Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Only in vitro test were available.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Septembre 19, 2013
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial gene mutation assay
Target gene:
Histidine gene mutation :
- his G46: TA 1535, TA 100
- his C 3076: TA 1537
- his D 3052: TA 98
- His G 428: TA 102

The two strains TA1535 and TA100 show a base pair substitution in the his G gene (G-C instead of A-T). These two strains can be used to detect test items which cause reverse base pair substitution. In return, the strain TA102 contains an ochre (T-A-A) mutation in the his G gene permitting to detect several types
of mutation not only reverse base pair substitution but also small deletions.
The strain TA1537 has, in the his C gene, a loop of several cytosines which gives rise to a frameshift mutation. The strain TA98 contains, in the his D gene, a sequence of four base pairs of cytosine-guanine (CG) which also give rise to a frameshift mutation. These two strains can be used to detect products causing reverse frameshift mutation. The five strains also carry two other types of mutation: they are deficient in DNA repair mechanisms (uvrB-) - apart from the strain TA102 which conserves an intact excision repair system - and show a change in the structure of cell-wall lipopolysaccharides (LPS) (rfa-). These two mutations make the bacteria more sensitive to genotoxic agents and more permeable to cell penetration by large molecules. The R factor (present in strains TA98, TA100 and TA102), which is a pKM 101 ampicillin resistance plasmid, increases sensitivity to mutagenic agents via error-prone repair mechanisms. In addition, the strain TA102 contains the multicopy plasmid, pAQ1, which carries the his G428 mutation and a tetracycline resistance gene (Maron and Ames, 1983).

Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
The S9 fraction was prepared at Institut Pasteur de Lille (IPL). This preparation is carried out using the method described by Ames et al. (1975) in male rat Sprague Dawley OFA induced by Aroclor 1254.
Test concentrations with justification for top dose:
0, 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
The test item Hydrate de carbone et sucres, hexitol, anhydro - LAB4623 was dissolved in distilled water (Fresenius, batch 13ECP201) at the maximal initial concentration of 50 mg/mL taking into account a purity of 85% in order to obtain the top dose of 5000 μg/plate when added at 100 μL/plate.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-anthramine
Details on test system and experimental conditions:
PRELIMINARY TOXICITY TEST
In order to choose the range of doses for the test, the toxic activity of the test item was determined. The toxicity assay was carried out in all the strains to be tested under the same conditions as the mutagenicity test with and without metabolic activation but using only one plate per dose instead of 3. The plates were incubated for approximatively 48 hours at 37°C, and the revertants were counted. Toxicity was checked by microscopic examination of the background growth and was noted as follows:
- : non-toxic
+ : slightly toxic
++ : moderately toxic
+++ : strongly toxic
N : no bacterial growth


MUTAGENICITY TEST
1. Without metabolic activation
The following technique was performed for each one of the strains used in the test: 0.1 mL of a bacterial suspension from a culture agitated overnight at 37°C and 0.1 mL of the test item at the relevant initial concentrations were successively added to 2 mL of top agar to which 10 % of 0.5 mM biotin histidine solution, maintained in a state of superfusion at 45°C, has been added. The content of each tube was agitated, then spread out in a Petri plate containing 20 mL of minimum agar. Three plates were used per treatment. The plates were then incubated at 37°C for approximately 48 h. At the end of the expression time, colonies of revertants were counted for each plate.
CONTROLS
• Solvent controls, Positive controls
At the same time, solvent controls (0.1 mL solvent/plate) were performed under the same conditions but using 6 plates per solvent . Appropriate positive reference controls were also performed.
• Sterility of the media
At each assay, 3 Petri plates containing 20 mL of minimal agar receive 2 mL of top agar only and were incubated under the same conditions of assay for the control of media sterility. No colony was observed after 48 hours at 37°C. The results were satisfactory.


2. With metabolic activation
The method was the same as the one described above except that immediately before spreading in the plates, 0.5 mL of the S9 mix metabolic activation system was added in soft agar.
CONTROLS
Solvent controls, positive controls and assay for the control of media sterility were performed like in the mutagenicity assay without metabolic activation.

3. Repeat test
1. Without metabolic activation
The test was subsequently repeated in an independent assay. The same method was used but the test dose range was modified, depending on the results obtained during the first test.

