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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

bacterial mutation assay: negative

mammalian cell chromosome aberration assay: negative

mammalian cell mutation assay: negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 7, 1992 - July 31, 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 4 S. typhimurium strains were used
Qualifier:
equivalent or similar to guideline
Guideline:
other: EEC Directive 79/831/EEC, Annex V (Directive 84/449/EEC), Method B.14: Salmonella typhimurium- Reverse Mutation Assay
Qualifier:
equivalent or similar to guideline
Guideline:
other: US EPA, Method: HG-Gene Muta (40CFR798.5265)-S.typhimurium; The Salmonella typhimurium reverse mutation assay
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- batch No.of test material: 5007
- sample No.:1505E
- Expiration date of the lot/batch: March 1993

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4°C in the dark
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Professor B.N. Ames, University of California, U.S.A.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Nutrient broth (DAB 7, Merck)
- Properly maintained: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
other: deficient in normal DNA repair processes; strains TA 98 and 100 possess a plasmid (pKM101) which introduces an error-prone repair process, resulting in increased sensitivity to mutagens
Cytokinesis block (if used):
no
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from rat liver stimulated with Aroclor 1254
Test concentrations with justification for top dose:
50, 150, 500, 1500 and 5000 µg/plate
At the highest dose of 5000 µg/plate no toxicity was observed in the preliminary dose range finding study. This dose is recommended by the regulatory guidelines with which this study complies.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Prior to commercing testing the solubility of the test material in ethanol was assessed. The test material was found to be soluble in ethanol at 50 mg/mL so ethanol was used as the solvent for the assay.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding: 2x10E9 cells per mL

DURATION
- Preincubation period:
An aliquot of 0.1 mL of a 10 h bacterial culture and 0.5 mL S9-Mix or 0.1 M phoshate buffer (pH 7.4) were placed in glass bottles. An aliquot of 0.1 mL of the test solution was added, followed immediatly by 2 mL of histidine deficient agar. The mixture was overlaid onto previously prepared petri dishes containing 25 mL minimal agar. Three petri dishes were used for each dose level. A set of plates were also prepared containing only bacterial culture and S9-Mix or phosphate buffer (0 µg/plate). Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9-Mix and phosphate buffer. The strains were also tested for the presence of the following genotypic markers: rfa,uvrB and pKM101. Plates were incubated at 37°C for 3 days. After this period revertant colonies were counted.



Evaluation criteria:
For the test to consider valid, the following criteria must have been met:
- the presence of the relevant genotypic markers should have been confirmed
- the mean revertant colony counts for the solvent control for each strain should have been within the ranges HRC´s historical data base:
TA 1535 5-20
TA 1537 5-20
TA 98 15-40
TA 100 70-160

The mean number of reverant colonies for all treatment groups was compared with those obtained for the solvent controls.
Statistics:
A test substance is considered to show evidence of mutagenic activity if a reproducible statistically significant increase (p<0.05) in revertant colony numbers is observed. The statistical procedures are those described by Mahon et al. (1989) and analysis of variance followed by Dunnett´s test was used.
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:
valid
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:
valid
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:
valid
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:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
No visible effect on the background bacterial lawn or decrease in revertant colony numbers was observed at any dose level in either the presence or absence of S9-Mix in a preliminary toxicity test. No precipitation of the test material up to the highest concentration used was observed.



The genotypic markers rfa, uvrB and pKM101 were shown to be present in the appropriate strains in both mutation tests. All the revertant colony counts for the solvent controls were within the stated ranges for a valid assay. The test substance was stable in the solvent for at least 4 h. The test substance stock solution was analyzed for each test for achieved concentration and was found to be within 3% of the stated concentration of the active ingredient in all tests.

Conclusions:
The test substance MCPP-P 2-EHE shows no evidence of mutagenic activity in the bacterial system.
Executive summary:

A study similar to OECD 471 was performed to assess the in vitro mutagenic potential of MCPP-P 2-EHE using histidine-dependent auxotrophic mutants of S.typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100). Strains were exposed to the test material diluted in ethanol. In a preliminary dose range finding study no toxicity was observed up to the highest concentration of 5000 µg/plate. Five concentrations (50, 150, 500, 1500 and 5000 µg/plate) were used in the plate incorporation mutation assay. Bacteria were incubated with the test substance or solvent control ethanol in the presence or absence of rat liver S9-Mix for 3 days. No evidence of mutagenic activity was seen at any dose level of MCPP-P 2-EHE. The concurrent positive control compounds demonstrated the sensitivity of the assay and metabolizing activity of the liver S9-Mix.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 3, 1992 – November 15, 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted 26 May 1983
Qualifier:
according to guideline
Guideline:
other: US EPA Method: HG-Chrome-in vitro. In vitro mammalian cytogenetics (1982)
GLP compliance:
yes
Type of assay:
other: lymphocyte chromosomal aberration
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- batch No.of test material: 5007
- sample No.:1505E
- Expiration date of the lot/batch: March 1993

