Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

MIPA did not cause gene mutations in Salmonella typhimurium (Ames test) or in Chinese hamster ovary cells (CHO/HGPRT) according or similar to OECD 471 or OECD 476, repectively. No chromosomal aberrations were induced in rat lymphocytes according to OECD 473. All studies were performed in the absence and presence of metabolic activation. Thus, MIPA is not considered to be genotoxic.

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Haworth S, Lawlor T, Mortelmans K, Speck W, Zeiger E (1983): Salmonella mutagenicity results for 250 chemicals. Environ Mutagen S[Suppl 1]:3-142.
Deviations:
yes
Remarks:
No E.coli strain or TA102 tested
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich
- Purity: 96.4%
Target gene:
Strain Target Gene:
TA1537 hisC3076
TA1535 hisG46
TA100 hisG46
TA98 hisD3052
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
The S9 fractions were prepared from the liver of Aroclor 1254-induced male Sprague-Dawley rats and male Syrian hamsters.
Test concentrations with justification for top dose:
33 - 4000 ug/plate
Vehicle / solvent:
water
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: For strains tested with S9: All strains, 2-aminoanthracene.
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 2 days
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
Evaluation criteria:
According to Haworth et al. (1983).
Species / strain:
S. typhimurium, other: TA98, TA100, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slightly toxic at highest dose in the 3 strains.
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slightly toxic at highest dose
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slightly toxic at highest dose
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid

Table 1-4: Results for the Ames-testing.

Strain TA100

Dose

No Activation
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

Dose units

Mean

± SEM

Mean

± SEM

Mean

± SEM

0         

135

2

141

6.8

139

5.6

33         

152

4

144

8.7

132

14.4

100         

141

3.8

140

12.2

138

5.8

333         

148

4.2

140

5.5

149

11.1

1000         

155

0.9

128

6.4

124

4.3

3333         

130S

7.8

159S

7

156S

6.5

Positive Control

1298

63.2

1452

83.5

947

33.3

Strain TA98

Dose

No Activation
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

Dose units

Mean

± SEM

Mean

± SEM

Mean

± SEM

0         

18

2

30

0.6

24

1.2

33         

19

2.6

36

1.5

25

4.4

100         

25

2.9

43

1.9

30

6.3

333         

17

2.3

25

2.1

33

0.7

1000         

21

3.3

31

4.5

26

4.1

3333         

8T

3.5

20S

4.3

20S

1.8

Positive Control

1475

103.9

935

33.9

637

42.8

Strain TA1537

Dose

No Activation
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

Dose units

Mean

± SEM

Mean

± SEM

Mean

± SEM

0         

8

1.7

10

1.7

8

0.9

33         

7

2.9

12

1.5

11

2.9

100         

7

0.3

13

1.7

9

0.9

333         

6

1.8

7

1.2

7

1

1000         

6

0.6

7

1.3

8

0.3

3333         

4S

1.2

4S

0.3

5S

0.3

Positive Control

87

12.3

124

6.1

97

2.9

Strain TA1535:

Dose

No Activation
(Negative)

No Activation
(Negative)

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Equivocal)

10% HLI
(Weak Positive)

10% HLI
(Negative)

10% HLI
(Positive)

10% RLI
(Negative)

10% RLI
(Positive)

10% RLI
(Negative)

10% RLI
(Positive)

Dose units

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0         

27

5

54

6.4

7

1.2

36

3.6

9

0.9

11

3.2

9

2.5

5

1

9

1.5

9

0.7

7

1.5

11

0

33         

30

1.5

 

 

 

 

 

 

11

2.2

 

 

 

 

 

 

7

2.6

 

 

 

 

 

 

100         

30

4.4

51

4.2

7

0.9

34

0.9

11

1

9

1

8

1.2

10

2.3

11

1.2

10

0.6

7

1.9

11

1.3

333         

27

4.4

45

2.2

6

1.5

32

5

14

0.9

14

1.2

6

0.7

11

2

9

0.9

14

2.8

6

0.7

7

0.3

1000         

20

1.5

47

3.8

4

0.7

23

4.8

10

1.2

9

0.3

7

1.3

11

4.2

7

1.8

14

0.7

5

1.5

9

0.7

1800         

 

 

34

3.8

5

1.3

28

1.3

 

 

24

1.9

10

1.3

29

0.7

 

 

15

1.9

5

1.7

13

3.2

2800         

 

 

25S

0.9

4S

1

23S

2

 

 

30S

2.9

5

0.6

28

6.6

 

 

33S

4.3

8

2

32

2.1

3333         

12S

3.8

25S

4.5

4S

0.9

16S

3.2

27S

1.7

20S

1.8

8S

2.1

24S

2.7

17S

2.5

32S

2.6

6S

2.2

32S

2.2

3500         

 

 

16S

0.6

3S

0.6

21S

0.3

 

 

18S

2.7

6S

1.5

25S

4.7

 

 

20S

1.5

6S

1

26S

2.2

4000         

 

 

13S

1.7

2S

0.6

17S

2.1

 

 

21S

0.9

3S

0.3

15S

2.4

 

