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)

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Cyclopropanecarboxyl​ic acid, 2,​2-​dimethyl-​3-​(2-​methyl-​1-​propen-1-yl)​-​, (3-​phenoxyphenyl)​methyl ester, (1R)​-

Cas Number:

188023-86-1

Molecular formula:

C23H26O3

IUPAC Name:

Cyclopropanecarboxyl​ic acid, 2,​2-​dimethyl-​3-​(2-​methyl-​1-​propen-1-yl)​-​, (3-​phenoxyphenyl)​methyl ester, (1R)​-

Test material form:

liquid

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.

Method

Target gene:

thymidine kinase

Species / strain

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

Controls

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

Results and discussion

Test results

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.

Any other information on results incl. tables

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

 

 

Applicant's summary and conclusion

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.