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4°C in the dark
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: pooled human blood taken from healthy male donors
- Whether whole blood or separated lymphocytes were used: lymphocytes
- lymphocytes incubated with phytohaemagglutinin (PHA) for approximately 48 h

MEDIA USED
- Type and identity of media including CO2 concentration: RPMI 1640 tissue culture medium+20% foetal calf serum+PHA, 175 µg/ml; incubated at 5% CO2, 37°C
- Properly maintained: yes
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
yes /colchicine
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from rat liver stimuated with Aroclor 1254
Test concentrations with justification for top dose:
10, 20, 40, 80, 160 and 320 µg/mL
The highest final concentration chosen for this test was 320 µg/mL, because at higher concentrations precipitations of the test substance in tissue culture medium occurred.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: MCPP-p EHE was found to be soluble in ethanol at 500 mg/ml.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding:1x10E6 cells/mL

PROCEDURE FOR PRELIMINRY TOXICITY TEST:
Approximately 48 h after cultures were established, 50 µL of MCPP-p 2-EHE were added to one set of single cultures to give final concentrations of 10, 20, 40, 80, 160 and 320 µg/ml. Ethanol as solvent control was added (50µL) at two cultures.
1.25 mL S9-Mix was added to each culture in a second set followed by 62.5 µL aliquots of the various dilutions of MCPP-p 2-EHE giving the same series of final concentrations. Ethanol as solvent control (62.5 µL) was added to two cultures. Three hours after dosing, the cultures containing S9-Mix were centrifuged and the resulting cell pellets resuspended in fresh medium. 50 µL of 5- bromodeoxyuridine was added to each culture in both the presence and absence of S9-Mix to give a final concentration of 10 µg/ml. Cultures were returned to the incubator for further 21 h. This procedure and all subsequent procedures were carried out in yellow light.
Twenty-two hours after initiation of treatment with test substance, mitotic activity was arrested by addition of colchicine (0.25 µg/mL final concentration) for 2 h.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Cell suspensions were centrifuged and pellets were treated with 20% Hanks balanced salt solution. After a 10 min period of hypotonic incubation the suspensions were centrifuged and pellets were fixed using methanol/glacial acetic acid (3:1) for 2 h. Two or three drops of the cell suspensions were dropped onto pre-cleaned microscope slides and air-dried. Slides were stained by immersion in Hoechst 33258 at a concentration of 0.5 µg/mL in phosphate buffer and exposed to UV light for 1 h. After 30 min incubation at 60°C slides were stained with 4% Giemsa solution for 3-4 min, air-dried and mounted in DPX.

NUMBER OF CELLS EVALUATED: 1000 cells in each culture using light microscopy at a magnification of x160. The proportion of mitotic cells per 1000 cells in each culture was recorded. The proportion of cells in first and second division in 50 cells per culture was also recorded. From these results, dose levels and the most appropriate harvest times were selected for the main test.

PROCEDURE FOR MAIN TEST
48 h after cultures of lymphocytes were established, 1.25 mL S9-Mix was added to each culture followed by addition of MCPP-p 2EHE. 50 µL aliquots of the test substance and controls were added in the absence of S9-Mix and 62.5 µL aliquots in its presence. To the cultures to be harvested in the absence of S9-Mix after 13 h, MCPP-p 2-EHE was added to duplicate cultures to give final concentrations of 40, 20 and 10 µg/mL. For the 21 h harvest final concentrations of 80, 60 and 40 µg/mL were used. For the test in the presence of S9-Mix final concentrations were 320, 160, 80 and 40 µg/mL. Ethanol as solvent control was added to quadruplicate cultures. Ethylmethanesulphonate was added to duplicate cultures in the abscence of S9-Mix at final concentrations of 750 µg/mL and cyclophosamide in the prescen of Sß-Mix at final concentriaons of 15and 19 µg/mL (both positive controls). Three hours after dosing the cultures containing the S9-Mix were centrifuged and the cell pellets resuspended in fresh medium and incubated for further 13 h.

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.25 µg/mL) was added to each culture 2 h prior harvest

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
same procedure as preliminary test

NUMBER OF CELLS EVALUATED: The proportion of mitotic cells per 1000 cells in each culture using light microscopy at a magnification of x160 was recorded. Metaphase figures were identified at a magnification of x1000. Approximately 100 metaphase figures were examined, where possible, from each culture, with a maximum of 25 from each slide. Only cells with 46 chromosomes were analyzed. The Vernier readings of all aberrant metaphase figures were recorded and aberrations were scored according to the classification of the ISCN (ISCN 1985).