 

26S

5.1

4S

1.7

23S

3.3

Positive Control

1005

61

1484

111

869

23.8

1124

41.3

124

3.5

99

2.3

85

2.3

231

16.1

98

6.4

95

11.4

83

4.7

103

13.6

Abbreviations:
RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9
s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; T = Toxic; c = Contamination
Conclusions:
Under the conditions tested the test substance was not mutagenic in the strains TA98, TA100 and TA1537.
However, with S9-activation the TA1535 strain presented some weak positive results. Therefore the test substance was rated as ambiguous.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Ames et al., Mut Res 31, 347-364, 1975
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Analytical purity: 97%
- Supplier: Tokyo Kasei Kogyo Co. LTD. Tokyo, Japan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: S. typhimurium provided by B.N. Ames, U.S .A.; E. coly provided by M. Ishizawa, Japan

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
- S9 mix or sodium phosphate buffer (pH 7.4) for preincubation
- minimal glucose agar
- 37°C
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells:
S. typhimurium provided by B.N. Ames, U.S .A.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
- S9 mix or sodium phosphate buffer (pH 7.4) for preincubation
- minimal glucose agar
- 37°C
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from the liver of male Sprague-Dawley rats, pretreated with polychlorinated biphenyl (KC 500) at a dose of 500 mg/kg body weight five days before sacrifice.
Test concentrations with justification for top dose:
1 - 5000 ug/plate
Vehicle / solvent:
water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h


Species / strain:
other: TA98, TA100, TA1535, TA1537, E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

 Dose (µg/plate  TA100     TA1535     WP2 uvrA    TA98      TA1537     TA1535   
   +S9  -S9   +S9   -S9   +S9   -S9   +S9   -S9   +S9   -S9   +S9   -S9
1  177  183  38  12  34  30  21  43  7  10  18  28
 5  160  187  41  14  35  34  18  36  8  14  18  24
 10  164  181  38  14  41  44  19  45  9  13  20  29
 50  167  183  36  18  33  37  22  37  9  13  23  28
 100  175  165  41  12  31  32  21  34  10  10  24  30
500  144  189  38  18  34 37   16  34  8 19   21  24
 1000  163  183  44  19  35  42  24  31  9  8  22  30
5000  94*  106*  17*  18  36  37 15  26*  4* 12  19  30
 H20 149±17.1  161±16.2   28±6.9   15±3.6  32±7.3  33±10.3  29±6.2  39±8.6  16±6.4  21±8.1  21±5.5  28±7.0
AF2  501 ± 84.7  -  -  -  1082 ± 293.7  -  278 ± 64.8  -  -  -  -  -
 ENNG  -  -  1101 ± 683.1  -  -  -  -  -  -  -  -  -
 9AC  -  -  -  -  -  -  -  -  889 ± 275.7  -  -  -
 4NQO  -  -  -  -  -  -  -  270 ± 66.2  -
 B(a)P  -  1084  ± 236.3  -  -  -  -  -  809 ± 108.4  -  313 ± 48.6  -  354 ± 89.4
 2AA  -  -  -  440 ± 198.6  -  359 ± 127.0  -  -  -  -  -  -

 * growth inhibition was observed

AF2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine, 9AC: 9-aminoacridine, 4NQO: 4-nitroquinoline-l-oxide, B(a)P: benzo(a)pyrene, 2AA: 2-aminoanthracene

Conclusions:
The reverse bacterial gene mutation test for MIPA is negative.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The Dow Chemical Co., Midland, MI, lot MM930105
- Purity: 99.63%
- Purity test date: 1994
Target gene:
HGPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
-K1-BH-4
Details on mammalian cell type (if applicable):
Proficiences: low spontaneous mutation frequency at the HGPRT locus.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : purchased from Sitek Research Laboratories, Rockville, MD, USA
- method of preparation of S9 mix : liver homogenates prepared from Aroclor-1254 treated (500 mg/kg) male Sprague-Dawley rats
- concentration or volume of S9 mix and S9 in the final culture medium : 10% (v/v)
Test concentrations with justification for top dose:
156.3-2500 µg/ml
Number of replicates: 2
Analytical method: GC/HPLC
Vehicle / solvent:
water
Untreated negative controls:
no
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: with S9: 20-Methylcholanthrene, final conc.: 4µg/mL
Details on test system and experimental conditions:
HGPRT assay
- Application: Cells were treated for approximately 4 hours. Then, cells were subcultured into petri dishes (1x10E6/dish). This was repeated every 2-3 days up to 7-8 days. Then the cells were trypsinised and plated in selection medium (2x10E5 cells/dish). Mutant frequency was determined 8-10 days later.
Evaluation criteria:
Based on statistical evaluation.

DESCRIPTION OF FOLLOW UP REPEAT STUDY: repeat study was similar to first study.

