Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1,3 -Dichloropropanol has been found to be mutagenic in numerous strains of Salmonella typhimurium in the Ames test (Ziegler et al; Hahn et al), further in vitro testing was not conducted.

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
no
Remarks:
The study
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
purchased from Aldrich chemical
Label purity 95%
analysed purity 94%
Target gene:
HIstidine loci
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
Test concentrations with justification for top dose:
0 to 6666 µg/plate- The substance was initially tested in half log dose intervals upto the dose that elcited toxicity in the initial toxicity screen. At least 5 doses were tested in tripplicate.
Vehicle / solvent:
DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine TA98 without metabolic activation; 2-aminoanthracene all strains with metabolic activaiton
Details on test system and experimental conditions:
Cultures were grown overnight with shaking at 37°C in Oxoid No. 2 broth, and their phenotypes were analyzed prior to their use for mutagenicity assays.
Preparation of Liver S-9 Fractions The S-9 (9,OOOg supernatant) fractions of Aroclor 1254-induced, male Sprague- Dawley rat and male Syrian hamster livers were prepared. The S-9 mixes were prepared immediately prior to use and contained 10% S-9; The substance was tested in the absence of metabolic activation and with rat and hamster S-9 fractions.

A preincubation assay was performed . The test chemical (0.05 ml), Salmonella culture (0.10 ml), and S-9 mix or buffer (0.50 ml) were incubated at 37"C, without shaking, for 20 min. Chemicals known or suspected to be volatile were incubated in capped tubes. The top agar was added and the contents of the tubes were mixed and poured onto the surface of petri dishes containing Vogel-Bonner medium. The histidine-independent (his') colonies arising on these plates were counted following two days incubation at 37°C. Plates were machine counted (New Brunswick, Edison, NJ; Artek, Farmingdale, NY) unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the background agar. At the discretion of the investigators, plates with low numbers of colonies were counted by hand.
1) Testing in strains TA97, TA98, TA100, and TA1535, 10% S-9 was used. 2) The first test of a chemical was without activation and with 10% S-9 in the S-9 mix. If a positive result was obtained the test was repeated. If the tests were negative they were repeated without S-9 and with 30% S-9. 3) The order of use of 10% and 30% S-9 was reversed. 4) Initial testing was in strains TA98 and TA100 without activation and with 30% rat and hamster S-9s. If a positive result was obtained in one of these two strains it was repeated and the other strains were not used. If the tests were negative, the other strains were used with 30% and 10% S-9. A chemical was not designated nonmutagenic unless it had been tested in strains TA98, TA100, TA1535, and TA97 and/or TA1537, without activation and with 10% and 30% rat and hamster $9. Occasionally, 5% S-9 was also used in all protocol variations.
All chemicals were tested initially in a toxicity assay to determine the appropri- ate dose range for the mutagenicity assay. The toxicity assay was performed using TA1OO. Toxic concentrations were those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.
Rationale for test conditions:
Standard for the reverese mutation assay
Evaluation criteria:
Evaluations were made at both the individual trial and overall chemical levels. Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or nonmutagenic (-), depending on the magnitude of the increase of his+ revertants, and the shape of the dose-response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “ +W,” if only a single dose was elevated over the control, or if the increase seen was not dose related. During the study the standard evaluaiton critieria fro a positive result were not applied: 3 fold increase overcurrent solvent control for TA1535 and TA97 and 2 fold increase over solvent control for TA 98, TA100. Thes ehave been applied in the current analysis.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid

Dose

TA100

TA1535

TA97

TA98

 

