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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2013-07-08 to 2013-12-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with the GLP and according to agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 487
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)
Remarks:
2013-03-03
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Method

Species / strain
Species / strain / cell type:
other: cultured human lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor1254-induced S9 from rat livers
Test concentrations with justification for top dose:
39.06 to 2500 µg/mL
Vehicle / solvent:
Cells : Human peripheral blood lymphocytes
Culture medium : RPMI 1640 containing inactivated fetal calf serum, glutamine solution, antibiotics (penicillin, streptomycin), heparin and phytohaemagglutinin A solution.
Cytochalasin B concentration : 6 μg/mL
Solvent used : DMSO (Merck, batch K42958678 231)
Stability in solvent : unknown (dilutions were prepared extemporaneously)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: griseofulvin 10 μg/mL
Details on test system and experimental conditions:
GENOTOXICITY ASSAY
Carried out both without and with metabolic activation using Aroclor1254-induced S9 from rat livers.

Number of assays : 3
Number of cultures/concentration : 2
Number of analyzed cells :
+2000 to 4000 binucleated cells / concentration depending the experimental conditions
+ 2000 to 4000 mononucleated cells / concentration depending the experimental conditions
Factor limiting the maximum concentration analyzed : cytotoxicity

Concentrations tested expressed as μg/mL Rhodafac AAP XLP
Assay with 4 h treatment and 24 h recovery period:
- Without S9-mix : 2500 – 2174 – 1890 – 1644 – 1429 – 1243 – 1081
- With S9-mix (5% S9-mix) : 2500 – 2174 – 1890 – 1644 – 1429 – 1243 – 1081
Assay with 24 h treatment and no recovery period:
- Without S9-mix : 1429 – 1243 – 1081 – 940 – 817 – 545 – 363

In bold, concentrations actually assessed
Positive controls
- Short treatment
+ Without S9-mix : mitomycin C 0.15 μg/mL
+ With S9-mix : cyclophosphamide 10 μg/mL
- Continuous treatment : mitomycin C 0.075 μg/mL, griseofulvin 10 μg/mL
Evaluation criteria:
In the solvent control group, the number of micronucleated cells per binucleated and mononucleated cells were within the limits of values generally observed under our experimental conditions and were lower than 16 per 1000 (Van Hummelen and Kirsch-Volders, 1992).
Concurrently to the main assays, tests were carried out with reference mutagenic compounds (mitomycin C in the absence of metabolic activation and cyclophosphamide in the presence of metabolic activation via S9-mix) and with a reference aneugenic compound (griseofulvin in the continuous treatment in
absence of metabolic activation), in order to demonstrate the sensitivity of the cells and the effectiveness of the metabolic activation system. As expected statistically significant increases in the number of micronucleated cells were observed in the presence of mitomycin C, cyclophosphamide and griseofulvin.
The test item was toxic, but the highest concentration of the test item presented for the 2 subjects a CBPI equals or above 50 % compared with the control.
The acceptance criteria for the results were thus fulfilled.
Statistics:
Statistical analysis of the results obtained in the cells treated at each concentration level is performed using the χ2 test in comparison with those in control groups.

Results and discussion

Test results
Species / strain:
other: cultured human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity assay using a 4-hour treatment without metabolic activation, a strong but acceptable cytotoxicity was observed at 2500 μg/mL, with 55% of cytostasis (replication index of 45%). An important strong hemolysis was observed at this dose as well as a marked decrease in cell density. Under these conditions for the corresponding main assay, the concentration of 2500 μg/mL was retained as the maximum concentration to be tested with a narrowed range of doses.

In the preliminary toxicity assay using a 4-hour treatment with metabolic activation, a strong cytotoxicity was observed at 2500 μg/mL, with 77.7% of cytostasis (replication index of 22.3%). Moreover, a slight hemolysis was observed at this dose as well a marked decrease in cell density. In return, the concentration of 1250 μg/mL induced no cytotoxicity. Under these conditions for the corresponding main assay, the concentration of 2500 μg/mL was kept as the maximum concentration to be tested but a narrowed range of doses was used.