Statistics:
- frequency of mutants per 10E6 clonable cells: ANOVA
- actual plate counts: follow a poisson distribution
- mean plate count is used as an estimate of variance
- linear trend test and lack of fit test are employed (α=0.05) as an omnibus test to compare treated groups to the negative control
- if significant increasing trend or a significant lack of fit: Dunnett’s t-test is conducted, comparing each treated group and the positive control to the negative control (α=0.05, one-sided)
- additional comparison of the positive control to the negative control (α=0.05): linear contrast statement

- acceptance criteria: the mutation frequency in positive controls should be significantly higher than the negative controls, and the negative controls should be within reasonable limits of laboratory historical controls and literature values
- chemical considered positive: statistically significant, dose related, reproducible increase in mutation frequency induced
- final interpretation of the data: mutation frequency and cloning efficiencies in the negative controls
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
at the highest concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
Highest dose was based on changes in pH/osmolality. pH at 2500 ug/mL was 9.19. This was lowered to 7.40 (pH of control was 7.05). Osmolality 307 mOsmol/kg water versus 257 in the control. Higher concentrations needed more HCl to adjust the pH, revealing an unacceptably high osmolality.

Table 1: Survival of CHO cells (passage 29) treated with the test chemical.

 

Without S-9

 

With S-9

 

No. of Colonies Per Dish

 

No. of Colonies Per Dish

Treatment(µg/ml)

1

2

3

RCS(%) a

1

2

3

RCS(%) a

Neg. control b

166

163

147

100.0

102

148

142

100.0

39.06

166

147

152

97.7

111

125

123

91.6

78.13

131

132

136

83.8

115

114

131

91.8

156.25

171

157

163

103.2

120

120

106

88.3

312.50

124

142

136

84.5

146

123

121

99.5

625.00

142

160

139

92.6

128

106

130

92.9

1250.00

137

162

123

88.7

111

91

133

85.5

2500.00

140

107

122

77.5

124

122

112

91.3

a: Relative cell survival (%) = (mean number of colonies / dish in the treated*100) / (Mean number of colonies / dish in the negative control)

b: 1% water

Table 2: Results of the gene mutation assay in the abscence of S-9, Assay 1. Positive control: 621 µg/mL EMS. Negative control: 1% water.

Toxicity Assay

Mutation Assay

Cloning Efficiency (CE)

 

 

Colonies/Dish

 

TGr Colonies/Dish b

Colonies/Dish

 

TGr Muatants per 1E6 Clonable Cells

Treatment (µg/mL)

1

2

3

RCS(%)a

1

2

3

4

5

6

7

8

9

10

Total

1

2

3

CE(%) c

 

Neg. Control

122

123

125

104.4

0

0

0

2

1

0

1

0

1

0

5

156

187

163

84.3

3.0

Neg. Control

128

112

99

95.6

2

0

2

1

0

0

0

1

0

0

6

123

162

144

71.5

4.2

156.3

129

136

102

103.5

0

0

0

0

0

1

1

0

1

1

4

114

165

137

69.3

2.9

156.3

125

123

119

103.5

0

0

0

0

0

1

1

1

1

0

4

166

129

185

80.0

2.5

312.5

94

83

97

77.3

4

1

0

1

0

1

1

0

0

0

8

222

251

204

112.8

3.5

312.5

109

109

109

92.2

0

2

0

1

3

2

0

1

1

2

12

123

155

128

67.7

8.9

625.0

107

128

107

96.5

0

0

0

1

0

2

0

0

0

0

3

144

102

146

65.3

2.3

625.0

89

94

94

78.1

0

0

0

0

1

0

0

0

0

0

1

137

133

111

63.5

0.8

1250.0

106

118

112

94.8

0

0

0

0

0

0

1

0

1

1

3

134

149

145

71.3

2.1

1250.0

105

108

115

92.5

2

1

0

0

1

1

1

0

0

1

7

153

147

157

76.2

4.6

2500.0

74

68

58

56.4

0

0

0

0

0

0

0

0

0

0

0

135

119

158

68.7

0.0

2500.0

80

53

62

55.0

2

0

1

3

0

3

4

1

2

3

19

134

149

132

69.2

13.7

Pos. Control

15

25

26

18.6

20

17

16

18

28

26

27

30

20

21

223

42

28

41

18.5

602.7 d

Pos. Control

22

21

18

17.2

17

13

12

17

23

22

20

14

14

9

161

42

56

62

26.7

301.9 d

a: Relative cell survival (%)= (Mean number of colonies / dish in the treated) / (Mean number of colonies/ dish in the negative control [avg. of replicates]) * 100

b: TGr = 6-Thioguanine resistant

c: CE(%) = (Mean number of colonies/dish) / (No. of cells seeded/dish) * 100

d: The frequency of TGr mutants is significantly higher than the concurrent negative control value (alpha=0.05).

Table 3: Results of the gene mutation assay in the abscence of S-9, Assay 2. Positive control: 621 µg/mL EMS. Negative control: 1% water.

