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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 June to 3 July 1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Non-GLP study, evaluation criteria, historical control data not reported
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
evaluation criteria, historical control data not reported
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Remarks:
pre-GLP but considered to be equivalent to GLP standard
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan gene for Salmonella typhimurium and Escherichia coli, respectively.
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10 % S9 mix; S9 from liver of SD rats induced with Aroclor 1254
Test concentrations with justification for top dose:
- Test: 10, 33.3, 100.0, 333.3, 1000 and 3300 µg/plate in S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and in E.coli WP2 uvrA-, with and without S9-mix
- Retest (at higher dose levels): 1000, 2000, 4000, 6000 and 8000 µg/plate in S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, with and without S9-mix
- Test to deduce if exposure to air, light and humidity affect the mutagenic activity of test item: 100, 500, 1000, 2000, 4000 and 6000 µg/plate in S. typhimurium strain TA 100, with and without S9-mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: The test compound was dissolved and diluted in Dimethylsulphoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
with and without S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: Two days at 37 °C

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of microcolony growth.

OTHERS:
- After two days incubation at 37 °C the colonies counted using a New Brunswick Inc. (New Brunswick, N.J.) Biotran II automated counter set for maximum sensitivity (colonies of 0.1 mm or more in diameter counted). The plates were also examined for precipitates and, microscopically, for microcolony growth.
Evaluation criteria:
No data
Statistics:
No data
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Cytotoxicity: no cytotoxicity.

Results of mutagenicity:
The substance was mutagenic in strain TA 100 both with activation at a lowest effective dose of 3300 µg/plate and without activation at a lowest effective dose of 1000 µg/plate. The compound was not mutagenic in E. coli WP2 uvrA. Exposure to air, light and humidity did not affect its mutagenic activity in strain TA 100

None

Conclusions:
Under the test condition, the test item is mutagenic in S. typhimurium TA 100 with and without metabolic activation.
Executive summary:

In a reverse gene mutation assay, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and Escherichia coliWP2 uvrA- were exposed to the test item at 10, 33.3, 100.0, 333.3, 1000 and 3300 µg/plate. The compound was also tested at 1000, 2000, 4000, 6000 and 8000 µg/plate to obtain a mutagenic dose response and at 100, 500, 1000, 2000, 4000 and 6000 µg/plate to assess the effect of air, light and humidity.

 

The substance was mutagenic in S. typhimurium TA 100 at a lowest effective dose of 1000 µg/plate in the absence of the activation system and 3300 µg/plate in the presence of the activation system. Even though the compound was known to decompose in the presence of air and humidity, exposure of the compound to these effects did not alter its mutagenic activity. The compound was not mutagenic in E. coli WP2 uVrA-.

 

The vehicle control plates gave counts of revertant colonies within the normal range. Positive control induced marked increases in the frequency of revertant colonies indicating the validity of the study. 

 

Under the test condition, the test item is mutagenic in S. typhimurium TA 100 with and without metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 July to 28 August 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study performed equivalent or similar to OECD test guideline No. 476 with deviations: purity of test item, mycoplasma contamination details, survival (relative cloning efficiencies), historical negative (solvent/vehicle) and positive control data not reported.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
purity of test item, mycoplasma contamination details, survival (relative cloning efficiencies), historical negative (solvent/vehicle) and positive control data not reported
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Remarks:
pre-GLP but considered to be of a standard equivalent to GLP
Type of assay:
other: in vitro mammalian cell gene mutation assay
Target gene:
tk locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
heterozygous at the TK locus
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: D. Clive, Research Triangle Park, North Carolina, USA.

MEDIA USED
- Type and identity of media:
The basic medium (F0P) comprised of Fischer's medium supplemented with penicillin (100 units/mL), streptomycin (100 µg/mL), sodium bicarbonate (1.125 g/L), sodium pyruvate (2 mM), pluronic acid (0.5% v/v) and glutamine (2 mM).
The addition of horse serum (10% v/v) to F0P gave F10P which was the medium used for all cell growth and culture maintenance in liquid suspension.
The addition of donor horse serum (20% v/v) and sodium pyruvate to 4 mM gave cloning medium (CM) , the medium required for colony formation during experiments.
- Properly maintained: Yes
During routine culture maintenance, cells were grown in Nunc tissue culture flasks at 37 °C and in an atmosphere of 5% CO2 : 95% air (v/v). Cell density was calculated daily using a haemocytometer and cultures diluted with F10P to a concentration of 3 x 10^5 cells/mL.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10% S9 fraction; S9 fraction was obtained from the liver homogenates of male rats induced with Aroclor 1254
Test concentrations with justification for top dose:
Toxicity test: 47, 94, 187.5, 375 and 750 µg/mL
Mutation test: 111, 167, 250, 375 and 750 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-acetamidofluorene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; Fischer's medium supplemented with penicillin (100 units/mL), streptomycin (100 µg/mL), sodium bicarbonate (1.125 g/L), sodium pyruvate (2 mM), pluronic acid (0.5% v/v) and glutamine (2 mM).
- Cell density at seeding:
Toxicity test: 10 mL samples of mouse lymphoma culture containing 3 x 10^6 cells were exposed to test item.
Mutation test: 3 x 10^6 exponentially growing TK mouse lymphoma cells were dispensed to sterile plastic universal bottles, and harvested by centrifuging at 1000 rpm for 5 min in an MSE Chilspin centrifuge. The supernatant fluid volume was reduced to 5 mL and a further 5 mL of F0P were added to make the total volume 10 mL.

DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 7-10 days
- Selection time (if incubation with a selection agent): 7-10 days
- Incubation: 5% CO2 : 95% air (v/v) at 37 °C

SELECTION AGENT (mutation assays): Trifluorothymidine

NUMBER OF REPLICATIONS:
Single culture for test item, vehicle and positive controls

NUMBER OF CELLS EVALUATED:
No. of TFT colonies / 10^5 survivors were calculated

DETERMINATION OF CYTOTOXICITY
- Cell density was obtained by hand counting with a haemocytometer, each day for the next 3 days until the toxic effects of the chemicals could be estimated.
- Survival assay: Survival assay was carried out immediately after dosage of cells with test item. Colonies were counted by hand 7-10 days later.
Evaluation criteria:
The criterion used for a significant positive effect in this test was a doubling of the trifluorothymidine induced mutant frequency over the solvent treated control.
Statistics:
No data
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TOXICITY STUDY:
- The LC50 of test item was estimated to be 65 µg/mL. This value was calculated from the data in Table 7.6.1/1.

MUTATION STUDY:
- In the absence of S-9 mix, the mutation frequencies induced by all 5 concentrations of test item were below or just at background level.
- In the presence of S-9, cells treated with the top 3 concentrations of test item were below background and one other dose was slightly above background. Only one dose gave an induced mutant frequency higher than background, but this was not as high as twice background level.

Table 7.6.1/1:Cytotoxicity Test

Test item (µg/mL)

Haemocytometer Counts Total Visible Cells x 106

Day 1

Day 2

Day 3

750

1.1

1.7

2.3

375

1.2

2.4

3.2

187.5

1.9

3.0

3.7

94

2.3

3.5

4.5

47

3.3

1.7

9.0

DMSO

2.3

6.8

10.5

EMS 400

1.8

3.2

3.6

EMS 200

1.8

3.5

6.0

 

Table 7.6.1/2: Mutation test - plate counts

Test item (µg/mL)

Day 0

Day 3

TFT+ colonies / 105 survivors

Colonies/dish

Average

Survival % control

Survival

TFT+

Colonies/dish

Average

Colonies/dish

Average

750

28, 30, 40

33

23

53, 49, 42

48

46, 72, 78, 52, 60, 40

58

48

375

inf, inf, inf

-

-

53, 71, inf

62

106, 98, 142, 56, 66, 86

92

59

250

57, 66, 38

54

38

50, 62, 62

58

106, 106, 122, 70, 64, 66

89

61

167

82, 68, 80

77

54

66, 101, inf

84

88, 124, 144, 104, 116, 80

109

52

111

58, 80, 80

73

51

65, 68, 54

62

62, 84, 96, 54, 30, 56

64

41

DMSO

140, 138, 148

142

100

41, 60, inf

51

82, 84, 70, 76, 68, 76

76

60

EMS 400

100, 68, 34

67

47

29, 36, 28

31

118, 94, 88, 70, 104, 106

97

125

EMS 200

80, 102, 64

82

58

50, 56, 35

47

72, 92, 100, inf, 86, 84

87

74

Inf: infected

Conclusions:
Under the test conditions, test item is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells, in the presence and absence of metabolic activation.
Executive summary:

In an in vitro mammalian cell gene mutation test performed similarly to OECD Guideline 476, L5178Y tk mouse lymphoma cells were exposed to test item for 3 h at the following concentrations: 111, 167, 250, 375 and 750 µg/mL. 

Vehicle and positive control groups were also included in each mutagenicity test. Metabolic activation system used in this test was 10 % S9 mix. S9 fraction was prepared from liver homogenates of rats treated with Aroclor 1254. Based on the results of toxicity test (47, 94, 187.5, 375 and 750 µg/mL), the dose levels were selected for main study. 

 

In the absence of S-9 mix, the mutation frequencies induced by all 5 concentrations of test item were below or just at background level. In the presence of S-9, cells treated with the top 3 concentrations of test item were below background and one other dose was slightly above background. Only one dose gave an induced mutant frequency higher than background, but this was not as high as twice background level.

 

Clear increases in mutation were induced by the positive control chemicals ethylmethane sulphonate (without S-9) and 2-acetamidofluorene (with S-9). The study was therefore accepted as valid.

 

Under the test conditions, test item is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells, in the presence and absence of metabolic activation.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Qualifier:
no guideline available
Principles of method if other than guideline:
Test item was previously reported to yield positive results in the GADD45- Gaussia Luciferase 'Blue Screen HC' (BSHC) reporter gene assay and in the Ames test. As a follow-up, genotoxic potential of the test item was evaluated in the Turkey Egg Genotoxicity Assay (TEGA) using single cell gel (comet) and 32P-nucleotide postlabeling (NPL) assays for DNA strand breaks and adduct formation, respectively.
GLP compliance:
no
Type of assay:
other: Turkey Egg Genotoxicity Assay (TEGA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Firmenich SA / 1000903286
Target gene:
Not applicable
Species / strain / cell type:
other: Fertilized broad-breasted white turkey (Meleagris gallopavo) eggs
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
100 and 120 mg per egg (for 3 daily injections)