no S9

Hamster S9

Rat S9

no S9

Hamster S9

Rat S9

no S9

Hamster S9

Rat S9

no S9

Hamster S9

Rat S9

µg/plate

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

133

9.1

89

1.5

125

4

27

4.4

9

2.9

11

2.5

132

6.6

150

11.5

184

12.7

15

0.9

28

3.4

26

1.2

100

147

4.7

177

3.5

139

3.8

28

4.2

27

1.5

14

1.3

126

1.5

148

3.1

174

9.7

17

3.3

32

3.8

31

2.1

333

182

7.2

359

11.8

173

21.5

59

5.5

34

2.4

23

3.3

140

4.9

151

10.7

183

13.2

17

3.8

31

2.8

28

3.2

1000

272

7.5

725

13.5

247

20.9

195

12.8

218

18.9

59

7.9

138

10.5

259

11.7

209

4.4

19

4.2

39

0.6

40

6.2

3333

511

15.1

1936

30.7

545

9.4

517

12.6

525

12.7

236

15.8

151

11.8

485

10

227

8.7

23

4

59

3.1

37

1.5

6666

934

9.9

2350

18.5

852

34.4

828

12.9

734

29.6

324

9.2

148

7

749

12.2

265

3.5

20

1.5

64

3.3

49

7.2

POS

521

10.7

446

25.7

801

34.9

336

16.2

43

3.1

176

13.3

238

16.2

293

10.3

1438

46.7

157

9.6

143

9.4

247

8

Conclusions:
Based upon the positive responses observed in all four strains tested, it can be concluded that the substance is a potential mutagen.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting 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)
Deviations:
yes
Remarks:
only 2 strains (TA100 and TA 1535 used), No plate incorporation assay conducted.
GLP compliance:
no
Remarks:
Data from openliterature study
Target gene:
histidne loci
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat S9
Test concentrations with justification for top dose:
1-62.8 µmol/plate (TA100)
1-78.6 µmol/plate (TA 1535)
Vehicle / solvent:
DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
other: 2-aminoanthracene
Details on test system and experimental conditions:

The 30-min preincubation test was performed according to Maron and Ames
using S~ typhimurium strain TA100 and strain TA1535, kindly provided by Professor B.N. Ames, with and without addition of
S9 mix (30.5 mg protein ml-1) prepared from livers of Wistar rats pretreated
with arochlor. The mean values of at least
two independent duplicate determinations were calculated. Differences in colony
counts were in all cases less than 20%. Colonies were counted using a Biotran
III automatic colony counter. The substances were dissolved in dimethyl sulphoxide
(10 µl). The average number of spontaneous revertants per plate was 190 for
strain TA100 and 22 for TA1535. 660 revertants per µg NaN3 per plate were
determined in the positive control for strain TA100 and 600 for TA1535. 1161
revertants per plate were obtained as positive control in the presence of S9 mix
with 2-aminoanthracene (1 µg per plate).
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

The results of the mutagenicity testing of 1,3-DCP-OH with S. typhimurium TA100 in the 30-min preincubation assay are shown in Table I. There are no significant differences between the results obtamed with and without addition of the metabolizing enzymes S9 mix or two different concentrations of ADH and cofactor NAD +. In order to examine whether the ADH present in S9 mix could cause activation, we added the cofactor NAD ÷ instead of NADP ÷ to S9 in an additional set of experiments (Table I). Here too no significant differences were observed. When using the S. typhimurium test strain TA1535 (Table II) a clear decrease in revertants was observed after addition of $9 mix or $9 mix and ADH whereas

addition of ADH and cofactor NAD ÷ without S9 mix did not influence the revertant numbers. In principle, an activating effect by ADH is possible and could be demonstrated with 2-chloropropenol. We have recently shown that 2-chloropropenol is metabolically transformed to 2-chloropropenal, an extremely strong mutagen . A comparison of the mutagenicity of 1,3-dichloroacetone and 1,3-DCP-OH shows that 1,3-dichloroacetone possesses a much higher activity than 1,3-DCP-OH. We found 23 875 revertants/#mol for 1,3-dichloroacetone in strain TA100 and 1137 in strain TA1535 but only about 33 revertants for 1,3-DCP-OH in both strains. Therefore activation should be

also expected if ADH converts 1,3-DCP-OH to 1,3-dichloroacetone. An activation by ADH and cofactors was, however, not observed in this case. In further mutagenicity studies we investigated the influence of the incubation time and whether preincubation of 1,3-DCP-OH without bacteria, in analogy to the SOS chromotest (see later), also leads to activation. A 90-min preincubation without S9 mix resulted only in marginally higher mutagenicity than was obtained with a 30-min preincubation. An additional incubation of the substance without bacteria for 30 min before the 90 rain preincubation with bacteria had also practically no effect.

TABLE I MUTAGENICITY OF 1,3-DCP-OH IN S. TYPHIMURIUM TA100 WITH AND WITHOUT S9 MIX AND WITH ADH IN REVERTANTS/PLATE AFTER 30-MIN PREINCUBATION

µmol/plate -S9 +S9 NADP+ +S9 NAD+ ADH + NAD+ 5µl ADH +NAD+ 50µl
1 239 258 245 224 210
2.6 241 281 281 248 282
5.2 323 375 326 324 358
7.9 351 441 379 371 360
10.5 418 480 418 431 395
15.7 484 559 473 499 504
26.2 630 696 589 689 609
36.7 793 843      
52.4 818 889 812 792 796
62.8 827 743 822 781 835

TABLE II MUTAGENICITY OF 1,3-DCP-OH IN S. TYPHIMURIUM TA1535 WITH OR WITHOUT ADDITION OF THE METABOLIZING ENZYMES $9 MIX OR ADH IN REVERTANTS/PLATE AFTER 30 MIN PREINCUBATION