Toxicity Assay

Mutation Assay

Cloning Efficiency (CE)

 

 

Colonies/Dish

 

TGr Colonies/Dish b

Colonies/Dish

 

TGr Muatant per 1E6 Clonable Cells

Treatment (µg/mL)

1

2

3

RCS(%) a

1

2

3

4

5

6

7

8

9

10

Total

1

2

3

CE(%) c

 

Neg. Control

116

102

137

97.7

1

1

0

2

3

2

0

0

0

0

9

221

192

199

102.0

4.4

Neg. Control

124

113

135

102.3

0

0

0

1

0

1

0

0

0

0

2

186

160

189

89.2

1.1

156.3

108

123

97

90.2

0

1

0

1

0

0

0

0

0

0

2

206

174

196

96.0

1.0

156.3

122

144

123

107.0

2

1

1

2

1

1

0

4

0

2

14

187

177

171

89.2

7.9

312.5

118

128

106

96.8

2

2

2

2

3

2

3

4

2

1

23

207

191

188

97.7

11.8

312.5

143

127

97

101.0

0

1

1

0

0

1

1

0

0

1

5

163

195

178

89.3

2.8

625.0

141

161

142

122.1

0

2

0

5

1

8

1

1

0

0

18

229

241

223

115.5

7.8

625.0

145

126

103

102.9

1

1

1

0

0

5

1

1

0

3

13

214

184

190

98.0

6.6

1250.0

179

146

131

125.4

2

0

0

0

0

0

1

0

0

1

4

211

221

198

105.0

1.9

1250.0

84

117

8.5

78.7

3

2

3

1

1

3

2

1

2

1

19

214

205

214

105.5

9.0

2500.0

106

119

140

100.4

0

0

2

2

0

1

0

3

1

0

9

208

207

171

97.7

4.6

2500.0

124

121

134

104.3

1

0

0

0

1

1

1

1

1

0

6

178

182

198

93.0

3.2

Pos. Control

45

43

48

37.4

40

41

26

30

37

32

34

40

29

24

333

82

91

98

45.2

368.6 d

Pos. Control

36

47

34

32.2

41

47

41

49

38

37

26

55

35

33

402

112

100

97

51.5

390.3 d

a: Relative cell survival (%)= (Mean number of colonies / dish in the treated) / (Mean number of colonies/ dish in the negative control [avg. of replicates]) * 100

b: TGr = 6-Thioguanine resistant

c: CE(%) = (Mean number of colonies/dish) / (No. of cells seeded/dish) * 100

d: The frequency of TGr mutants is significantly higher than the concurrent negative control value (alpha=0.05).

Table 4: Results of the gene mutation assay in the presence of S-9, Assay 1. Positive control: 4 µg/mL 20-MCA. Negative control: 1% water.

Toxicity Assay

Mutation Assay

Cloning Efficiency (CE)

 

 

Colonies/Dish

 

TGr Colonies/Dish b

Colonies/Dish

 

TGr Muatant per 1E6 Clonable Cells

Treatment (µg/mL)

1

2

3

RCS(%) a

1

2

3

4

5

6

7

8

9

10

Total

1

2

3

CE(%) c

 

Neg. Control

81

68

70

103.8

0

0

0

1

0

0

0

0

0

0

1

104

126

102

55.3

0.9

Neg. Control

67

70

66

96.2

2

2

1

0

1

2

2

0

1

1

12

127

143

118

64.7

9.3

156.3

88

66

61

101.9

0

0

0

0

0

1

1

0

0

1

3

134

112

107

58.8

2.5

156.3

98

74

78

118.5

0

0

1

1

1

0

0

1

1

1

6

123

119

143

64.2

4.7

312.5

90

89

78

121.8

1

2

1

0

1

2

0

0

1

0

8

119

156

114

64.8

6.2

312.5

84

70

72

107.1

1

0

1

0

0

0

0

0

1

0

3

141

148

154

73.8

2.0

625.0

58

63

64

87.7

1

0

2

1

1

1

0

1

0

1

8

139

120

129

64.7

6.2

625.0

74

69

61

96.7

0

0

0

0

1

0

0

0

0

1

2

114

138

173

70.8

1.4

1250.0

70

68

74

100.5

- d

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

1250.0

74

61

57

91.0

0

1

1

0

0

1

0

0

0

0

3

155

123

143

70.2

2.1

2500.0

58

45

55

74.9

0

0

0

0

0

0

1

0

0

0

1

144

135

135

69.0

0.7

2500.0

70

74

79

105.7

0

0

0

0

0

0

0

0

0

0

0

123

130

150

67.2

0.0

Pos. Control

65

68

76

99.1

16

20

14

16

12

16

17

6

17

15

149

108

106

111

54.2

137.5 e

Pos. Control

63

61

67

90.5

14

20

11

13

12

21

24

12

18

19

164

90

97

105

48.7

168.5 e

a: Relative cell survival (%)= (Mean number of colonies / dish in the treated) / (Mean number of colonies/ dish in the negative control [avg. of replicates]) * 100

b: TGr = 6-Thioguanine resistant

c: CE(%) = (Mean number of colonies/dish) / (No. of cells seeded/dish) * 100

d: Data lostdueto technical error.

e: The frequency of TGr mutants is significantly higher than the concurrent negative control value (alpha=0.05).

Table 5: Results of the gene mutation assay in the presence of S-9, Assay 2. Positive control: 4 µg/mL 20-MCA. Negative control: 1% water.