2. With metabolic activation
According to the results obtained in the first assay in the presence of metabolic activation, the second assay can either be performed using a standard plate-incorporation technique, or be extended by the use of the pre-incubation modification of the assay. As negative responses were observed in the first assay, the method used in a second assay was the pre-incubation test.
On a technical point of view, this method is the same as the one using agar plate incorporation with, however, the following modifications: The following solutions were added in this order: the bacterial strain to be tested (100 μL), S9-mix (500 μL) and at the end the test item solution (100 μL as aqueous solvent was used). The mixture was preincubated with stirring at 37°C for 60 minutes prior to adding soft agar and spreading out in a Petri plate.
Evaluation criteria:
After about 48 hours of incubation at 37°C, the prototrophic mutant colonies that have developed in the plates were counted, eventually using a colony counter. The results are expressed as the mean number of revertants per plate and, for each concentration of the test product, the following ratio was established: Mean number of revertants per plate in presence of the test product / Mean number of revertants per plate without the test product (solvent control).


Validity criteria for the test
After spreading out under the conditions of the bacteriostatic test and after 48-hour incubation at 37°C, no colony was visible.
Concurrently to the main assays, tests were carried out on reference mutagenic test compounds in order to show the sensitivity of the strains tested and the efficiency of the metabolic activation system.
Statistically and biologically significant increases in the numbers of revertants were observed in the presence of positive reference test substances. The values observed were within the limits of historical controls. The frequencies of spontaneous revertants (solvent controls) were within the limits generally observed under our experimental conditions.
The validity criteria for the test were fulfilled.
Statistics:
Data were analysed by means of Dunnett's method allowing the comparison of the mean value for each dose to the mean value for the corresponding solvent control.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TOXICITY ASSAY
The test item Hydrate de carbone et sucres, hexitol, anhydro - LAB4623 induced no toxicity whatever the dose and the strain tested, both with and without metabolic activation. Therefore, the maximum doses retained for the first mutagenicity assay was 5000 μg/plate.

TOXICITY IN MUTAGENICITY ASSAYS
In both assays, no toxicity was noted, either with or without metabolic activation.

In the first assay in strain TA100 in absence of metabolic activation, statistically significant increases were observed at all the doses tested of from 50 to 5000 μg/plate, except at the intermediary dose of 500 μg/plate with induction ratios of 1.4 and 1.5, i.e. clearly below the threshold ratio for a biologically significant response (set at 2 in this strain) and no dose-response relationship was observed. Moreover, this effect was not reproducible as in the second assay performed in the same experimental condition, no statistically or biologically significant increase was observed at any of the doses
tested ranging from 50 to 5000 μg/plate.
The test item was thus considered as not mutagenic in this condition. In the first assay in strain TA102 in absence of metabolic activation, a statistically significant increase was observed at the intermediate dose tested of 500 μg/plate with an induction ratios of 1.7, i.e. below the threshold ratio for a biologically significant response (set at 2 in this strain) and no doseresponse relationship was observed. Nevertheless, this effect was not reproducible as in the second assay performed in the same experimental condition with a narrowed range of doses, no statistically or biologically significant increase was observed at any of the doses tested ranging from 50 to 5000 μg/plate.
The test item was thus considered as not mutagenic in this condition.
In the second assay in strain TA1537 in presence of metabolic activation, a statistically significant increase in the number of revertants was noted at the intermediary dose of 150 μg/plate with an induction ratio of 1.8. This value was far from the threshold ratio for a biologically significant response
set at 3 in this strain and there was no relation-dose effect.
The test item was thus considered as not mutagenic in this condition.
In the second assay in strain TA98 in presence of metabolic activation, non-statistically significant increases were observed at both the lowest and the highest doses tested of 50 and 5000 μg/plate with induction ratios of 1.7 and 1.8, respectively, i.e. below to the threshold ratio for a biologically
significant response (set at 2 in this strain). However, the mean numbers of revertants at these doses were below the highest value already observed in historical data for negative control (i.e. 32.3 and 35.7vs. 47). Furthermore, no dose-response relationship was observed.
The test item was thus considered as not mutagenic in this condition.
In two independent assays performed either with or without metabolic activation (the second assay with S9-mix was performed according to the pre-incubation protocol), no biologically significant increase in the mean number of revertants was noted in the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested, in the presence of Hydrate de carbone et sucres, hexitol, anhydro - LAB4623.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

BACTERIAL REVERSE MUTATION TEST RESULT ON ASSAY 1

 

TA 1535

TA 1537

TA 98

 

DOSES inµg/plate

revertants/plate

Induction

Ratio (a)

DOSES in µg/plate

revertants/plate

Induction

Ratio (a)

DOSES inµg/plate

revertants/plate

Induction

Ratio (a)