Justification: The tested doses were selected based on the dose range findings from the previous experiments. Thus, due to the previous negative outcomes in the comet assay (initial TEGA) for total doses of 7.5,15, 30, 60 and 120 mg/egg, a pilot experiment was conducted with total doses of 100, 120 and 150 mg/egg in order to confirm that previously tested doses were not too low to elicit a response. The total dose of 150 mg/egg caused mortality of 73%, and this group was not evaluated in the pilot and confirmatory studies.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Deionized water
- Test item was dissolved in deionized water to obtain serial dilutions of 33.3 to 40 mg per 200 µL resulting in total doses of 100 and 120 mg per egg (for 3 daily injections).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionized water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: hydrogen peroxide
Details on test system and experimental conditions:
TEST SYSTEM
- Fertilized broad-breasted white turkey (Meleagris gallopavo) eggs of undetermined sex were purchased from Aviagen Turkeys, Inc. (Lewisburg, WV, USA). Eggs were shipped overnight by United Parcel Service. Upon arrival, all eggs were numbered on the eggshell with pencil, and weighed.
- Prior to incubation, eggs were randomly divided into control and dosed groups and stored at room temperature for no more than 3 days. The day on which incubation commenced was designated as Day 0. Eggs were incubated in 2GIF Styrofoam incubator (Murray McMurray Hatchery, Webster City, lA, USA) at 37 ± 0.5 °C and 60 ± 5 % humidity, and were manually rotated at least once a day. Viability was assessed on day 19 by transillumination with a strong flashlight (Model L6, Lenser, Solingen, Germany). Eggs that did not developed were recorded and eliminated. Control and dosed eggs were separated to avoid any possible airborne cross contamination.

METHOD OF ADMINISTRATION:
- Test item and vehicle was administered in total volume of 0.6 mL/egg via 3 daily injections into the air sac on days 22 through 24 of incubation. The injection site for dosing of usable eggs was marked with a 1-2 cm cross above the blunt end of the egg (over the air sac) with a pencil, sterilized using alcohol swabs, and pierced with sterilized 10 cm long FST scissors.

DURATION
- Exposure duration: 3 h

TERMINATION
- At termination, three hours after the last injection, egg shells were opened, fetuses were removed and decapitated. Fetal livers were harvested and processed immediately for the enhanced, enzyme-linked, comet assay with two enzymes: formamidopyrimidine DNA glycosylase (FPG) and human 8-hydroxyguanine-DNA glycosylase (hOGG1). Fetal body and liver weights as well as viability were recorded. Groups with viability lower than 50% were excluded from analysis.

ENZYME-LINKED COMET ASSAY
- The enhanced comet assay of liver tissue was performed to assess general and oxidative DNA strand breaks according to OECD guidelines (OECD, 2014), as previously reported by Tice and Strauss (1995) and Williams et al. (2014).
- After electrophoresis, slides were covered with neutralization buffer (0.4 M Tris-HCI, pH 7.5) for 5 min twice. Slides were dried with ethanol and 50 µL of ethidium bromide (10 µg/ml) was added onto each slide. Slides were then covered with a coverslip and kept for 5 min in the dark. DNA migration was analyzed by fluorescence microscopy using a Nikon OPTIPHOT microscope. The percentage of DNA-in-tail was determined using the Comet Score software v 1.5 (TriTek Corp, Sumerduck VA), counting >150 cells.
- Median percentage of DNA-in-tail for each slide was determined and the mean of the median values was calculated for each sample. Group mean was calculated as an average of individual means. The obtained mean comet value from each of the dose groups was then compared to the corresponding negative (vehicle) control group, and a ratio value was obtained, which reflected the fold increase over the control value.
Evaluation criteria:
The evaluation criteria for determining positive results include the following: a statistically significant increase in the percentage of DNA-in-tail in dose group(s) compared to corresponding vehicle control group; a dose-related increase in DNA damage determined by linear regression analysis; the percentage of DNA-in-tail in dose group(s) should be outside of the historical vehicle control range established in the laboratory (for deionized water a 95% CI is 10.92-5.09); the results should be reproducible, in case a repeat assay is conducted. In cases where only some of the criteria are met, the results are judged as equivocal. In cases where none of the criteria are met, the results are judged as negative (vander Leede et al., 2014).
Statistics:
Results are presented as mean ± SD. Statistical analyses were performed using SigmaStat software version 3.11.0 (Systat Software Inc., Chicago, IL), with the most appropriate statistical analysis of the data sets, which included the one-way analysis of variance (ANOVA) with the pairwise multiple comparison being made by the Bonferroni method. Linear regression analysis was used to determine dose-related trends. p-values <0.05 were considered significant.
Key result
Species / strain:
other: Fertilized broad-breasted white turkey (Meleagris gallopavo) eggs
Metabolic activation:
not applicable
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PILOT EXPERIMENT:
In the pilot experiments, 3 dose levels i.e., 100, 120 and 150 mg/egg were tested. Two dose levels i.e., 100, 120 mg/egg was selected for the pilot analysis and the confirmatory study based on viability and limitations in the number of samples which can be analyzed simultaneously. Test item at the total doses of 100 and 120 mg/egg yielded positive response in the enhanced comet assay with FPG digestion, but not hOGG1 enrichment.

CONFIRMATORY EXPERIMENT
Viability of control eggs dosed with deionized water was 100%. A dose-related decrease in viability of eggs occurred reaching 83% at the high dose of test item.