µmol/plate -S9 +S9 NADP+ ADH + NAD+ 5µl ADH +NAD+ 50µl
1 69 33 40  
2.6 119 56 63 114
5.6 222 88 85 227
7.9 305 137 144 295
10.5 317 137 168 328
26.2 506 277 278 503
52.4 631 435 402 607
62.8   366 431  
Conclusions:
Exposure of TA100 and TA1535 strains of salmonella to the substance results in a clear positive result in the reverse mutaiton assay.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Subsequent to the positive results found in the Ames test, standard somatic testing in mouse bone marrow erythrocytes did not demonstrate genotoxic potential.Similarly no genotoxic potential was found in a Drosophila spp wing spot test. A COMET assay conducted by oral gavage in mice found a positive dose dependant indication of genotoxicity in the glandular stomach. This is indicative of a reactive, point of contact genotoxin which is not available for systemic exposure.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
mammalian comet assay
Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals and environmental conditions:
Age 8-10 weeks
body weight 20-35 g
supplier Taconic NIEHS colony
Food: Purina Certified 5002 Pelleted Diet, ad libitum

Housed singly
Temperature 18-26 celsius
relative humidity 30%
Light period 600-1800
Route of administration:
oral: gavage
Vehicle:
0.9% w/v saline
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: prepared in 0.9 % saline
at 2.5mg/ml, 5 mg/ml and 10 mg/ml
Frequency of treatment:
single dose
Post exposure period:
72 hours
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
No. of animals per sex per dose:
5 (five)
Control animals:
yes, concurrent vehicle
Positive control(s):
ethyl methanesulfonate 150 mg/kg/day
Tissues and cell types examined:
blood; colon; liver,left lobe; glandular stomach
Details of tissue and slide preparation:
tissues were lysed overnight and DNA samples prepared on Trevigen slides in 0.5% agarose. The DNA was unwound for 20 minutes at a pH of 13.2. Electrophoresis : 25V, 300 mA starting current at 28% relative humidity/21C temperature in a GTX5 COMET electophoresis system. DNA stained with SYBR gold stain.
Evaluation criteria:
COMET tails assessed using Perceptive Instruments software verison 4.11
Sex:
male
Genotoxicity:
positive
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: glandular stomach
Sex:
male
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: blood, colon, left liver lobe
Additional information on results:
All assessments were completed based on COMET tail length.

Mean COMET tail lengths in tested tissues

Dose Level COMET tail length mean COMET tail length (SEM) pairwise P value Organ Pairwise Test
Vehicle Control 26.52 0.85 Blood
Low Dose 29.45 0.82 0.154 Blood Williams
Mid Dose 26.21 0.93 0.186 Blood Williams
High Dose 30.36 0.91 0.004 Blood Williams
Positive Control 58.20 2.31 0.005 Blood Dunn
Vehicle Control 44.88 2.68 Colon
Low Dose 46.94 3.23 0.384 Colon Williams
Mid Dose 45.36 2.88 0.452 Colon Williams
High Dose 50.18 3.59 0.164 Colon Williams
Positive Control 65.58 5.11 0.004 Colon Williams
Vehicle Control 56.12 2.26 Liver
Low Dose 60.19 3.00 0.781 Liver Williams
Mid Dose 43.21 2.13 0.857 Liver Williams
High Dose 57.18 3.01 0.493 Liver Williams
Positive Control 71.95 2.25 0.001 Liver Williams
Vehicle Control 42.69 2.03 Stomach
Low Dose 64.31 4.92 0.038 Stomach Williams
Mid Dose 66.35 10.15 0.032 Stomach Williams
High Dose 75.73 11.37 0.006 Stomach Williams
Positive Control 84.25 6.99 0.000 Stomach Williams

Test for linear trend:

Colon LinearTrend 0.138105
Blood LinearTrend 0.030227
Stomach LinearTrend 0.007354
Liver LinearTrend 0.629107
Conclusions:
A significant increase in COMET tail length was observed in the glandular stomachs of male mice treated by oral gavage with the substance; the increase had a significant linear trend. Significant dose dependant increases in tail length were not observed in the other tissues tested. The investigator concluded that the substance was genotoxic in the glandular stomach, indicating a point of contact, rather than systemic genotoxin.
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
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 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
CB6F1
Sex:
male
Details on test animals and environmental conditions:
Age 8-10 weeks
body weight 20-35 g
supplier Taconic NIEHS colony
Food: Purina Certified 5002 Pelleted Diet, ad libitum