Positive control

(b)

209.3

24.1

(b)

182.0

55.2

(b)

278.0

20.6

TEST ITEMWITHOUT S9-mix

0

8.7

-

0

3.3

-

0

13.5

-

50

10.0

1.1

50

4.7

1.4

50

16.7

1.2

150

9.0

1.0

150

5.0

1.5

150

15.7

1.2

500

11.0

1.3

500

5.3

1.6

500

18.0

1.3

1500

5.7

0.7

1500

5.7

1.7

1500

16.7

1.2

5000

12.7

1.5

5000

5.0

1.5

5000

19.0

1.4

Positive control

(c )

308.7

33.2

(c )

166.0

26.8

(c )

1461.3

55.1

TEST ITEMWITH S9-mix

WITH PRE-INCUBATION

0

9.3

-

0

6.2

-

0

26.5

-

50

10.7

1.2

50

4.3

0.7

50

24.0

0.9

150

9.3

1.0

150

6.3

1.0

150

26.0

1.0

500

9.0

1.0

500

6.7

1.1

500

17.7

0.7

1500

7.7

0.8

1500

8.7

1.4

1500

21.3

0.8

5000

12.3

1.3

5000

5.7

0.9

5000

35.0

1.3

 

 

TA 100

TA 102

 

DOSES inµg/plate

revertants/plate

Induction

Ratio (a)

DOSES inµg/plate

revertants/plate

Induction

Ratio (a)

Positive control

(b)

313.3

5.0

(b)

1312.0

13.0

TEST ITEMWITHOUT S9-mix

0

63.2

-

0

101.3

-

50

89.0

1.4

50

126.7

1.3

150

88.3

1.4

150

120.7

1.2

500

82.0

1.3

500

175.3

1.7

1500

91.7

1.5

1500

138.0

1.4

5000

96.3

1.5

5000

136.0

1.3

Positive control

(c )

1232.0

12.6

(c )

1546.7

5.9

TEST ITEMWITH S9-mix

WITH PRE-INCUBATION

0

97.7

-

0

261.7

-

50

79.3

0.8

50

262.0

1.0

150

97.0

1.0

150

285.3

1.1

500

113.3

1.2

500

313.3

1.2

1500

99.7

1.0

1500

306.7

1.2

5000

97.3

1.0

5000

301.3

1.2

 

 (a) Induction Ratio = number of revertants in the treated / number of revertants in the control

Reference positive compounds (μg/plate):

(b) TA1535 and TA100: Sodium azidew 1; TA1537: 9-amino-acridined 50; TA98: 2-nitrofluorened 2; TA102: Mitomycin Cw 0.125

(c) TA1535, TA1537, TA98, TA100: 2-anthramined 2; TA102: benzo(a)pyrened 2

Solvents used for positive controls: d DMSO; w distilled water


 

BACTERIAL REVERSE MUTATION TEST RESULT ON ASSAY 2

 

TA 1535

TA 1537

TA 98

 

DOSES inµg/plate

revertants/plate

Induction

Ratio (a)

DOSES in µg/plate

revertants/plate

Induction

Ratio (a)

DOSES inµg/plate

revertants/plate

Induction

Ratio (a)

Positive control

(b)

254.0

28.9

(b)

716.0

137.7

(b)

361.3

22.6

TEST ITEMWITHOUT S9-mix

0

8.8

-

0

5.2

-

0

16.0

-

50

7.7

0.9

50

5.0

1.0

50

15.7

1.0

150

12.3

1.4

150

7.7

1.5

150

14.0

0.9

500

12.3

1.4

500

8.0

1.5

500

14.3

0.9

1500

6.7

0.8

1500

4.3

0.8

1500

10.7

0.7

5000

7.3

0.8

5000

7.0

1.3

5000

8.7

0.5

Positive control

(c )

290.0

36.3

(c )

220.7

31.5

(c )

2410.7

123.6

TEST ITEMWITH S9-mix

WITH PRE-INCUBATION

0

8.0

-

0

7.0

-

0

19.5

-

50

11.3

1.4

50

10.0

1.4

50

32.3

1.7

150

9.7

1.2

150

12.7

1.8

150

24.3

1.2

500

10.3

1.3

500

5.7

0.8

500

19.3

1.0

1500

9.7

1.2

1500

6.7

1.0

1500

29.7

1.5

5000

7.0

0.9

5000

8.3

1.2

5000

35.7

1.8

  

 

TA 100

TA 102

 