Toxicity Assay

Mutation Assay

Cloning Efficiency (CE)

 

 

Colonies/Dish

 

TGr Colonies/Dish b

Colonies/Dish

 

TGr Muatant per 1E6 Clonable Cells

Treatment (µg/mL)

1

2

3

RCS(%) a

1

2

3

4

5

6

7

8

9

10

Total

1

2

3

CE(%) c

 

Neg. Control

122

148

133

87.3

1

1

1

0

0

4

1

1

0

1

10

172

195

170

89.5

5.6

Neg. Control

112

200

208

112.7

3

0

0

0

0

0

0

0

2

2

7

166

111

125

67.0

5.2

156.3

218

184

189

128.1

1

0

1

1

2

0

1

1

0

2

9

185

223

231

106.5

4.2

156.3

159

138

162

99.5

1

1

0

1

1

2

0

1

1

0

8

171

180

206

92.8

4.3

312.5

137

152

150

95.1

1

0

0

1

1

2

1

5

1 ,

0

12

213

205

201

103.2

5.8

312.5

104

125

122

76.1

0

0

0

0

0

0

0

1

1

0

2

180

200

209

98.2

1.0

625.0

109

120

138

79.5

0

0

0

0

1

2

0

0

1

0

4

225

226

193

107.3

1.9

625.0

139

114

133

83.6

3

1

1

3

1

2

0

1

1

0

13

217

209

210

106.0

6.1

1250.0

105

95

97

64.4

0

3

1

1

0

0

0

1

0

0

6

227

198

187

102.0

2.9

1250.0

99

81

95

59.6

0

0

1

1

2

0

0

0

1

1

6

227

223

225

112.5

2.7

2500.0

81

81

98

56.3

0

0

2

0

1

1

0

0

0

4

8

183

189

206

96.3

4.2

2500.0

75

79

74

49.4

1

3

1

2

2

3

6

2

1

2

23

207

186

206

99.8

11.5

Pos. Control

84

87

98

58.3

42

42

41

35

30

38

41

34

24

23

350

184

154

165

83.8

208.7d

Pos. Control

97

90

93

60.7

32

43

24

38

29

41

32

24

21

38

322

176

178

155

84.8

189.8d

a: Relative cell survival (%)= (Mean number of colonies / dish in the treated) / (Mean number of colonies/ dish in the negative control [avg. of replicates]) * 100

b: TGr = 6-Thioguanine resistant

c: CE(%) = (Mean number of colonies/dish) / (No. of cells seeded/dish) * 100

d: The frequency of TGr mutants is significantly higher than the concurrent negative control value (alpha=0.05).

Conclusions:
The forward mammalian gene mutation assay in the chinese hamster overay cell/Hypoxanthine-Guanine-Phosphoribosyl transferase (CHO/HGPRT) for MIPA is negative.
Under the conditions tested the substance was not mutagenic in a HGPRT assay in CHO cells.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
OECD (1983)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The Dow Chemical Co., Midland, MI, lot MM930105
- Purity: 99.63%
- Purity test date: 1994
Species / strain / cell type:
lymphocytes: rat lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: blood sample collection from Sprague-Dawley rats
- Suitability of cells: The laboratory rat is widely used for toxicologieal studies and hence the results of the in vitro cytogenetic tests can be compared with other toxicological end points. Since rats can be maintained in well controlled environments, the influence of environmental factors on the end point being measured ean be controlled. The karyotype of cultured rat lymphocytes (2N=42) is stable as opposed to the relative karyotypic instability of established cell lines. Furthermore, with the rat lymphocyte system, S-9 preparations from the same species ean be used for metabolite activation.

For lymphocytes:
- Sex, age and number of blood donors: male, 17-18 weeks, number of animals not specified
- whole blood cultures used
- blood from different donors were pooled

MEDIA USED
- Type and composition of media, CO2 concentration, temperature:
- RPMI 1640 medium (with 25 mM HEPES, GIBCO, Grand Island, NY) + 10% heat-inactivated fetal bovine serum (GIBCO) + Fungizone 0.25 Ilg/ml; penicillin G, 100 u/ml; and streptomycin sulfate, 0.1 mg/ml (GIBCO) + 20 Ilg/ml PHA (HA17, Murex Diagnostics Ltd.,Dartford, England) + 2 mM L-glutamine (GIBCO)
- 37°C
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: purchased from Sitek Research Laboratories, Rockville, MD, USA
- method of preparation of S9 mix: liver homogenates prepared from Aroclor-1254 treated (500 mg/kg) male Sprague-Dawley rats
- concentration or volume of S9 mix and S9 in the final culture medium : 10% (v/v)
Test concentrations with justification for top dose:
Assay 1, presence of S-9
2.5, 8.3, 25, 83.3, 250, 833, 2500 µg/mL
Assay 1, abscence of S-9
2.5, 8.3, 25, 83.3, 250, 833, 2500 µg/mL
Assay 2, presence of S-9
250, 833, 2500 µg/mL
Assay 2, abscence of S-9
83.3, 250, 833, 1500 µg/mL