ENZYME-LINKED COMET ASSAY
- Exposure to test item at both dose levels (100 and 120 mg/egg) resulted in no DNA damage in the undigested liver samples, confirming previous negative findings in the conventional comet assay. In contrast, there was a dose-dependent (R2=0.7), significant 2.0 fold increase in the percentage of DNA-in-tail in the turkey fetal livers digested with FPG enzyme. However, no positive outcome was detected in the liver samples digested with hOGG1 enzyme.
- Significantly higher levels of DNA strand breaks were detected in the groups incubated with H2O2 (positive control) compared to corresponding vehicle control groups, with highest (4.4 fold) increase detected in the samples with FPG digestion, followed by hOGG1 digestion (2.7 fold increase) and undigested control (1.6 fold increase).
- No statistically significant difference was detected between undigested and digested samples, with either FPG or hOGG1 enzymes, in the vehicle control group.

Table 7.6.1/1: Summary of the results in confirmatory study

Items

Dose (mg/egg)

Viability (%)

Undigested

hOGG1

FPG

Tail DNA (%)

Results

Tail DNA (%)

Results

Tail DNA (%)

Results

Vehicle (Deionized water)

0

100

10.03 ± 2.07

-

8.35 ± 1.99

-

7.31 ± 1.80

-

Test item

100

100

12.18 ± 2.88

-

12.69 ± 3.14

-

14.79 ± 2.55*

+

120

83

12.61 ± 0.24

-

10.19 ± 0.89

-

14.61 ± 3.88*

+

Positive

control (H2O2)

 

 

16.44 ± 2.49*

+

22.72 ± 2.72*

+

32.14 ± 4.30*

+

Values are expressed as the mean ± SD (n=3/per group)

- negative; + positive

* denotes a significant (p < 0.05) difference from vehicle control group

Significant linear trend in dose-response was observed in the groups exposed to test item after digestion with FPG enzyme (R2= 0.67, p=0.0073)

Conclusions:
Based on the results of the study, test item can produce DNA strand breakage through indirect mechanisms that involve oxidative DNA damage, which can explain the positive findings in the bacterial mutation assay.
Executive summary:

The genotoxic potential of the test item was evaluated in the Turkey Egg Genotoxicity Assay (TEGA) using single cell gel (comet) and 32P-nucleotide postlabeling (NPL) assays for DNA strand breaks and adduct formation, respectively.

In this first experiment, the test item was negative in both NPL and comet assays at the cumulative dose of up to 120 mg/egg (maximal dose which was not lethal to ≥50% of the cells). Based on these findings, the hypothesis was made that positive results in other assays could be caused by oxidative stress, rather than direct genotoxicity. Thus, the primary objective of the current study was to evaluate the ability of test item to cause oxidative DNA damage, using enhanced comet assay with oxidative DNA damage repair enzyme digestion.

 

In the present experiment, fertilized medium white turkey eggs were administered 3 consecutive daily injections with test item (100 and 120 mg/egg) on day 22 to 24 of incubation. Three hours after the last injection, fetal livers were harvested for enhanced, enzyme-linked, comet assay with two enzymes: formamidopyrimidine DNA glycosylase (FPG), which detects general oxidative DNA damage, and human 8-hydroxyguanine DNA glycosylase (hOGG1), an 8-oxo-deoxyguanine (8-oxodG) excision enzyme which specifically excises 8-oxodG damage.

 

In the groups dosed with test item, no significant increase in DNA damage was observed in undigested samples, similar to previous results from the conventional comet assay. Dosing of turkey eggs with test item at both dose levels resulted in positive outcomes in the enzyme-linked comet assay only with FPG enzyme enhancement, but not with hOGG1 enrichment, indicating possible oxidative DNA damage, but not 8-oxodG adduct formation. Liver samples from the positive control group incubated with hydrogen peroxide (H2O2) were strongly positive in the assay, both undigested and enhanced with enzymes.

 

Based on the results of the study, the test item can produce DNA strand breakage through indirect mechanisms that involve oxidative DNA damage.

Genetic toxicity in vivo

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 March to 26 June 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed equivalent or similar to OECD test guideline No. 474 without deviation.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 22 January 1996/ signed on 27 February 1996)
Type of assay:
other: micronucleus assay
Species:
mouse
Strain:
CD-1
Details on species / strain selection:
The test system was chosen because the mouse has been shown to be a suitable model for this type of study and is recommended in the test method.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent.
- Age at study initiation: ca.5-7 weeks
- Weight at study initiation: Males: 22-30 g; females: 20-26 g
- Assigned to test groups randomly: Yes; after a minimum acclimatisation period of five days the animals were selected at random.
- Housing: Animals were housed in groups of five by sex in solid-floor polypropylene cages furnished with woodflakes
- Diet: Food (Rat and Mouse Expanded Diet No.1, Special Diets Services Limited, Witham, Essex, UK), ad libitum
- Water: Mains drinking water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-23 °C
- Humidity: 50-51 %
- Air changes: Rate of air exchange was ca.15 changes per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 20 March to 26 June 1996
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Distilled water
- Concentration of test material in vehicle: 18.75, 37.5, 75, 150 and 175 mg/mL
- Amount of vehicle (gavage): 10 mL/kg bw
- Lot/batch no.: Steripak, 501041
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Test item formulation: For the purpose of this study the test item was freshly prepared as required as a solution at the appropriate concentrations in distilled water. Initially the test item was formulated as a suspension in arachis oil. However, it was apparent that poor homogeneity of the formulations was giving inconsistent results in the initial range-finding investigation. Following discussion with the sponsor the vehicle was changed to distilled water for the final range-finding study and main study.

