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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP lab following strict internal protocols

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995
Reference Type:
study report
Title:
Unnamed
Year:
1982
Report date:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
The V-79 Chinese hamster lung cell mutagenesis system measures mutations at the hypoxanthine-guanine phosphoribosyl transferase (hpt) locus as resistance to the purine analog 6-thioguanine (6-TG). Cells that have lost functional activity of the salvage enzyme HGPRT are resistant to 6-TG, form clones in its presence, and are scored as mutants. This assay measures forward mutations so that most small deletion, nonsense, missense, regulatory, base-pair substitution, or frame-shift mutation at that locus should be detected within the limits of sensitivity of the assay. V-79 cells possess little metabolic activation capacity; however, they can be coupled to rat liver S-9 preparations for metabolic activation as is standard for this and other mammalian and bacterial mutagenesis assays. The assay scheme includes: (1) Establishing cell cultures for treatment from frozen cultures of V-79 that have been cleansed of all pre-existing hpt' mutants by passage through HMT medium before freezing. (2) Range-finding studies to determine appropriate drug doses. (3) Mutagenesis assay: (a) Treatment with test compounds of exponentially growing cultures to determine toxicity and mutagenicity, (b) Growth and expression periods, (c) Mutant selection with 6-TG. (d) Staining and counting of colonies.
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
Glycerol formal
IUPAC Name:
Glycerol formal
Test material form:
other: liquid
Details on test material:
L-584,735-000X008, Glycerol Formal
Sigma Chemical Co., Lot #063H1020
Drug Class: Excipient for Ivermectin formulations
Purity: -60% 5-hydroxy-1,3-dioxane, -40% 4-hydroxymethyl-l,3-dioxolane by Gas Chromatography

Method

Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Hypoxanthine-methotrexate-thymidine stock HMT cleansed cells frozen in liquid N2- Recovered for use in assay.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Test concentrations with justification for top dose:
3, 10, 30 and 100 mM with S-9
Vehicle / solvent:
water
Controls
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
not specified
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Evaluation criteria:
Number of Scorable Dose Levels: In order to be considered valid, a V79 mutagenesis assay must have three scorable dose levels. A scorable dose level is defined as a dose level allowing the survival of at least 10E6 cells from each dosed culture. At the time of treatment, a culture contains 1-2 x 10E7 cells so survival must be > 5-10%. Cell survival is estimated by two techniques. Plates are treated concurrently with flasks and survival estimated by clone forming ability (plating efficiency). In addition, when toxicity appears high by clone forming ability, the growth of the treated cultures is monitored over the eight days of recovery required to allow for phenotypic expression of any treatment induced genotypic change. The initial number of viable cells in the culture is estimated by growth curve extrapolation. In this procedure, the log relative growth of the culture, as determined by counting cultures on passage days, is plotted and extrapolated to day 0, yielding an estimate of the number of viable cells remaining after treatment (DeLuca et al, 1983, Mutat. Res. 107: 347-370). Since data collected in this laboratory has indicated that, in the V79 system, toxicity data generated by clone forming ability often overestimates toxicity relative to growth curve extrapolation, estimates derived from the latter technique will take precedence over those from the former when deciding the scorability of a particular dose level.
Statistics:
The results of the V-79 mutagenesis assay are analyzed for statistical significance by the NOSTASOT trend analysis procedure (Biometrics, 41: 295-301). Mutant colony counts are divided by their corresponding plating efficiencies to yield a corrected mutant colony count (CMC). The CMC is then subjected to a Freeman-Tukey transformation to stabilize the variance (Annal. Math. Stat., 21: 607-611) using the following formula: transformed CMC - SQRT (CMC) + SQRT (CMC +1). The transformed CMC is then used to compute the NOSTASOT significance level for positive trend and using three different dose scalings. The lowest of these p values (i.e., the most significant) is then corrected to account for the comparison of multiple treatment groups to a single set of controls. All calculations are performed by a set of computer programs, running on the VAX mainframe, written by the Department of Biostatistics at MSDRL. This is the same type of analysis that is performed on all animal toxicology studies in Safety Assessment at MSDRL.
Results are expressed as a p value for the lowest dose level at which a significant ( P <= 0.05) positive trend is observed. If no significant positive trend is observed, the p value for the highest dose level tested is reported.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
1. Cytotoxicity after Treatment (Table 1):

In the V-79 mutagenesis assay, TT #95-8502, the relative survival after treatment with Glycerol Formal ranged from 56 percent to 24 percent over the dose range of 3 to 100 mM with S-9 activation (see Table 1 for exact results). Survival at all dose levels was sufficient to fulfill our criterion for a scorable dose level, i.e. greater than or equal to 106 cells surviving treatment.