Analytical method: GC/HPLC
Vehicle / solvent:
- Vehicle used: water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
ADMINISTRATION:
-Treatment time/harvest time:
-S9-mix: 24/24 hours and 48/48 hours (second test)
+S9-mix: 4/24 hours and 4/48 hours (second test)
- Dosing;
-S9-mix: 2.5, 8.33, 25.0, 83.3, 250, 833, 2500 ug/mL
(first test)
-S9-mix: 83.3, 250, 833, 1500 ug/mL (second test)
+S9-mix: 2.5, 8.33, 25.0, 83.3, 250, 833, 2500 ug/mL
(first test)
+S9-mix: 83.3, 250, 833, 2500 ug/mL (second test)
-Doses used for evaluation
-S9-mix: 83.3, 250, 833 ug/mL (first test)
-S9-mix: 250, 833, 1500 ug/mL (24 h harvest)
1500 ug/ml (48 h harvest) (second test)
+S9-mix: 250, 833, 2500 ug/mL (first test)
+S9-mix: 250, 833, 2500 ug/mL (24 h harvest)
2500 ug/mL (48 h harvest) (second test)
- Number of replicates: 2

Mitotic index: based on 1000 cells.
No. of metaphases analyzed: 100 metaphases/replicate (200/dose).
Evaluation criteria:
- Statistically significant increase in the aberrant cells frequency, with or without S9-mix
Statistics:
- At each dose level, data from the replicates were pooled
- A 2-way contingency table was constucted to analyze the frequencies of cytogenetic abnormalities. An overall Chi-square statistic, based on the table, was partitioned into components of interest.
- Two global hypotheses: (1) no differences in average number of cells with aberrations among the dose groups, and (2) no linear trend of increasing number of cells with aberrations with increasing dose
- ordinal metric used for the doses in the statistical evaluation
- If either statistic was found to be significant at α=0.01 versus a one-sided increasing alternative, pairwise tests (i.e., control vs treatment) were performed at each dose level and evaluated at α=0.01 again versus a one-sided alternative.
- Acceptance criteria: The chromosomal aberration frequency in the positive control cultures should be significantly higher than the negative controls, the aberration frequency in the negative control should be within reasonable limits of the laboratory historical values.
- considered positive in this assay if it induces a significant dose-related, and reproducible increase in the frequency of cells with aberrations
Species / strain:
lymphocytes: primary cultures from the rat
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Without metabolic activation: 833 ug/mL (Assay 1), 1500 µg/mL (Assay 2); With metabolic activation: no toxicity up to 2500 ug/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
GENOTOXIC EFFECTS:
- with metabolic activation: no effects
- without metabolic activation: no effects
MITOTIC INDEX:
- Without metabolic activation (shown as % change of MI compared to controls)
- 1500 ug/mL (24h): 78% of the control
- 1500 ug/mL (48 h): 86% of the control
- With metabolic activation
- 2500 ug/mL (24h and 48h): no significant reduction.

TEST-SPECIFIC CONFOUNDING FACTORS:
Highest dose was based on changes in pH/osmolality. pH at 2500 ug/mL was too high and was lowered to approximately 7.4. Osmolality at 2500 ug/mL was at an acceptable level (55-57 mOsmol/kg water above the control). Higher concentrations needed more HCl to adjust the pH, revealing an unacceptably high osmolality.

The following tables only show the confirmation assays (second round assays).

Table 1: Mitotic indicies (M.I.) of cell cultures treated with MIPA in the abscence of S-9

 

% M.I. Harvested 24 h After Treatment

% M.I. Harvested 48 h After Treatment

 

Replicate

Replicate

Average

Replicate

Replicate

Average

Dose µg/ml

A

B

A+B

A

B

A+B

 

Negative control a

 

7.3

 

9.3

 

8.3

 

6.3

 

5.8

 

6.1

83.3

10.2

8.0

9.1

8.8

6.9

7.9

250.0

8.5

10.3

9.4

7.3

6.8

7.1

833.0 b

7.9

5.9

6.9

3.4

3.7

3.6

1500.0 b

7.0

6.0

6.5

4.1

6.3

5.2

Positive control c

3.9

4.1

4.0

ND

ND

ND

Positivecontrol d

3.2

4.0

3.6

ND

ND

ND

a: 1% Water

b: 2N HCl was added to adjust the pH of the treatment medium.

c: MMC (0.05 µg/ml)

d: MMC (0.075 µg/ml)

ND: Not Done

Table 2: Mitotic indicies (M.I.) of cell cultures treated with MIPA in the presence of S-9

 

% M.I. Harvested 24 h After Treatment

% M.I. Harvested 48 h After Treatment

 

Replicate

Replicate

Average

Replicate

Replicate

Average

Dose µg/ml

A

B

A+B

A

B

A+B

Negative control a

9.2

13.0

11.1

4.5

6.2

5.4

250.0

6.0

7.6

6.8

8.6

6.2

7.4

833.0 b

9.1

11.0

10.1

9.0

9.1

9.1

2500.0 b

11.5

12.1

11.8

5.9

7.8

6.9

Positive control c

1.6

1.8

1.7

ND

ND

ND

a: 1% Water

b: 2N HCl was added to adjust the pH of the treatment medium.