DOSE VOLUME: 10 mL/kg bw
Duration of treatment / exposure:
Single treatment (One day)
Frequency of treatment:
Aall animals were dosed once only at the appropriate dose level by gavage.
Post exposure period:
24 and 48 h after administration of test item
Dose / conc.:
187.5 mg/kg bw/day (nominal)
Dose / conc.:
375 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
Dose / conc.:
1 500 mg/kg bw/day (nominal)
Dose / conc.:
1 750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: Cyclophosphamide
- Route of administration: Oral (gavage)
- Concentration: 5 mg/mL
- Dose volume: 10 mL/kg bw
- Dose: 50 mg/kg bw
- For the purpose of this study the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water.
Tissues and cell types examined:
- The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored.
- Number of normochromatic erythrocytes (NCE) associated with 1000 erythrocytes were counted; the ratio of polychromatic to normochromatic erythrocytes was calculated as a measure of bone marrow toxicity.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Range-finding Toxicity Study:
- A range-finding toxicity study was performed to determine a suitable dose level for the micronucleus study. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of cytotoxicity up to a maximum recommended dose of 2000 mg/kg bw.
- Dose levels for range-finding toxicity study: 1500, 1750 and 2000 mg/kg bw
- Animals were observed one hour after dosing and subsequently once daily for up to two days. Any deaths and evidence of overt toxicity were recorded at each observation. Animals were killed 24 or 48 hours after dosing, femurs were removed and slides prepared for cytotoxicity evaluation.
- Based on the results of the range-finding study the vehicle to be used in the main study was distilled water which gave a homogenous solution of the test item. Though no premature deaths were observed at 2000 mg/kg bw using distilled water as the vehicle, it was decided following discussion with the sponsor that the toxic effects at 2000 mg/kg bw were excessive and to limit the maximum dose level to 1750 mg/kg bw. The other dose levels to be used in the main study were 187.5, 375, 750 and 1500 mg/kg bw.

TREATMENT AND SAMPLING TIMES
- All animals were dosed once only at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of dosing.
- One group of mice from each dose level was killed by cervical dislocation 24 h following treatment and a second group from each dose level at 48 h. The vehicle controls were killed 24 or 48 h following dosing and positive control group animals were killed 24 h following dosing.

DETAILS OF SLIDE PREPARATION:
- Immediately following sacrifice (i.e., 24 or 48 h following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and resuspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grunwald/Giemsa.

METHOD OF ANALYSIS:
- Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification.
- The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining.
- In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes were counted; these cells were also scored for incidence of micronuclei.
- The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values.

OTHER:
- All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to sacrifice.
Evaluation criteria:
- A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the corresponding vehicle control group.
- A positive mutagenic response was demonstrated when a statistically significant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48 h kill times when compared to their corresponding control group.
- If these criteria were not demonstrated, then the test item was considered to be non-genotoxic under the conditions of the test.
- A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.
Statistics:
All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.
Key result
Sex:
male/female
Genotoxicity:
positive
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1500, 1750 and 2000 mg/kg bw
- Solubility: Test item was dissolved in distilled water.
- Clinical signs of toxicity in test animals: There were no premature deaths in animals dosed with the test item formulated in distilled water. Clinical signs were observed in the 48 h dose group animals as follows: hunched posture, lethargy, ataxia, splayed gait, decreased respiratory rate and ptosis. Animals used to obtain 24 h PCE/NCE ratios exhibited the following clinical signs: hunched posture, lethargy, ataxia, decreased respiratory rate, laboured respiration, splayed gait and ptosis.
- Harvest times: 24 and 48 h
- Evidence of cytotoxicity in tissue analyzed: A summary of the mean PCE/NCE ratio values for the 24 and 48 h dose groups were presented in Table 7.6.2/1.
- Other: As in the arachis oil formulated test item range-finding study some animals were observed to have an elevated frequency of micronucleated polychromatic erythrocytes.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test item at ≥ 750 mg/kg bw in the 24 h groups and at ≥1500 mg/kg bw in the 48 h groups, these were as follows: lethargy, ataxia, ptosis and hunched posture.
- Induction of micronuclei (for Micronucleus assay): There were dose-related statistically significant increases in the frequency of micronucleated PCEs in the 24 h test item dose groups at ≥ 1500 mg/kg bw when compared to their concurrent vehicle control group. In both the 1500 and 1750 mg/kg bw 24 h groups there were several animals which had micronucleated polychromatic erythrocyte values similar to those normally seen in vehicle control animals. However, the majority of animals had values far greater than those seen in control animals. A possible hypothesis for the low values is that inhibition of erythropoiesis due to the toxicity of the test material to the bone marrow cells, delayed erythrocyte production and any accompanying micronucleated erythrocytes. This theory is supported by the cytotoxic response observed in the 24 h test item groups. There was no evidence of a statistically significant increase in the frequency of micronucleated PCEs in the 48 h test item dose groups. There were statistically significant decreases in the PCE/NCE ratio in the 24 h test item groups when compared to their concurrent vehicle control group. This would indicate that a cytotoxic response in the target tissue, bone marrow, had been achieved.
- Ratio of PCE/NCE (for Micronucleus assay): Table 7.6.2/2

Table 7.6.2/1: Range finding study

Dose group (mg/kg bw)

48 h PCE/NCE ratio

24 h PCE/NCE ratio

Mean

SD

Mean

SD

1500

1.53

0.42

1.40

0.22

1750

1.59

0.69

1.37

0.40

2000

1.85

0.69

1.68

1.20

 