2. V-79 Mutagenesis (Tables 2 and 3):

The mutagenesis results are shown in Table 2 and the results of the NOSTASOT statistical analysis and induced mutant fractions (IMF) are shown in Table 3. These results were analyzed for statistical significance as follows. Mutant colony counts were divided by their corresponding plating efficiencies to yield a corrected mutant colony count (CMC). The CMC is then subjected to a Freeman- Tukey transformation in order to stabilize the variance (Annal. Math. Stat., 21:607-611) using the following formula: transformed CMC = sqrt(CMC) + sqrt(CMC+l). The transformed CMC is then used to compute the NOSTASOT significance level for positive trend and these values corrected for the comparison of multiple treatment groups to a single set of controls. This is done by a series of computer programs written by the Department of Biostatistics at MRL. The results are expressed as the lowest dose level for which a significant positive trend is observed and the P value is reported next to this "NOSTASOT" dose level. If no significant trend is observed, the P value is reported at the highest dose level tested (see Table 3). The P value reported represents the lowest (i.e., most significant) P value obtained using three different theoretical dose scalings (Biometrics, 41: 295-301).

It can be seen that, for Glycerol Formal, the trend was negative and all IMFs were zero.
The positive control, 3-methylcholanthrene at 37.3 jiM with S-9 activation, produced an IMF of 33.88 x 10E-6. This value was highly significantly different from the negative controls (P < 0.0001), indicating the assay was working as expected.

The current historical negative control mean for this assay is 4.71 x 10-6 with a standard deviation of 2.20 x 10E-6 (Appendix II). Thus, the mean concurrent negative control value was within three standard deviations of the mean historical negative control value, thereby fulfilling the criteria for a valid assay.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1.Glycerol Formal: V-79 Mammalian Cell Mutagenesis Assay. TT #95-8502

Cytotoxicity at Time of Treatment.

Treatment

S-9

Metabolic

Activation

Precipitate at Dosing

Colony

Count

X

Absolute

Plating

Efficiency

Relative

Plating

Efficiency[1]

Negative Control - 5% Water

+

-

261,

248

255

0.85

1.00

 

+

-

270,

253

262

0.87

1. 03

Negative Control - 5% Water[1]

-

-

240,

253

247

0. 82

0 . 97

 

-

-

255,

247

251

0. 84

0.99

Positive Control

+

-

189,

199

194

0. 65

0 . 77

3-methylcholanthrene, 3 7.3 JIM

+

 

205,

173

189

0. 63

0.75

3.0 mM

+

-

144,

153

149

0. 50

0. 59

 

+

-

121,

147

134

0.45

0. 53

10.0 mM

+

-

115,

130

123

0 .41

0.48

 

+

-

140,

137

139

0.46

0.55

30.0 mM

+

-

102,

112

107

0.36

0.42

 

+

-

70,

113

92

0. 31

0. 36

100.0 mM

+

-

50,

69

60

0.20

0. 23

 

+

-

68,

60

64

0.21

0.25

1 - Without S-9 controls were run for a larger assay of which the data reported is a part; relative plating efficiencies are calculated based on the mean of all four negative controls 


Table 2.   Glycerol Formal: V-79 Mammalian Cell Mutagenesis Assay. TT #95-8502

Mutant Fraction in V-79 Cells.

Treatment

S-9 Metabolic Activation

Plating Efficiency Colonies/Plate

Absolute Plating Efficiency1

6-Thioguanine Resistant Colonies

Total 6-TG Resistant Colonies²

Mutant Fraction (x 10-6)

Negative Control

+

149

131

130

0. 91

2

2

2

4

1

2

0

2

1

16

6 . 50

5% Water

+

13 8

135

141

0 .92

2

4

0

2

1

1

2

2

2

16

6.44

Negative Control

-

141

140

142

0. 94

2

2

1

1

3

3

2

2

2

18

7.09

5% Water

-

154

141

130

0.94

2

1

4

1

2

2

2

1

2

17

6. 67

Positive Control

+

136

142

132

0 . 91

11

6

8

14

9

14

12

8

13

95

38. 62

3-methylcholanthrene, 37.3 µM

+

14 6

154

151

1.00

17

13

10

10

20

14

11

11

9

115

42 . 50

3.0 mM

+

140

139

137

0.92

0

0

0

0

1

0

0

0

0

1

0 . 40

 

+

140

155

176

1.05

0

2

0

0

1

0

0

0

1

4

1.42

10 .0 mM

+

132

135

160

0.95

0

0

0

1

0

0

1

0

0

2

0 . 78

 

+

139

135

133

0. 90

1

1

0

1

1

0

2

1

0

7

2.87

30.0 mM

+

153

146

153

1.00

0

0

0

1

0

0

0

0

0

1

0.37

 

+

150

151

138

0. 98

0

0

0

0

0

1

1

1

0

3

1.14

100.0 mM

+

147

163

15 9

1.04

1

0

1

1

0

0

0

1

0

4

1.42

 

+

144

135

168

0.99

0

1

1

0

0

0

0

1

1

4

1.49

1. Total seed number for plating efficiency is 450 cells (150/plate)

2. Total seed number for 6 -thioguanine resistance is 2.7 x 10E6 cells (3 x 10E5/plate)


Table 3.   Glycerol Formal: V-79 Mammalian Cell Mutagenesis Assay. TT #95-8502

Summary of the effects of L-584,735 on Mutant Fraction in V-79 Cells

Treatment

S-9

Metabolic

Activation

Concentration

Mean Mutant Fraction (x 10-6)

IMF (x 10-6)

(Mean MF-Mean Neg. Control MF)1

NOSTASOT

Significance

Level2

Solvent Negative

Control - Water

+/-

5%

6.68

.