Rationale for test conditions:
derived from preliminary toxicity test (harvest times and concentrations)
Evaluation criteria:
The proportion of aberrant cells in solvent control cultures should not exceed 5% (excluding gap damage). The minimum percentage of aberrant cells in positive control cultures should not be 10%. At least one of the dose levels should cause a reduction in mitotic index of 40-80%. This does not apply if the maximum advisable concentration (5mg/mL) or the limit of solubility of the test substance has been reached without toxic effects.
Statistics:
The number of aberrant metaphase figures in each treatment group was compared with the solvent control value using Fisher´s Test. A positive response is claimed if a clear treatment-related and statistically significant increase in the number of cells containing chromosome aberrations is observed. A negative response is claimed if no statistically significant increases are observed. An equivocal response is claimed if a statistically significant increase is observed which does not fulfil the criteria for a positive or negative response.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
Significant increase to 4.5% aberrant cells at highest dose level of 320 µg/mL, but this was in the historical control range of 0-5.25%
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: decrease in mitotic index observed (cell cycle delay)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the preliminary toxicity test, in the presence of S9-Mix, no toxicity and no cell cycle delay was seen. On the other hand, MCPP-P 2-EHE showed a dose- dependent decrease in mitotic index in the absence of S9-Mix. As cell cycle delay was observed in the absence of S9-Mix, two harvest times were chosen for the main test (13 h and 21 h; approximately 1x and 1.5x the cell cycle). The lower dose levels, where no cell cycle delay was observed, where chosen for the early harvest and the higher dose levels, where cell cycle delay was evident, were chosen for the later harvest. Where cell cycle delay was only slight, the dose level used in both early and late harvesting. In the presence of S9-Mix, where no cell cycle delay was seen, only one harvest time, at 1x the cell cycle was chosen. This was 16 h, as the cell cycle time was longer in the presence of S9 -Mix than in its absence.

The dose levels selected for metaphase analysis in absence of S9-Mix were 80 µg/mL, from the 21 h harvest as well as 40 and 10 µg/mL, from the 13 hour harvest. At 10 µg/mL no decrease in cell cycle delay was observed, while at 40 µg/mL and at 80 µg/ml a cell cycle delay occurred (see table 1 and 2). The solvent control cultures from the 13 h harvest were used for the metaphase analysis and the 500 µg/mL ethylmethanesulphonate positive control was used for analysis. In the presence of the S9-Mix, no cell cycle delay was observed (see table 3). Here, the dose levels selected for the metaphase analysis were 320, 160 and 40 µg/mL. Cyclophosphamide at 10 µg/mL was chosen for analysis as positive control.

Table 1: Main test-mitotic indices of cultured human lymphocytes treated with MCPP-P 2-EHE (without S9-Mix, 13 harvest)

Test agent

Concentration [µg/mL]

Mitotic index

Relative mitotic index (%)

Incidence           % mean

Ethanol (solvent)

10 µL/mL

92/1000

65/1000

97/1000                   8.2

75/1000

100

MCPP-p 2-EHE

10

85/1000

77/1000                   8.1

99

20

100/1000                 

80/1000                   9.0

110

40

67/1000

58/1000                   6.3

77

Table 2: Main test-mitotic indices of cultured human lymphocytes treated with MCPP-P 2-EHE (without S9 -Mix, 21 h harvest)

Test agent

Concentration [µg/mL]

Mitotic index

Relative mitotic index (%)

Incidence           % mean

Ethanol (solvent)

10 µL/mL

51/1000

54/1000

48/1000                   5.2

55/1000

100

MCPP-p 2-EHE

40

35/1000

35/1000                   3.5

67

60

25/1000                 

26/1000                   2.6

50

80

26/1000

19/1000                   2.3

44

Table 3: Main test-mitotic indices of cultured human lymphocytes treated with MCPP-P 2-EHE (with S9 -Mix, 16 h harvest)

Test agent

Concentration [µg/mL]

Mitotic index

Relative mitotic index (%)

Incidence           % mean

Ethanol (solvent)

10 µL/mL

80/1000

99/1000

110/1000                   9.7

99/1000

100

MCPP-p 2-EHE

40

86/1000

87/1000                     8.7

90

80

98/1000                 

70/1000                     8.4

87

160

87/1000

91/1000                     8.9

92

320

73/1000

109/1000                   9.1

94

Note: a precipitate was seen in cultures treated with 320 and 160 µg/mL on dosing.

Conclusions:
MCPP-P 2-EHE has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.
Executive summary:

In this GLP study according OECD TG 473 (Version 1983), the ability of MCPP-P 2-EHE to induce chromosomal aberrations in human lymphocytes was assessed in vitro. In a preliminary toxicity test, of MCPP-P 2-EHE was found to be toxic and caused cell cycle delay in the absence of S9-Mix. In the presence of S9-Mix it was non-toxic and no cell cycle delay was observed. Based on these, suitable harvest times for the main study were chosen: 13 h and 21 h in the absence of S9-Mix and 16 h for the presence of S9 -Mix. Precipitation in the medium at high dose level occured, therefore 320 µg/mL was chosen to be the highest concentration used in the main test. In the absence of S9-Mix 80, 60 and 40 µg/mL (21 h harvest) or 40, 20 and 10 µg/mL (13 h harvest) were utilized. In the presence of S9-Mix, 320, 160, 80 and 40 µg/mL was chosen. The positive controls induced significant clastogenic effects and demonstrated the sensitivity of the test system and the activity of the S9 -Mix used. In both, the presence and the absence of S9 -Mix, no biological significant increase in the proportion of aberrant cells were seen at any dose level when compared with the solvent control.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 18, 1992 - November 3, 1992
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 4 April, 1984
Qualifier:
according to guideline
Guideline:
other: US EPA, Method: HG-Gene Muta-Somatic Cells: Detection of gene mutations in somatic cells in culture.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell transformation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- batch No.of test material: 5007
- sample No.:1505E
- Expiration date of the lot/batch: March 1993

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4°C in the dark
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1-BH4
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells:British industrial Biological Research Association

MEDIA USED
- Type and identity of media including CO2 concentration: Ham´s F12 medium, supplemented with 200 mM glutamine and 50 µg/mL gentamicin= the resulting medium is referred to as H0 medium; H0 medium supplemented with 5% heat-inactivated FCS = H5 medium was used for general cell culture purposes
- selection medium, in which only HGPRT deficient cells grow: H5 medium supplemented with 6-TG at a final concentration of 10 µg/mL
- the week prior treatment, spontaneous mutants were eliminated from stock cultures by growing cells in H5 medium containing 15 µg/mL hypoxanthine, 0.3 µg/mL amethopterin and 4 µg/mL thymidine (=H5-HAT medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
Cytokinesis block (if used):
no
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from rat liver stimuated with Aroclor 1254
Test concentrations with justification for top dose:
Preliminary toxicity test: 5, 10, 20, 40, 80, 160, 240, 320 µg/mL
Due to precipitation in the culture medium, 320 µg/mL in culture medium (32 mg/mL ethanol) was chosen as the top concentration to be used in the
preliminary test.
Mutation tests:
-S9-Mix
Test 1: 2.5, 5, 10, 20, 40, 50, 65, 80 µg/mL
Test 2: 5, 10, 15, 30, 60, 125, 250 µg/mL
+S9-Mix:
Test 1: 10, 20, 50, 125, 250, 375, 500 µg/mL
Test 2: 15, 30, 60, 125, 250, 375, 500 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The maximum solubility of MCPP-P 2-EHE in ethanol was found to be 500 mg/mL.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: 20-Methylcholanthrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 7.5 x 10E5 cells/mL in H5 medium
PRELIMINARY TOXICITY TEST
After seeding in flasks (80 cm2), cells were incubated at least 20 h prior exposure to test substance. Then, 2 mL of H0 medium or S9-Mix was added to one flask per treatment group followed by 120 µL of test substance (at 100 times the desired final concentration) or solvent. After 4 h, cells were harvested, washed and three 60 mm dishes per culture seeded with 200 cells in H5 medium. Plates incubated for 7 days after which time colonies growing in the plates were fixed, stained and counted. Cell survival was expressed as the plating efficiency relative to that of the solvent controls.

MAIN TEST
Cell suspensions were prepared as above except that duplicate cultures were used for each treatment. Following treatment with the test substance as described above, cells were again seeded (200 cells/plates) in 60 mm culture plates in H5 medium to determine cytotoxicity. In addition, 10E6 viable cells from each culture, estimated on the basis of toxicity data, were seeded into a 175 cm2 flask containing H5 medium and incubated for 7 days to allow for expression of the mutant phenotype. The cultures were sub-cultured once during the expression period on day 4 or 5 and, after a total of 7 days, were harvested by trypsination. For each treatment group, three 60 mm culture plates were each seeded with 200 cells in H5 medium to determine plating efficiency, and five 100 mm plates with 2x10E5 cells in selective medium, to determine the number of mutant colonies. Incubation was for further 7 days. At the end of incubation, plates were fixed, stained and counted. Two independent tests in the absence of S9-Mix and two independent tests in the presence of S9-Mix were carried out.
Rationale for test conditions:
derived from preliminary toxicity test (concentrations)
Evaluation criteria:
For an assay to be accepted as valid the following criteria should be met:
- The plating efficiency of the solvent control group should be 50% or greater
- For the solvent group, the mean mutant frequency per 10E6 viable cells should not exceed 15
- For the positive control group, the mean mutant frequency per 10E6 viable cells should exceed 100
At least one of the dose levels should show a cytotoxic effect such that the relative cell survival is 40% or less. This does not apply if the maximum advisable concentration (500 mg/mL) or the limit of solubility of the test substance has been reached without toxic effect.
Statistics:
The statistical significance of the data was analyzed by weighted analysis or variance following the methods described by Arlett et al (1989). The criteria for a positive response were:
- Demonstration of statistically significant increase in mutant frequency in cultures treated with test substance
- Evidence of dose relationship over at least two consecutive doses, in the increase in mutant frequency
- Demonstration of reproducibility of any increase in mutant frequency in treated cultures
- Response must lie outside historical control range of 15 mutant colonies over 10E6 surviving cells
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1-BH4
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1-BH4
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
A statistically significant increase in mutant frequency was observed in Test 1 at 125 µg/mL but considered as not biologically significant.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

