1	0	1	1
MMC, 0.5	1	0.39	34%	22	1	1DL, 3D, 8E, 1F	36	20
	2	0.59	51%	43	0	2DL, 2D, 18E, 2F, 2R	69	24
MMC = mitomycin C, F= Fragment, E=Exchange, DL=Deletion, D=Dicentric, Cb=Centromeric break, R=Ring
Table 4: Phase III - 4 h exposure, with 5% S9 mix
Concentration [µg/mL]	Replicate	Mitotic index [%]	Mitotic index [% DMSO control]	No. of aberrant cells		Others	Total No. Of aberrations	% aberrant cells
				Chromatid type	Chromosome type
0 (water)	1	1.87	88%	0	0	0	0	0
	2	1.69	79%	1	0	0	1	1
0 (DMSO)	1	1.51	71%	1	0	0	1	1
	2	1.38	65%	2	0	0	2	2
0.01875	1	1.37	64%	2	0	0	2	2
	2	1.25	59%	2	0	1DL	3	2
0.0375	1	1.02	48%	1	0	0	1	1
	2	1.15	54%	1	1	0	2	2
0.075	1	0.10	5%	0	0	0	0	0
	2	0.19	9%	0	0	0	0	0
CP, 25	1	0.87	41%	7	2	1DL, 4E, 1F	15	14
	2	0.69	32%	13	5	3DL, 1D, 4E, 1F	27	16
CP = cyclophosphamide, F= Fragment, E=Exchange, DL=Deletion, D=Dicentric, Cb=Centromeric break, R=Ring

 

Conclusions:

The test substance does not cause chromosomal aberrations in CHO-K1 cells, with and without metabolic activation.

Executive summary:

This study was conducted to determine the chromosomal aberration induction potential of d-Phenothrin in the CHO-K1 cell line both in the presence and in the absence of metabolic activation. The methods followed were as per the guidelines of OECD N° 473 (1997).

The test system employed was the CHO-K1 cell line. This cell line was grown in tissue culture flasks as adherent cultures using Ham’s F-12 medium supplemented with 10% foetal bovine serum in a CO2 incubator at 37 °C and 5% CO2 level. The experiment was conducted in the absence and in the presence of metabolic activation depending on the treatment phase. The cells were treated with d-Phenothrin and replaced with complete medium at the end of the treatment. Culture flasks were incubated for a period of 18 - 21 h (1.5 normal cell cycle). Four hours prior to harvesting, cells were arrested in the metaphase state with colchicine.

After the completion of the incubation period the cultures were trypsinised and processed for the preparation of chromosomes. The Giemsa stained metaphase preparations were analysed microscopically for chromosome aberrations. A minimum number of 1000 cells per slide were counted and the numbers of metaphases were recorded in different fields to determine the mitotic index. The number of aberrant cells with one or more aberrations excluding gaps and polyploidy were recorded to calculate the per cent aberrant cells.

Before conducting the chromosomal aberration study, d-Phenothrin was evaluated for cytotoxicity, both in the absence and in the presence of metabolic activation (1% v/v S9 mix). The test concentration for cytotoxicity was selected based on the solubility and precipitation tests. Cytotoxicity was performed at the dose levels of 0.075, 0.15, 0.3 and 0.6µL of d-Phenothrin/mL of culture medium, both in the absence and in the presence of metabolic activation. The cytotoxicity was assessed based on the mitotic index.

A differential cytotoxicity pattern was observed in the absence and in the presence of metabolic activation. Based on the results of the cytotoxicity test, d-Phenothrin was evaluated for chromosome aberrations in the absence and in the presence of metabolic activation at the dose levels of 0.025, 0.05 and 0.1µL of d-Phenothrin/mL of culture medium and 0.01875, 0.0375 and 0.075µL of d-Phenothrin/mL of culture medium, respectively.

Phase 1:

In Phase 1 of the experiment cultures were exposed to d-Phenothrin for a short period of time (4 hours) both in the presence and in the absence of metabolic activation (1% v/v S9 mix). The results did not show any statistical significance in either the mitotic index or percent aberrant cells at the dose levels of 0.025, 0.05 and 0.1µL of d-Phenothrin/mL of culture medium in the absence of metabolic activation and 0.01875, 0.0375 and 0.075µL of d-Phenothrin/mL of culture medium in the presence of metabolic activation (1% v/v S9 mix) when compared with the vehicle control.

Phase 2

In Phase 2 of the experiment, cultures were exposed to d-Phenothrin for a prolonged period of time (continuous exposure for 18 - 21 h until sampling at 1.5 normal cell cycle) in the absence of metabolic activation. The results did not show any statistical significance in either mitotic index or percent aberrant cells at dose levels of 0.025, 0.05 and 0.1µL of d-Phenothrin/mL of culture medium in the absence of metabolic activation when compared with the vehicle control.

Phase 3

The negative results obtained with metabolic activation (1% v/v S9 mix) in Phase 1, were subsequently confirmed by increasing the concentration of S9 mix to 5% v/v in PhaseI 3 of the experiment, cultures were exposed to d-Phenothrin for a short period of time (4 hours) in the presence of metabolic activation (5% v/v S9 mix). The results did not show any statistical significance in the mitotic index and per cent aberration at the dose levels of 0.01875 and 0.0375µL of d-Phenothrin/mL of culture medium when compared with the vehicle control. A significant reduction in the mitotic index was observed at the dose level of 0.075µL of d-Phenothrin/mL of culture medium when compared to the vehicle control. However, this reduction is attributed to the toxicity caused by the increased metabolism of the test substance due to the increased concentration of the S9 mix (5% v/v).

The results revealed a clear increase in per cent aberrant cells and a clear decrease in mitotic index in the cultures treated with positive controls, mitomycin-C at the concentration of 0.5µg and cyclophosphamide at the concentration of 25µg per mL of culture medium in the absence and in the presence of metabolic activation respectively, when compared with the vehicle control. This demonstrated the sensitivity of the test system and suitability of procedures employed in the study.

From the results of this study, it is concluded that d-Phenothrin at the dose levels of 0.025, 0.05 and 0.1µL/mL of culture medium in the absence of metabolic activation and 0.01875, 0.0375 and 0.075µL/mL of culture medium in the presence of metabolic activation (1% v/v and 5% v/v S9 mix) does not possess chromosomal aberration induction potential to the CHO-K1 cell line.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on negative outcome from three different in vitro mutagenicity studies, the substance d-Phenothrin is not considered mutagenic and thus, is not subject to classification for mutagenicicity according to CLP (Regulation EC No 1272/2008)