Table 7.6.2/2: Micronucleus study

Dose group (mg/kg bw)

Sampling

Number of PCE with micronuclei per 2000 PCE

PCE/NCE ratio

Group Mean

SD

Group Mean

SD

Vehicle control

48 h

2.4

2.2

1.29

0.34

Vehicle control

24 h

2.6

1.7

1.44

0.51

Positive control

24 h

50.1***

28.7

1.22

0.50

Test item 1750 mg/kg bw

48 h

2.7

2.8

1.09

0.37

Test item 1500 mg/kg bw

2.8

1.9

1.47

0.43

Test item 750 mg/kg bw

3.5

4.8

1.62

0.92

Test item 375 mg/kg bw

3.2

1.3

1.36

0.48

Test item 187.5 mg/kg bw

3.4

3.0

1.46

0.42

Test item 1750 mg/kg bw

24 h

18.4**

15.4

0.79**

0.34

Test item 1500 mg/kg bw

12.2***

8.9

0.92*

0.30

Test item 750 mg/kg bw

3.0

3.0

0.85**

0.27

Test item 375 mg/kg bw

4.0

3.6

1.36

0.58

Test item 187.5 mg/kg bw

2.2

1.4

1.21

0.36

 

PCE-Polychromatic erythrocytes 

NCE-Normochromatic erythrocytes 

SD-standard deviation

*** -p < 0.001

**-P < 0.01

*-P < 0.05

Conclusions:
The test item was found to produce a dose related significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice at dose levels greater than 750 mg/kg bw 24 hours after dosing. Therefore, test item was considered to be genotoxic under the conditions of the test.
Executive summary:

In an in vivo bone marrow micronucleus test, groups of CD-1 mice (5/sex/dose) were exposed to test item in distilled water at doses of 187.5, 375, 750, 1500 and 1750 mg/kg bw by gavage. Further groups of mice were given a single oral dose of distilled water or cyclophosphamide, to serve as vehicle and positive controls respectively. Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei.

 

There was evidence of a dose-related increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item in the 24 h harvest group when compared to the concurrent vehicle control group but no statistically significant increases in the incidence of micronucleated cells at 750 mg/kg bw and below. There was no evidence of a statistically significant increase in the frequency of micronucleated PCEs in the 48 h test item dose groups. Statistically significant decreases in the PCE/NCE ratio were observed in the 24 h 750, 1500 and 1750 mg/kg bw test item dose groups when compared to their concurrent control group. This confirms that systemic absorption was achieved and that a cytotoxic response was achieved in the target tissue, bone marrow, even at the 750 mg/kg bw dose level.

 

The positive control item produced a marked increase in the frequency of micronucleated polychromatic erythrocytes.

 

Test item was found to produce a dose related significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice at dose levels greater than 750 mg/kg bw 24 hours after dosing. The dose response exhibited marked evidence of a clear threshold between 750 and 1500 mg/kg bw. The test item was considered to be genotoxic (clastogenic) to somatic cells under the conditions of the test.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
dominant lethal assay
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 October 2007 to 6 February 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
GLP study conducted similarly to OECD Guideline 478 without deviation.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
rodent dominant lethal assay
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The animal model, the Crl:CD(SD) rat, is recognized as appropriate for genetic toxicity studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina.
- Age and weight at study initiation: At the initiation of dose administration (study day 0), the males were approximately 9 weeks old; body weights ranged from 256 to 333 g. Males were approximately 11 and 18 weeks old at the initiation of mating with Phase I and II females, respectively. The Phase I females were approximately 12 weeks old when paired with the males on study day 13; body weights ranged from 217 to 288 g on gestation day 0. The Phase II females were approximately 12 weeks old when paired with the males on study day 62; body weights ranged from 226 to 283 g on gestation day 0.
- Assigned to test groups randomly: Yes; At the conclusion of the respective acclimation periods, all available males and females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements (250-450 g for males, 170-287 g for Phase I females and 170-278 g for Phase II females) was selected for use in the computerized randomization procedure. At that time, the individual body weights and corresponding animal identification numbers were entered into the WIL Toxicology Data Management System (WTDMS™). A printout containing the animal numbers, corresponding body weights and individual group assignments was generated based on body weight stratification randomized in a block design. The animals then were arranged into groups according to the printout.
- Housing: Upon arrival and until pairing, all rats were individually housed in clean, stainless steel, wiremesh cages suspended above cage board. The cage-board was changed at least 3 times per week. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the animals were returned to individual cages. Nesting material was not required, as euthanasia was scheduled prior to the date of expected parturition.
- Diet: Basal diet (PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002), ad libitum
- Water: Reverse osmosis-purified (on-site) drinking water, ad libitum
- Acclimation period: The males were housed for an acclimation period of 12 days prior to the first day of treatment. The phase I and phase II females were housed for an acclimation period of 12 days through the first day of pairing.