In a preliminary toxicity test, cytotoxicity was observed in the absence of S9-Mix at 80 µg/mL (3 % cell survival compared to control) and at higher concentrations no cell survived (0%). In the presence of S9-Mix, lower cytotoxic effects were observed, but at 160 µg/mL and 320 µg/mL reductions of cell survival of about 50% compared to the solvent control were observed

Conclusions:
MCPP-P 2-EHE did not demonstrate mutagenic potential in this in vitro mammalian gene mutation assay.
Executive summary:

In a this GLP study according OECD TG 476 (Version 1984), the mutagenic potential of MCPP-P 2-EHE in an in vitro mammalian cell mutation assay using CHO cells was assessed. Four independent tests were carried out, two in the presence and two in the absence of exogenous activation in form of Aroclor 1254-induced rat liver S9-Mix. In a preliminary toxicity test, concentrations ranging from 5-320 µg/mL were used, both in the presence and absence of S9 -Mix. In the absence of S9 -Mix, cytotoxicity was observed at 80 µg/mL (3 % cell survival compared to control) and at higher concentrations no cell survived. In the presence of S9 -mix, lower cytotoxic effects were observed, but at 160 µg/mL and 320 µg/mL reductions of cell survival of about 50% compared to the solvent control were observed. Based on this, concentrations for the mutagenic test were chosen. In the absence of S9-Mix, treatment of cells with 2.5-80 µg/mL in Test 1 and 5-250 µg/mL in Test 2 resulted in mean cell survivals of 98-47% and 100-0% respectively. Cultures treated with 20, 40, 50, 65 and 80 µg/mL in Test 1 and 5, 10, 15, 30 and 60 µg/mL in Test 2 were assessed for viability and induced mutation. No significant increase in mutant frequency was observed in any of the tests in the absence of S9-Mix. The positive control EMS induced significant increase in mutant frequency in both tests. In the presence of S9-Mix, treatment of cells with 10-500 µg/mL in Test 1 and 15-500 µg/mL in Test 2 resulted in mean cell survivals of 166-70% and 142-38% respectively. Cultures treated with 50, 125, 250, 375 and 500 µg/mL in Test 1 and 60, 125, 250, 375 and 500 µg/mL in Test 2 were assessed for viability and induced mutation. A statistically significant increase in mutant frequency was observed in Test 1 at 125 µg/mL, which was not considered biologically significant. No increase was observed in Test 2. The positive control 20-Methylcholanthrene induced significantly increases in mutant rate and proved the sensitivity of the test system and the activity of the metabolic activation system.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

micronucleus test: 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:
June 2, 1992 - September 9, 1992
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:
1982
Qualifier:
according to guideline
Guideline:
other: EEC Annex V Committee (EEC 1984)
Qualifier:
according to guideline
Guideline:
other: JEPA/MITI joint directive (JEPA,MOHW,MITI 1987)
Qualifier:
according to guideline
Guideline:
other: Guideline of the JMOHW
Version / remarks:
JMOHW 1989
Qualifier:
according to guideline
Guideline:
other: US EPA TSCA
Version / remarks:
1987
GLP compliance:
yes
Type of assay:
other: micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- batch No.of test material: 5007
- sample No.:1505E
- Expiration date of the lot/batch: March 1993

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4°C in the dark
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River U.K. Limited, Margate,Kent, England
- Age at study initiation: approximately 35 days
- Weight at study initiation: 22-24 g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: plastic disposable cages, sexes separated
- Diet: ad libitum, pelleted Biosure LAD 1 rodent diet
- Water: ad libitum, tap water
- Acclimation period:11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%):
- Air changes (per hr): 30
- Photoperiod (hrs dark / hrs light): 12 h/ 12h