c: CP (6 µg/ml)

ND: Not Done

Table 3: Results of the chromosomal aberration assay 24h after treatment in the abcence of S-9

Test Chemical: MIPA, Negative Control: 1% Water, Positive Control: (0.075 µg/ml MMC)

 

Neg. Control

250 µg/ml

833 µg/ml

1500µg/ml

Pos. Control

 

 

A

B

 

A+B

 

 A

B

A+B

A

B

A+B

A

 B

A+B

A

B

A+B

No. of cells scored

100

100

200

100

100

200

100

100

200

100

100

200

100

100

200

 

Chromatid Gaps

 

0

 

4

 

4

 

4

 

4

 

8

 

6

 

6

 

12

 

7

 

7

 

14

 

10

 

11

 

21

Chromosome Gaps

0

2

2

1

0

1

1

0

1

1

5

6

3

15

18

Chromatid Breaks

2

1

3

0

3

3

1

0

1

0

9

9

14

19

33

Chromatid Exchanges

0

0

0

0

0

0

0

0

0

0

0

0

15

7

22

Chromosome Breaks

0

0

0

0

0

0

0

0

0

1

0

1

8

9

17

Chromosome Exchanges

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Total Aberrations

2

1

3

0

3

3

1

0

1

1

9

10

37

36

73

(excluding gaps) a

 

 

(1.5)

 

 

(1.5)

 

 

(0.5)

 

 

(5.0)

 

 

(36.5)

No. of cells with Aberr.

2

1

3

0

3

3

1

0

1

1

8

9

24

32

56 b

(excluding gaps) a

 

 

(1.5)

 

 

(1.5)

 

 

(0.5)

 

 

(4.5)

 

 

(28.0)

Miscellaneous Aberr.

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

Cells with Multiple Aberr.

(5 or more aberr.)

0

0

0

0

0

0

0

0

0

0

0

0

1

0

1

a: Values in parentheses are percentages.

b: Significantly (alpha<0.01) different from the negative control.

Table 3: Results of the chromosomal aberration assay 24h after treatment in the presence of S-9

Test Chemical: MIPA, Negative Control: 1% Water, Positive Control: (6 µg/ml CP)

 

Neg. Control

250 µg/ml

833 µg/ml

2500 µg/ml

Pos. Control

 

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

No.of cells scored

100

100

200

100

100

200

100

100

200

100

100

200

100

100

200

 

Chromatid Gaps

 

2

 

0

 

2

 

1

 

4

 

5

 

3

 

0

 

3

 

1

 

0

 

1

 

42

35

77

Chromosome Gaps

0

1

1

1

1

2

1

1

2

1

1

2

15

22

37

Chromatid Breaks

2

1

3

0

1

1

1

1

2

0

0

0

29

24

53

Chromatid Exchanges

0

0

0

0

0

0

0

0

0

0

0

0

59

45

104

Chromosome Breaks

0

0

0

0

1

1

0

0

0

0

1

1

34

24

58

Chromosome Exchanges

0

0

0

0

0

0

0

0

0

0

0

0

0

2

2

Total Aberrations

2

1

3

0

2

2

1

1

2

0

1

1

122

95

217

(excluding gaps) a

 

 

(1.5)

 

 

(1.0)

 

 

(1.0)

 

 

(0.5)

 

 

(108.5)

No. of cells with Aberr.

2

1

3

0

2

2

1

1

2

0

1

1

61

60

121 b

(excluding gaps) a

 

 

(1.5)

 

 

(1.0)

 

 

(1.0)

 

 

(0.5)

 

 

(60.5)

Miscellaneous Aberr.

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Cells with Multiple Aberr.

0

0

0

0

0

0

0

0

0

0

0

0

2

5

7

(5 or more aberr.)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

a: Values in parentheses are percentages.

b: Significantly (alpha<0.01) different from the negative contral.

Conclusions:
The test material, MIPA, did not induce a significant increase in the frequency of cells with chromosomal abnormalities. Hence, it was concluded that under the experimental conditions used, MIPA was negative in this in vitro chromosomal aberration test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