Housed singly
Temperature 18-26 celsius
relative humidity 30%
Light period 600-1800
Route of administration:
oral: gavage
Vehicle:
0.9% saline
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: prepared in 0.9 % saline
at 2.5mg/ml, 5 mg/ml and 10 mg/ml
Duration of treatment / exposure:
72 hours
Frequency of treatment:
single
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 (five)
Control animals:
yes, concurrent vehicle
Positive control(s):
150 mg/kg ethyl methane sulfonate
Tissues and cell types examined:
bone marrow erythrocytes
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Blood
Start Date Sample Collection Time Sex Methodology Used Route Dosing Regimen
23/09/2008 28 hour Male Flow Cytometry Gavage Gavage x 4, 4 day
  Dose Animal Polychromatic Erythrocytes Normochromatic Erythrocytes
(mg/kg) Number Trend P: 0.9057418632200795 Trend P: 0.5630751976468024
    No. Examined Total MN Cells Percent PCE MN Cells No. Examined Total MN Cells Percent NCE MN Cells
    per 1000 per 1000
Vehicle Control Saline  0          1 20000 62 1.4 3.1 1428487 2095 98.6 1.5
   0          2 20000 57 1.1 2.9 1724165 2304 98.9 1.3
   0          3 20000 49 1.3 2.5 1522067 2273 98.7 1.5
   0          4 20000 51 1.4 2.6 1389274 2125 98.6 1.5
   0          5 20000 54 1.2 2.7 1669659 2434 98.8 1.5
Average ± SEM   1.285 ± 0.053 2.730 ± 0.115   0.000 ± 0.000 1.457 ± 0.033
 
Test Chemical 1,3-Dichloro-2-propanol 25          10 20000 44 1.6 2.2 1201091 1817 98.4 1.5
  25          6 20000 52 1.4 2.6 1437928 2142 98.6 1.5
  25          7 20000 48 1.3 2.4 1576941 2422 98.7 1.5
  25          8 20000 40 1.3 2 1543362 2215 98.7 1.4
  25          9 20000 62 1.3 3.1 1482301 2267 98.7 1.5
Average ± SEM   1.375 ± 0.069 2.460 ± 0.189   0.000 ± 0.000 1.501 ± 0.018
Pairwise P   0.817   0.349
 
Test Chemical 1,3-Dichloro-2-propanol 50          11 20000 45 1.3 2.3 1549017 2174 98.7 1.4
  50          12 20623 41 1.2 2 1662551 2475 98.8 1.5
  50          13 20000 38 1.1 1.9 1769073 2455 98.9 1.4
  50          14 20000 49 1.3 2.5 1475861 2145 98.7 1.5
  50          15 20000 62 1.6 3.1 1222994 1825 98.4 1.5
Average ± SEM   1.313 ± 0.082 2.338 ± 0.214   0.000 ± 0.000 1.445 ± 0.021
Pairwise P   0.885   0.415
 
Test Chemical 1,3-Dichloro-2-propanol 100          16 20000 56 1 2.8 1903261 2781 99 1.5
  100          17 20000 37 1.1 1.9 1757290 2554 98.9 1.5
  100          18 20000 46 0.8 2.3 2494753 3909 99.2 1.6
  100          19 20000 61 0.8 3.1 2391448 3396 99.2 1.4
  100          20 20000 33 0.9 1.7 2220912 3186 99.1 1.4
Average ± SEM   0.936 ± 0.063 2.330 ± 0.268   0.000 ± 0.000 1.467 ± 0.026
Pairwise P   0.909   0.443
 
Positive Control Ethyl Methane Sulfonate 150          21 20000 177 1 8.9 2020359 3163 99 1.6
  150          22 20000 147 1.2 7.4 1637685 2642 98.8 1.6
  150          23 20000 235 1.1 11.8 1806498 3315 98.9 1.8
  150          24 20000 234 1.2 11.7 1718822 2967 98.8 1.7
  150          25 20000 203 1 10.2 1947659 3185 99 1.6
Average ± SEM   1.090 ± 0.042 9.960 ± 0.846   0.000 ± 0.000 1.675 ± 0.048
Pairwise P   0.005   0.003
 
Conclusions:
1,3-dichloropropan-2-ol did not induce any micronuclei in the femur bone marrow of male B6C3F1 mice at 25-100 mg/kg bw when examined using the OECD 474 test guideline
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Justification for classification or non-classification

The weight of evidence presented by the results of the Ames studies (positive in a number of strains of S.typhimurium) indicate that the substance does have mutagenic potential. However, the negative somatic mouse micronucleus study and negative findings in the somatic tissues in the mouse COMET assay indictate that there is negiligible concern that the substance is a Germ Cell mutagen. The positive findings in the mouse glandular stomach following oral dosing indicate that there is a point mutagen potential; in conjunction with the known carcinogenic properties of the substance, it is therefore recommended that the substance be classified as Germ Cell Mutagen Category 2 in accordance with the criteria set out in 1272/2008/EC.