DOSES inµg/plate

revertants/plate

Induction

Ratio (a)

DOSES inµg/plate

revertants/plate

Induction

Ratio (a)

Positive control

(b)

866.7

9.1

(b)

1381.3

7.9

TEST ITEMWITHOUT S9-mix

0

95.0

-

0

174.0

-

50

93.3

1.0

50

196.7

1.1

150

93.3

1.0

150

171.3

1.0

500

92.0

1.0

500

188.0

1.1

1500

90.7

1.0

1500

220.7

1.3

5000

104.3

1.1

5000

220.0

1.3

Positive control

(c )

1664.0

21.0

(c )

1338.7

4.8

TEST ITEMWITH S9-mix

WITH PRE-INCUBATION

0

79.3

-

0

280.7

-

50

84.3

1.1

50

286.7

1.0

150

80.3

1.0

150

301.3

1.1

500

87.7

1.1

500

302.7

1.1

1500

92.0

1.2

1500

322.0

1.1

5000

92.3

1.2

5000

318.0

1.1

 (a) Induction Ratio = number of revertants in the treated / number of revertants in the control

Reference positive compounds (μg/plate):

(b) TA1535 and TA100: Sodium azidew 1; TA1537: 9-amino-acridined 50; TA98: 2-nitrofluorened 2; TA102: Mitomycin Cw 0.125

(c) TA1535, TA1537, TA98, TA100: 2-anthramined 1; TA102: benzo(a)pyrened 2

Solvents used for positive controls: d DMSO; w distilled water

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation system

The mutagenic activity of the test item Hydrate de carbone et sucres, hexitol, anhydro - LAB4623 was assessed by means of the Ames’ test in the
five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested either in presence or in absence of metabolic activation, in two independent assays. The validity criteria for the assay were fulfilled. The study was thus considered as valid.
Under these experimental conditions, no mutagenic activity was revealed.
Executive summary:

The search for any mutagenic activity of HYDRATE DE CARBONE ET SUCRES, HEXITOL, ANHYDRO - LAB4623 was studied by means of the Ames’ test (Salmonella his-/microsome system) in compliance with the Commission Regulation EC 440/2008 and the OECD Guideline 471.

5 strains of Salmonella typhimurium were tested : TA1535, TA1537, TA98, TA100, TA102, in distilled water at different dilutions : 0, 50, 150,500, 1500, 5000 µg/plate.

A Preliminary test of toxic activity was carried out on 5 strains for an incubation period of 48 h.The examination of the background growth demonstrated that Hydrate de carbone et sucres, hexitol, anhydro - LAB4623 induced no toxicity.

Therefore, the maximum doses retained for the first mutagenicity assay was 5000 μg/plate in all strains, both with and without S9-mix.

The mutagenicity assays were carried out on 5 strains both with and without metabolic activation using hepatic microsomes from rat livers induced by Aroclor 1254 – The incubation period was 48h.

2 assays were performed, the second assay with metabolic activation was performed according to pre-incubation method.

No biologically significant increase in the number of revertants was noted whatever the strain and the dose tested. The test item Hydrate de carbone et sucres, hexitol, anhydro - LAB4623 was thus considered as not mutagenic.

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

Additional information

Additional information from genetic toxicity in vitro:

The mutagenic activity of the test item Hydrate de carbone et sucres, hexitol, anhydro - LAB4623 was assessed by means of the Ames’ test in the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested either in presence or in absence of metabolic activation, in two independent assays. The validity criteria for the assay were fulfilled. The study was thus considered as valid.

Under these experimental conditions, no mutagenic activity was revealed. The search for any mutagenic activity of LAB 4623, was studied by means of gene mutation test at the TK locus in L5178Y mouse lymphoma cell culture in compliance with the Commission Regulation EC 440/2008 and the OECD Guideline 476, in 2 independent assays without and with metabolic activation. The acceptance criteria for the assay were fulfilled. The current study was considered as valid. Under these experimental conditions, the test item LAB 4623, induced no mutagenic activity being demonstrated at the TK locus in L5178Y mouse lymphoma cell culture.

The genotoxic activity of the test item LAB 4623 was assessed by means of the in vitro metaphase analysis test in human lymphocytes treated in presence and in absence of metabolic activation, either with a short or with a continuous treatment. The acceptance criteria for the assay were fulfilled. The current study was considered as valid. Under these experimental conditions, the test item LAB 4623 induced no clastogenic activity in human lymphocytes either in presence or in absence of metabolic activation.

Justification for classification or non-classification