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: methylcellulose
- Amount of vehicle (if gavage or dermal): 1%
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Suspensions/emulsions of MCPP-P 2-EHE were prepared in aquous 1 % methylcellulose on the morning of the test.
Duration of treatment / exposure:
24 h, 48 h and 72 h
Frequency of treatment:
single
Dose / conc.:
320 mg/kg bw (total dose)
Remarks:
=16 mg/mL test item in suspension, preliminary test
Dose / conc.:
800 mg/kg bw (total dose)
Remarks:
=40 mg/mL test item in suspension, preliminary test
Dose / conc.:
1 600 mg/kg bw (total dose)
Remarks:
=80 mg/mL test item in suspension, preliminary test (Phase II)
Dose / conc.:
2 000 mg/kg bw (total dose)
Remarks:
=100 mg/mL test item in suspension, preliminary test
Dose / conc.:
2 500 mg/kg bw (total dose)
Remarks:
=125 mg/mL test item in suspension, preliminary test (Phase II)
Dose / conc.:
3 128 mg/kg bw (total dose)
Remarks:
=156.4 mg/mL test item in suspension, preliminary test (Phase II)
Dose / conc.:
5 000 mg/kg bw (total dose)
Remarks:
=250 mg/mL test item in suspension, preliminary test
Dose / conc.:
782 mg/kg bw (total dose)
Remarks:
=39.1 mg/mL test item in suspension, micronucleus test
Dose / conc.:
1 564 mg/kg bw (total dose)
Remarks:
=78.2 mg/mL test item in suspension, micronucleus test
Dose / conc.:
3 128 mg/kg bw (total dose)
Remarks:
=156.4 mg/mL test item in suspension, micronucleus test
No. of animals per sex per dose:
15 per sex and dose
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C
- Route of administration: oral, gavage
- Doses / concentrations: prepared as a solution in 0.9% saline at a concentration of 0.6 mg/mL (dose: 12 mg/kg bw)
Tissues and cell types examined:
both femurs dissected and bone marrow used for smears
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on resulted from preliminary toxicity test

SAMPLING TIMES: 5 males and 5 females from dose group and negative control sacrificed 24, 48 and 72 h after dosing; positive control group 24 h after dosing

DETAILS OF SLIDE PREPARATION: direct bone marrow smear was made onto slide containing a drop of calf serum, one smear from each femur, after air-drying smears were stained and fixed in methanol (>10 min). The smears were air-dried and stained for 10 min in 10% Giemsa (prepared by 1:9 dilution of Gurr´s improved R66 Giemsa with distilled water). Following rinsing in distilled water and differentiation in buffered distilled water (pH 6.8), the smears were air-dried and mounted with coverslips using DPX.

METHOD OF ANALYSIS: The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 1000 polychromatic erythrocytes per animal.

Evaluation criteria:
Micronuclei are identified by the following criteria:
(i) large enough to discern morphological characteristics
(ii) should possess a generally rounded shape with a clearly defined outline
(iii) should be deeply stained and similar in colour to the nuclei of other cells-not black
(iv) should lie in the same focal plane as the cell
(v) lack internal structure i.e. they are pyknotic
(vi) there should be no micronucleus-line debris in the area surrounding the cell
The proportion of polychromatic erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated normochromatic erythrocytes observed during this assessment was also kept as recommended by Schmid (Schmid 1976). A positive response is normally indicated by a substantial, dose-related and statistically significant increase of micronucleated polychromatic erythrocytes compared to the incidence for the concurrent vehicle control group.
Statistics:
Non- parametric statistical methods, based on rank, are chosen for analysis of results. For comparison of an individual treated group with a concurrent control group, Wilcoxon´s sum of ranks test was used. For multiple group comparisons Kruskal-Wallis´ version of this test was used. Jonckheere´s test for trend was used to analyze for dose-effect relationships.
Key result
Sex:
male/female
Genotoxicity:
negative
Remarks:
at 24 h and 72 h sampling time
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
Mitomycin C caused large, highly significant increases in the frequency of mirconucleated polychromatic erythrocytes
Key result
Sex:
male/female
Genotoxicity:
negative
Remarks:
at 48 h sampling time after re-examination of slides
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
Mitomycin C caused large, highly significant increases in the frequency of mirconucleated polychromatic erythrocytes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 320, 800, 2000, 5000 mg/kg bw (Phase I); 1600, 2000, 2500, 3128 mg/kg bw (Phase II)
- Clinical signs of toxicity in test animals:
phase I: after dosing piloerection was observed in all animals; at 2000 mg/kg animals showed in addition hunched posture and lethargy until approximatedy 24 h after dosing; at 5000 mg/kg bw mortality occured (3 animals out of 4 died) and animals showed increased respiratory rate, piloerection, hunched posture and lethargy
phase II: no mortality occured, moderate piloerection was observed in all animals directly after dosing, at 2500 and 3128 mg/kg bw animals showed lethargy, hunched posture, twitching, staggering gait, ptosis and increased respiratory rate
- Other: 3128 mg/kg bw was the maximum tolerated dose and chosen as highest dose in the micronulceus test

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei:
MCPP-p 2EHE did neither cause any substantial increases in micronucleated polychromatic erythrocyte counts (mnp) nor of micronucleated normochromatic erythrocytes (mnn) at any sampling time. The test item failed to cause any significant decreases in the proportion of polychromatic erythrocytes.

OTHER
- Chemical analysis by high performance liquid chromatography using ultraviolet detection proved the stability and homogeneity of MCPP-p 2EHE in the vehicle. In addition, it was confirmed that the dose formulations were accurately prepared and the mean result was within +4% of the nominal concentration.