-

Positive Control

3-methylcholanthrene

+

37.3 µM

40.56

33 .88

<0.0001

GLYCEROL FORMAL

+

3.0 mM

0.91

0

 

+

10.0 mM

1. 82

0

 

+

30.0 mM

0. 75

0

 

+

100.0 mM

1. 46

0

NT

1. IMF of <0 is given as 0.

2. The lowest (i.e., most significant) P Value is shown next to the NOSTASOT dose level to which it applies.

NT = Negative Trend

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Glycerol Formal does not induce mutation in V-79 cells at the hpt locus under these testing conditions.
Executive summary:

TT #81 -8521:

The V-79 Chinese hamster lung cell mutagenesis system measures mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus as resistance to the purine analog 6-thioguanine (6-TG). Cells that have lost functional activity of the salvage enzyme HGPRT are resistant to 6-TG, form clones in its presence, and are scored as mutants. This assay measures forward mutations so that any deletion, nonsense, missense, regulatory, base-pair substitution, or frame-shift mutation at that locus will be detected within the limits of sensitivity of the assay.

Glycerol Formal was evaluated for mutagenic potential in the V-79 Chinese hamster lung cell system. It was tested at concentrations ranging from 3 to 100 mM in the absence and in the presence of a microsomal enzyme activation system prepared from rat liver. Methylnitrosurea (MNU, 0.5 mM) without metabolic activation and 3-methylcholanthrene (MCA,10 µg/ml) with metabolic activation were used as positive control substances. The protocol for this test is well-documented in the literature and is detailed in the report which follows.

Glycerol Formal did not produce three-fold or greater increases in mutation frequency at any dose relative to the mean of the negative controls, and thus is considered negative in this assay. The positive control agents were both mutagenic in this assay.

TT #95 -8502:

Glycerol Formal was evaluated for mutagenic potential in a V-79 Chinese hamster lung cell system. It was tested with a microsomal enzyme activation system prepared from rat liver; since this assay was done to investigate an equivocal result obtained in a previous assay (TT #81-8521) in the presence of S-9 only, no cultures were treated in the absence of S-9. The protocol for this test is well-documented in the literature.

 

Glycerol Formal identified as L-584,735-000X008, and in the therapeutic class of excipient for Ivermectin formulations, was used in this study as a solution in water. A formula weight of 104.10 was used for calculating molarity. Though, using our current protocol, a maximum testable dose level would be 10 mM, the same dose levels were used as had been tested in the original mutation assay, i.e. 3, 10, 30 and 100 mM. The known mutagen 3-methylcholanthrene was run concurrently as a positive control in this study. Cultures of V-79 Chinese hamster lung fibroblasts, cleansed of pre-existing mutants by hypoxanthine-methotrexate-thymidine (HMT) treatment, were exposed to the test and control compounds for three hours. Mutation at thehptlocus was measured as resistance to 6-thioguanine after an expression period of nine days. Survival at the top dose was 21%.

 

Total mutant colony counts, corrected by their corresponding plating efficiencies, were subjected to a Freeman-Tukey transformation (Annal. Math. Stat., 21:607-611) and then analyzed by the NOSTASOT procedure (Biometrics, 41:295-301). This is a highly sensitive procedure designed to determine the highest dose at which no statistically significant positive trend can be detected. We consider an assay positive if the NOSTASOT analysis detects a positive trend at a significance level of P <= 0.05, after correction for multiple dose comparison, and the induced mutant fraction (IMF) exceeds 2.21 times the standard deviation of the current negative control data base [i.e., is outside of the Student's t-test 95 percent confidence interval (C.I.)]. As of 10 FEB 95, the standard deviation of the current negative control data base (1989 to present) is 2.20 x 10E-6 and the 95 percent C.I. is, therefore, 2.21 x 2.20 x 10E-6 =4.86x 10E-6.

 

The NOSTASOT analysis of Glycerol Formal indicated no significan positive trend (trend was negative) up to the maximum dose tested, 100 mM. In addition, all IMF's were zero.

 

The positive control 3-methylcholanthrene at 37.3 µM was highly significantly different from the negative controls (P <= 0.0001) indicating that the assay was working properly.

 

We conclude that Glycerol Formal does not induce mutation in V-79 cells at the hpt locus under these testing conditions. This conclusion is based on the fact that at the dose levels tested with S-9 activation, Glycerol Formal did not meet the criteria for a positive test compound, which is induction of a statistically significant (P <= 0.05) positive trend at a dose level less than or equal to the top dose tested and an induced mutant fraction greater than 4.86 x 10E-6 (Student's t-test 95% C.I. based on the standard deviation of the current negative control data base).