In the preliminary toxicity assay using a 24-hour treatment without metabolic activation, strong cytotoxicities were observed at 1250 and 2500 μg/mL, with 79.6 and 92.5% of cytostasis, respectively (replication indexes of 20.4 and 7.5%, respectively) Moreover, an important hemolysis was observed at the dose of 2500 μg/mL. The lower concentration of 625 μg/mL induced a slight cytotoxicity with 25.8% of cytostasis. Under these conditions for the corresponding main assay, the concentration of 1429 μg/mL was retained as the maximum concentration to be tested with a narrowed range of doses.
Remarks on result:
other: other: cultured human lymphocytes
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

 

Test item

Conc. in

μg/mL

% cytostasis

BINUCLEATED CELLS

MONONUCLEATED CELLS

Nb of

MBNC

/ 2000 BNC

P

Nb of

MBNC

/ 2000 BNC

P

 

Assay S9- 4h/+24h

 

Solvent

0

0.0

18.5*

-

6.5*

-

Mitomycin C

0.075

20.7

69

<0.001

15

<0.05

Rhodafac AAP

XLP

1644

23.1

22*

N.S.

6.5*

N.S.

1429

22.8

18*

N.S.

7

N.S.

1243

14.8

12

N.S.

8

N.S.

   

Assay S9+ 4h/+24h

 

Solvent

0

0.0

16

-

7

-

Cyclophosphamide

10

33.7

58

<0.001

8

N.S.

Rhodafac AAP

XLP

2174

30.0

22

 

10

N.S.

1890

21.3

21

 

9

N.S.

1644

14.6

14

 

5

N.S.

 

Assay S9- 24h/+0h

 

Solvent

0

0.0

24.5

-

6

-

Mitomycin C

0.075

34.5

53

<0.001

14

N.S.

Griseofulvin

10

19.2

40

<0.05

11

N.S.

Rhodafac AAP

XLP

940

46.4

25

N.S.

13

N.S.

817

23.3

13

<0.05

7

N.S.

545

1.1

29

N.S.

9

N.S.

MBNC: MicroBiNucleated Cells; BNC: BiNucleated Cells; MMNC: MicroMonoNucleated Cells; MNC:

% cytostasis= 100 - 100 [(CBPItreated-1) / (CBPIcontrol-1)]

With CBPI(Cytokinesis-Block Proliferation Index) =

(Nb of mononucleated cells + 2 x Nb of binucleated cells + 3 x Nb multinucleated cells) / Total Nb of cells

Applicant's summary and conclusion

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

The genotoxic activity of the test item Rhodafac AAP XLP (batch SP3F03X01) provided by Rhodia UK Limited was assessed by means of the in vitro micronucleus 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 study is thus considered as valid.
Under these experimental conditions, no genotoxic activity was observed.
Executive summary:

In an in vitro mammalian cell micronucleus test , performed according to the OECD No.487 and in compliance with the GLP,  the reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate (purity of 96%) diluted in Dimethylsulphoxide (DMSO) was tested in a human lymphocyte culture in the presence and the absence of mammalian metabolic activation (S9).

In the short treatment either with or without metabolic activation followed by a 24-hour recovery period (assay S9- 4h/+ 24h), the substance induced neither statistically nor biologically significant increase in the number of micronucleated cells at all the concentrations analyzed.

In the continuous treatment without metabolic activation without recovery period (assays S9- 24h/+0h), the substance induced neither statistically nor biologically significant increase in the number of micronucleated cells at all the concentrations analyzed from 545 and 940 μg/mL. The highest dose tested of 940 μg/mL induced an increase in the number of micronuclei in mononucleated cells over the doubling when compared to the negative control. Nevertheless, no dose effect relationship was observed, the mean value was close to the historical data and a slight difference between the slides was observed. This increase was thus considered as devoid of mutagenic hazard. It should be noted that a statistically significant decrease in the number of micronucleated binucleated cells was observed at the intermediary concentration analyzed of 817 μg/mL, but this effect had no meaning for genotoxicity assessment.

In conclusion, under the conditions of the test, The genotoxic activity of the substance was assessed by means of the in vitro micronucleus 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 study is thus considered as valid.

Under these experimental conditions, no genotoxic activity was observed.