Table 1 Summary of results - group totals/means for the entire experiment and results of statistical analysis

Sampling time

Treatment

Dose (mg/kg bw)

p/p + n (mean %)C

Incidence mnp (mean %)A

Incidence mnn (total %)

24 hours

Vehicle control

 

 

MCPP-P 2-EHE

 

 

Mitomycin C

-

 

782

1564

3128

 

12

52

 

51 ns

49 ns

49 ns

 

40 **

0.03

 

0.10 ns

0.07 ns

0.08 ns

 

4.66 **

0.00

 

0.04

0.00

0.00

 

0.09

48 hours

Vehicle control

 

 

MCPP-P 2-EHE

-

 

782

1564

3128

 

50

 

51 ns

51 ns

43 ns

0.03

 

0.09 ns

0.10 ns

 0.17 * (j)

0.04

 

0.00

0.04

0.07

48 hours

re-examination

 

 

 

24 hours

re-examination

Vehicle control

 

 

MCPP-P 2-EHE

 

  

 Mitomycin CB

-

 

782

1564

3128

 

12

not examined

0.01

 

0.13 ns

0.14 ns

   0.13 ns (j)

 

4.38

not examined

72 hours

Vehicle control

 

 

MCPP-P 2-EHE

-

 

782

1564

3128

 

54

 

52 ns

54 ns

55 ns

0.07

 

0.06 ns

0.04 ns

0.15 ns

0.06

 

0.00

0.02

0.04

p/(p+n) % Proportion of polychromatic (immature) erythrocytes

mnp       Number of micronucleated cells observed per 100 polychromatic erythrocytes

mnn      Number of mirconucleated cells observed per 100 normochromatic erythrocytes (calculated total = number of mnn observed in the group / number of normochromatic erythrocytes examined X 100%)

A            Results of statistical analysis using Kruskal-Wallis´, Jonckheere´s and Wilcoxon´s test as appropriate:

               ns (P > 0.01), * (P< 0.01), ** P< (0.001) one-sided probabilities; j = result using Jonckheere´s test for trend only

B            Positive control slides from the 24 hour time point were combined with slides from the 48 hour time point for examination by second slide reader

C            Small apparent errors of ± 1% are due to rounding of individual values for presentation in tables

Conclusions:
MCPP-P 2-EHE did not show any evidence of chromosome-damaging activity in the in vivo micronucleus assay.
Executive summary:

An in vivo micronucleus test according to OECD TG 474 (Version 1982) and GLP was employed in male and female CD-1 mice to investigate a possible clastogenic or aneugenic effect in bone-marrow erythroblasts. Mice were administered by gavage single dosages of 782, 1564 or 3128 mg/kg bw. The negative control group received the vehicle (aqueous 1% methylcellulose) and the positive control group was treated with mitomycin C at 12 mg/kg bw. Bone marrow smears from five males and five females were obtained at three sampling times after dosing (24 h, 48 h and 72 h). One smear of each animal was examined for the presence of micronuclei in 1000 polychromatic erythrocytes. In addition, the proportion of polychromatic immature erythrocytes was assessed and the incidence of micronucleated normochromatic erythrocytes was recorded. Three male and four females died after treatment with the highest dose of MCPP-P 2-EHE in the micronucleus test, with non of these animals showing signs of misdosing.

At 24 h and 72 h, mice treated with MCPP-P 2-EHE did not show any significant increase in the frequency of micronucleated polychromatic erythrocytes. A significant increase was observed after 48 h. This increase was not confirmed in the re-examination of the slides and since all the original individual and mean values fell within the laboratory historical negative control range, it was concluded that the original increase was not treatment-related. The positive control mitomycin C induced a significant increase of micronucleated polychromatic erythrocytes and a decrease in the proportion of polychromatic erythrocytes. The negative control did not induce an increased frequency of micronucleated polychromatic erythrocytes. Therefore, MCPP-P 2-EHE has not shown any evidence of chromosome-damaging activity in the in vivo micronucleus assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro

A study similar to OECD TG 471 under GLP considerations was performed to assess the in vitro mutagenic potential of MCPP-P 2-EHE using histidine-dependent auxotrophic mutants of S.typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100). Strains were exposed to the test material diluted in ethanol. In a preliminary dose range finding study no toxicity was observed up to the highest concentration of 5000 µg/plate. Five concentrations (50, 150, 500, 1500 and 5000 µg/plate) were used in the plate incorporation mutation assay. Bacteria were incubated with the test substance or solvent control ethanol in the presence or absence of rat liver S9-Mix for 3 days. No evidence of mutagenic activity was seen at any dose level of MCPP-P 2-EHE. The concurrent positive control compounds demonstrated the sensitivity of the assay and metabolizing activity of the liver S9 –Mix (MCPP-p Task Force JEL 52/921059; 1993).