ENVIRONMENTAL CONDITIONS
- Temperature: 70.8-73.9 °F (21.5-23.3 °C)
- Humidity: 38.1-50.2%
- Air changes: Air handling units were set to provide a minimum of 10 fresh air changes per hour.
- Photoperiod: 12 hour light / 12 hour dark photoperiod

IN-LIFE DATES: 16 October 2007 to 6 February 2008
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Propylene glycol
- Concentration in vehicle: 10, 50 and 100 mg/mL
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The vehicle, propylene glycol, was transferred to an appropriate sized glass container approximately weekly for administration to the control group males (Group 1) and for preparation of the test item formulations; aliquots were prepared for daily dispensation to the control group. The vehicle was mixed throughout preparation, sampling and dose administration procedures. Dosing formulations for the fertility phase were prepared at the test item concentrations of 10, 50 and 100 mg/mL. The appropriate amount of the test item for each formulation was weighed into a calibrated glass container. Approximately 70% of the vehicle was added to each container, and the formulations were mixed until uniform using a magnetic stirrer. Vehicle was added to each container to bring the formulations to the calibration mark. The test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation and stored refrigerated, protected from light. The test item formulations were stirred continuously throughout the preparation, sampling and dose administration procedures. The test item formulations were visually inspected by the Study Director on 28 October 2007, and were found to be visibly homogeneous and acceptable for dose administration.

DOSE VOLUME: 10 mL/kg bw/day
Duration of treatment / exposure:
Males received 14 daily doses prior to mating with untreated Phase I females and 63 daily doses prior to mating with untreated Phase II females. Males were dosed throughout both mating periods through 1 day prior to euthanasia for a total of 91 93 doses. The females were not dosed.
Frequency of treatment:
Once daily
Post exposure period:
None
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1 (control) males
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2 males
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Group 3 males
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4 males
No. of animals per sex per dose:
Males: 25 males/dose
Females
Phase I females: 25, 27*, 25 and 27* females/paired with males dosed at 0, 100, 500, 1000 mg/kg bw/day, respectively
Phase II females: 30*, 26*, 27* and 25 females/paired with males dosed at 0, 100, 500, 1000 mg/kg bw/day, respectively
* Following no evidence of copulation during the first 7 days of mating, new females (not previously assigned to the study) were placed with the males for an additional 7 days.
Control animals:
yes, concurrent vehicle
Positive control(s):
No data
Tissues and cell types examined:
The uterus was opened, and the number of corpora lutea on each ovary was recorded. The number and location of all embryos, early resorptions and the total number of implantation sites were recorded. Viability of the embryos was determined with the aid of a dissecting stereomicroscope, if necessary. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to right distal uterine horn.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dosage levels were selected based on the results of the 5-day tolerability phase of this study. Animals were administered the test item, in the vehicle propylene glycol, orally by gavage once daily during study days 0-4 at dosage levels of 0, 500 and 1000 mg/kg bw/day. In this phase, there were no test item-related effects on clinical signs, body weights, body weight changes or food consumption at 1000 mg/kg bw/day (the highest dosage level). Based on these results, the dosage levels selected for administration to male rats for the fertility phase of the study were 0, 100, 500 and 1000 mg/kg bw/day.

EXAMINATION OF UTERINE CONTENTS
All females with evidence of mating were euthanized on gestation day 15. The thoracic, abdominal and pelvic cavities were opened by a ventral mid line incision and the contents were examined. The uterus and ovaries were then exposed and excised. The uterus was opened, and the number of corpora lutea on each ovary was recorded. The number and location of all embryos, early resorptions and the total number of implantation sites were recorded. Viability of the embryos was determined with the aid of a dissecting stereomicroscope, if necessary. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to right distal uterine horn.

ANALYSIS:
Intrauterine data were summarized using 2 methods of calculation:
Group Mean Litter Basis:
Postimplantation Loss/Litter = (No. Dead Embryos, Resorptions (Early)/Group) / (No. Gravid Females/Group)
Proportional Litter Basis:
Summation Per Group (%) = (Sum of Postimplantation Loss/Litter (%)) / (No. Litters/Group)
Where:
Postimplantation Loss/Litter (%) = [(No. Dead Embryos, Resorptions (Early)/Litter) / (No. Implantation Sites/Litter)] x 100
Evaluation criteria:
No data
Statistics:
All statistical tests were performed using appropriate computing devices or programs. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test item-treated group to the control group by sex. Each mean was presented with the standard deviation (S.D.), standard error (S.E.) and the number of animals (N) used to calculate the mean.
Copulation indices were analyzed using the Chi-square test with Yates’ correction factor (Hollander and Wolfe, 1999). Numbers of corpora lutea, implantation sites and viable embryos were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test item-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable embryos, fetal sex distribution, early and late resorptions, total resorptions and pre- and postimplantation loss) were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test item-treated groups to the control group.
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Additional information on results:
Intrauterine survival of the embryos was unaffected by administration of the test article to the males at dosage levels of 100, 500 and 1000 mg/kg bw/day when paired following 14 daily doses (Phase I) or following 63 daily doses (Phase II). No statistically significant differences from the control group were noted in mean litter proportions of pre- and postimplantation losses, mean numbers of corpora lutea, implantation sites and viable embryos.