MIPA was found not mutagenic in an in vivo Drosophila SLRL study upon feeding (24000 ppm MIPA) or injection (1900 ppm).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian germ cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 477 (Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster)
Version / remarks:
The guideline is not specified but given the scientific description of the study, performance similar to OECD 477 is suggested.
Principles of method if other than guideline:
Method: Woodruff, RC et al., Env. Mutagen., 6, 189-202.
Adult Canton-S males were subjected to a 3 day feeding exposure. They were mated to Basc females using a 2 to 3 day brooding pattern for a total of
three broods spanning 7 days. If the feeding sex-linked recessive lethal (SLRL) test was negative, an injection exposure was performed. As in the feeding exposure, a 2 to 3 day brooding pattern for three broods was used.
GLP compliance:
not specified
Type of assay:
Drosophila SLRL assay
Specific details on test material used for the study:
- Purity: 96.4 %
- Supplier: Aldrich (3803 LE)
Species:
Drosophila melanogaster
Strain:
other: Canton S
Sex:
male
Route of administration:
other: In feed. If the results of the feeding SLRL test were negative, an injection exposure was performed.
Vehicle:
Water
Duration of treatment / exposure:
feeding study: 3 days; If the results of the feeding SLRL test were negative, a single injection exposure was performed.
Frequency of treatment:
continuously in feed
Dose / conc.:
24 250 ppm
Remarks:
feeding study
Dose / conc.:
1 940 ppm
Remarks:
injection
No. of animals per sex per dose:
no data
Control animals:
yes, concurrent no treatment
Evaluation criteria:
A minimum of approximately 5,000 chromosomes were scored in each of the treated and concurrent control groups, unless the mutant frequency exceeded 1%. Clusters were identified using the Poisson distribution (Owen, 1962) and were removed before analysis.
Statistics:
The statistical evaluation of a SLRL test included a comparison with the concurrent solvent control using the normal approximation to the binomial distribution, as presented by Margolin et al. (1983), as well as a comparison with the historical control as described by Mason et al. (1992). In order to be considered mutagenic, the mutant frequency in the treated sample must exceed 0.15% with a P value of less than 0.05, or the treated frequency must exceed 0.1% with a P value of less than 0.01. If the treated frequency was between 0.1% and 0.15% and the P value was between 0.1 and 0.01; or if the treated frequency was higher than 0.15%, and the P value was between 0.1 and 0.05 the assay was considered equivocal. All other assays were considered negative.
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
not specified

Table 1: Results of the drosophila testing in Mono-isopropanolamin.

Dose (ppm)

ROA

Percent mortality

Percent sterility

Lethals

Tests

Total lethals

Total tests

Percent lethals

 

 

 

 

Br 1

Br 2

Br 3

Br 1

Br 2

Br 3

 

 

 

24,000

feeding

5

17

1

0

2

2,658

1,421

847

3

4,926

0.06

0

 

 

 

1

0

1

2,875

2,748

2,414

2

8,037

0.02

1,900

injection

14

8

2

2

2

1,911

1,675

1,562

6

5,148

0.12

0

 

 

 

0

2

0

1,739

1,493

1,079

2

4,311

0.05

One cluster of 3 in the injection control and one of 4 in the treated feeding experiment

Conclusions:
Under the conditions tested the test substance was not mutagenic in a Drosophila SLRL assay.
Thus, MIPA is not considered to be genotoxic.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No information available.

Additional information

IN VITRO TESTS

Bacterial mutation testing

MIPA was tested in the Ames reverse mutation assay using S. typhimurium strains TA98, TA100, TA1535 and TA1537 at 33 to 4000 µg/plate with and without metabolic activation. MIPA was slightly cytotoxic at the highest concentration in all 4 strains.

Under the conditions tested, MIPA was not mutagenic in strains TA98, TA100 and TA1537. However, with S9 -activation the TA 1535 strain presented some weak positive results. Therefore the test substance was rated as ambiguous. In contrast, in another Ames test MIPA was found not mutagenic in S. typhimurium strains TA98, TA100, TA 1535, TA1537 and TA 1538, and E. coli WP2 uvr A, incubated with 1 to 5000 µg MIPA/plate in the presence and absence of metabolic activation. Cytotoxicity was observed at the highest dose only. However, substance purity in the first Ames-test with ambigous result was 95% whereas substance purity in the second Ames-test with negative result was 97%. Therefore impurities are likely to be reponsible for the ambigous result of the test-substance with the lower purity. Taken together, the test-substance is considered to be not mutagenic.

Mammalian cell gene mutation testing

Induction of gene mutations in mammalian cells was investigated in an HGPRT assay (according to OECD guideline 476, under GLP) using Chinese hamster ovary (CHO) cells at 156 to 2500 µg/mL, with and without metabolic activation. The results indicate that MIPA did not result in gene mutations in this assay. No cytotoxicity was observed. Overall, MIPA is considered not to induce gene mutations.

MIPA tested at 83 - 1500 µg/mL (-S9) and 250 - 2500 µg/mL (+S9) did not induce significant increases in chromosomal aberrations using rat lymphocytes with and without metabolic activation (according to OECD guideline 473, under GLP). Cytotoxicity was observed without metabolic activation only, at 833 µg/mL. MIPA was judged non-clastogenic.

Since no genotoxic properties were revealed in the in vitro studies, no in vivo genotoxicity study is needed for MIPA.

IN VIVO TESTING

An in vivo sex-linked recessive lethal (SLRL) assay was performed in Drosophila melanogaster. Adult Canton-S males were subjected to a 3 day feeding exposure to 24000 ppm MIPA. They were mated to Basc females using a 2 to 3 day brooding pattern for a total of three broods spanning 7 days. A minimum of approximately 5000 chromosomes were scored per group unless the mutant frequency exceeded 1%. If the feeding test was negative, an injection exposure (1900 ppm) was performed. MIPA was found not mutagenic in the Drosophila SLRL test under the conditions tested (Foureman et al., 1994).


Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.