In a GLP study according OECD TG 473 (Version 1983), the ability of MCPP-P 2-EHE to induce chromosomal aberrations in human lymphocytes was assessed in vitro. In a preliminary toxicity test, of MCPP-P 2-EHE was found to be toxic and caused cell cycle delay in the absence of S9-Mix. In the presence of S9-Mix it was non-toxic and no cell cycle delay was observed. Based on these, suitable harvest times for the main study were chosen: 13 h and 21 h in the absence of S9-Mix and 16 h for the presence of S9 -Mix. Precipitation in the medium at high dose level occured, therefore 320 µg/mL was chosen to be the highest concentration used in the main test. In the absence of S9-Mix 80, 60 and 40 µg/mL (21 h harvest) or 40, 20 and 10 µg/mL (13 h harvest) were utilized. In the presence of S9-Mix, 320, 160, 80 and 40 µg/mL was chosen. The positive controls induced significant clastogenic effects and demonstrated the sensitivity of the test system and the activity of the S9 -Mix used. In both, the presence and the absence of S9 -Mix, no biological significant increase in the proportion of aberrant cells were seen at any dose level when compared with the solvent control (MCPP-p Task Force JEL 63/921577; 1993).

In a further GLP study according OECD TG 476 (Version 1984), the mutagenic potential of MCPP-P 2-EHE in an in vitro mammalian cell mutation assay using CHO cells was assessed. Four independent tests were carried out, two in the presence and two in the absence of exogenous activation in form of Aroclor 1254-induced rat liver S9-Mix. In a preliminary toxicity test, concentrations ranging from 5-320 µg/mL were used, both in the presence and absence of S9 -Mix. In the absence of S9-Mix, cytotoxicity was observed at 80 µg/mL (3 % cell survival compared to control) and at higher concentrations no cell survived. In the presence of S9 -Mix, lower cytotoxic effects were observed, but at 160 µg/mL and 320 µg/mL reductions of cell survival of about 50% compared to the solvent control were observed. Based on this, concentrations for the mutagenic test were chosen. In the absence of S9-Mix, treatment of cells with 2.5-80 µg/mL in Test 1 and 5-250 µg/mL in Test 2 resulted in mean cell survivals of 98-47% and 100-0% respectively. Cultures treated with 20, 40, 50, 65 and 80 µg/mL in Test 1 and 5, 10, 15, 30 and 60 µg/mL in Test 2 were assessed for viability and induced mutation. No significant increase in mutant frequency was observed in any of the tests in the absence of S9-Mix. The positive control EMS induced significant increase in mutant frequency in both tests. In the presence of S9-Mix, treatment of cells with 10-500 µg/mL in Test 1 and 15-500 µg/mL in Test 2 resulted in mean cell survivals of 166-70% and 142-38% respectively. Cultures treated with 50, 125, 250, 375 and 500 µg/mL in Test 1 and 60, 125, 250, 375 and 500 µg/mL in Test 2 were assessed for viability and induced mutation. A statistically significant increase in mutant frequency was observed in Test 1 at 125 µg/mL, which was not considered biologically significant. No increase was observed in Test 2. The positive control 20-Methylcholanthrene induced significantly increases in mutant rate and proved the sensitivity of the test system and the activity of the metabolic activation system (MCPP-p Task Force JEL 61/921471; 1993).

in vivo

Furthermore, an in vivo micronucleus test according to OECD TG 474 (Version 1982) and GLP was employed in male and female CD-1 mice to investigate a possible clastogenic or aneugenic effect in bone-marrow erythroblasts. Mice were administered by gavage single dosages of 782, 1564 or 3128 mg/kg bw. The negative control group received the vehicle (aqueous 1% methylcellulose) and the positive control group was treated with mitomycin C at 12 mg/kg bw. Bone marrow smears from five males and five females were obtained at three sampling times after dosing (24 h, 48 h and 72 h). One smear of each animal was examined for the presence of micronuclei in 1000 polychromatic erythrocytes. In addition, the proportion of polychromatic immature erythrocytes was assessed and the incidence of micronucleated normochromatic erythrocytes was recorded. Three male and four females died after treatment with the highest dose of MCPP-P 2-EHE in the micronucleus test, with non of these animals showing signs of misdosing.

At 24 h and 72 h, mice treated with MCPP-P 2-EHE did not show any significant increase in the frequency of micronucleated polychromatic erythrocytes. A significant increase was observed after 48 h. This increase was not confirmed in the re-examination of the slides and since all the original individual and mean values fell within the laboratory historical negative control range, it was concluded that the original increase was not treatment-related. The positive control mitomycin C induced a significant increase of micronucleated polychromatic erythrocytes and a decrease in the proportion of polychromatic erythrocytes. The negative control did not induce an increased frequency of micronucleated polychromatic erythrocytes. Therefore, MCPP-P 2-EHE has not shown any evidence of chromosome-damaging activity in the in vivo micronucleus assay (MCPP-p Task Force JEL 55/920980).

In conclusion, there was no indication of a mutagenic effect of MCPP-p 2EHE in any of the genetic toxicity tests performed (in vitro bacterial and mammalian mutation assay, in vitro mammalian chromosome aberration, and in vivo micronucleus test).


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. The substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.