Table 7.6.2/1: SUMMARY OF EMBRYONIC DATA AT SCHEDULED NECROPSY [% PER LITTER]

GROUP

0 mg/kg bw/day

100 mg/kg bw/day

500 mg/kg bw/day

1000 mg/kg bw/day

Phase I females

CORPORA LUTEA

MEAN

15.0

16.2

16.4

16.3

SD

2.72

1.84

2.06

2.17

SE

0.55

0.38

0.41

0.47

N

24

24

25

21

IMPLANTATION SITES

MEAN

14.4

15.4

15.0

15.7

SD

2.70

1.38

2.96

2.11

SE

0.55

0.28

0.59

0.46

N

24

24

25

21

VIABLE EMBRYOS (%)

MEAN

93.8

95.9

93.9

96.9

SD

6.65

4.59

10.92

4.91

SE

1.36

0.94

2.18

1.07

N

24

24

25

21

DEAD EMBRYOS (%)

MEAN

0.0

0.0

0.0

0.0

SD

0.00

0.00

0.00

0.00

SE

0.00

0.00

0.00

0.00

N

24

24

25

21

EARLY RESORPTIONS (%)

MEAN

6.2

4.1

6.1

3.1

SD

6.65

4.60

10.92

4..91

SE

1.36

0.94

2.18

1.07

N

24

24

25

21

LATE RESORPTIONS (%)

MEAN

0.0

0.0

0.0

0.0

SD

0.00

0.00

0.00

0.00

SE

0.00

0.00

0.00

0.00

N

24

24

25

21

TOTAL RESORPTIONS (%)

MEAN

6.2

4.1

6.1

3.1

SD

6.65

4.60

10.92

4..91

SE

1.36

0.94

2.18

1.07

N

24

24

25

21

PRE-IMPLANTATION LOSS (%)

MEAN

3.9

4.6

8.4

3.7

SD

5.54

8.04

14.04

5.36

SE

1.13

1.64

2.81

1.17

N

24

24

25

21

POST-IMPLANTATION LOSS (%)

MEAN

6.2

4.1

6.1

3.1

SD

6.65

4.60

10.92

4..91

SE

1.36

0.94

2.18

1.07

N

24

24

25

21

Phase II females

CORPORA LUTEA

MEAN

17.3

16.8

17.1

16.9

SD

1.87

2.09

1.90

1.89

SE

0.38

0.43

0.39

0.39

N

24

24

24

23

IMPLANTATION SITES

MEAN

16.4

15.8

16.5

16.2

SD

1.79

2.33

1.84

1.70

SE

0.37

0.48

0.38

0.35

N

24

24

24

23

VIABLE EMBRYOS (%)

MEAN

94.8

95.7

96.7

93.0

SD

5.52

3.89

6.83

6.55

SE

1.13

0.79

1.39

1.37

N

24

24

24

23

DEAD EMBRYOS (%)

MEAN

0.0

0.0

0.0

0.0

SD

0.00

0.00

0.00

0.00

SE

0.00

0.00

0.00

0.00

N

24

24

24

23

EARLY RESORPTIONS (%)

MEAN

5.0

4.4

3.3

7.0

SD

5.26

3.89

6.85

6.55

SE

1.07

0.79

1.40

1.37

N

24

24

24

23

LATE RESORPTIONS (%)

MEAN

0.3

0.0

0.0

0.0

SD

1.37

0.00

0.00

0.00

SE

0.28

0.00

0.00

0.00

N

24

24

24

23

TOTAL RESORPTIONS (%)

MEAN

5.3

4.4

3.3

7.0

SD

5.52

3.89

6.85

6.55

SE

1.13

0.79

1.40

1.37

N

24

24

24

23

PRE-IMPLANTATION LOSS (%)

MEAN

4.5

5.5

3.3

3.9

SD

7.30

8.49

4.53

5.11

SE

1.49

1.73

0.93

1.07

N

24

24

24

23

POST-IMPLANTATION LOSS (%)

MEAN

5.3

4.4

3.3

7.0

SD

5.52

3.89

6.85

6.55

SE

1.13

0.79

1.40

1.37

N

24

24

24

23

Proportional (%) data compared using Dunn’s test

Corpora lutea and implantation sites compared using Dunnett’s test

Modified statistics used. * indicates parametric analysis and + indicates non-parametric analysis.

None significantly different from control group

Conclusions:
Under the test conditions, intrauterine survival of the embryos was unaffected by administration of the test item to the males at dosage levels of 100, 500 and 1000 mg/kg bw/day when paired following 14 daily doses (Phase I) or following 63 daily doses (Phase II). No statistically significant differences from the control group were noted in mean litter proportions of pre- and postimplantation losses, mean numbers of corpora lutea, implantation sites and viable embryos. Therefore, test item is considered to be non-mutagenic in this system.
Executive summary:

A study was conducted to determine the dominant lethal effects in unexposed female rats after mating with exposed male rats. The test item was administered orally by gavage once daily to 3 groups of 25 male Crl:CD(SD) rats. Dosage levels were 100, 500 and 1000 mg/kg bw/day administered at a dosage volume of 10 mL/kg bw/day. A concurrent control group of 25 males received the vehicle (propylene glycol) on a comparable regimen. Males were approximately 9 weeks of age at the beginning of test item administration. Males received 14 daily doses prior to mating with untreated Phase I females and 63 daily doses prior to mating with untreated Phase II females. Males were dosed throughout both mating periods through 1 day prior to euthanasia for a total of 9193 doses. The females were not dosed. A laparohysterectomy was performed on gestation day 15 for each female with evidence of mating.

 

Intrauterine survival of the embryos was unaffected by administration of the test item to the males at dosage levels of 100, 500 and 1000 mg/kg bw/day when paired following 14 daily doses (Phase I) or following 63 daily doses (Phase II). No statistically significant differences from the control group were noted in the mean litter proportions of pre- and post-implantation losses, mean numbers of corpora lutea, implantation sites and viable embryos.

 

Based on the results, test item is considered to be non-mutagenic and not clastogenic to germ cells in this test system.

Additional information

Justification for classification or non-classification