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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Acclimation period, animals (2nd pre-experiment), relative humidity in the room 45 – 80 % , maximum temperature 20 – 25°C for about 6 hours; Supplier of the animals changed from Harlan Laboratories B.V. to Charles River Lab.(except 1st pre-experiment)
GLP compliance:
yes
Type of assay:
other: Micronucleus Assay in bone marrow of the Mouse

Test material

Reference
Name:
Unnamed
Test material form:
liquid
Details on test material:
- additional information as appropriate is presented in the respective study record
Specific details on test material used for the study:
Identity: Novares LS 500 (phenol, styrenated; CAS No 61788-44-1)
Batch No.: 28324
Composition of test material: composition is specified in IUCLID Sect. 13 - Assessment reports under Certificate of Analysis_Novares LS 500_phenol, styrenated
Stability in solvent: not indicated by the sponsor
Storage: At room temperature, protected from light
Expiration Date: unknown

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
Strain: NMRI
Source: Charles River Laboratories
Research Models and Services Germany GmbH
Sandhofer Weg 7, 97633 Sulzfeld, Germany
Number of Animals
used in the pre experiments: 2 males and 2 females (for each tested dose level)
used in the main experiment: 35 males
Initial Age at Start of
Experiment: 8 - 12 weeks
Acclimation: minimum 5 days
Initial Body Weight
at Start of Treatment: 1st application: mean value 40.3 g (SD  2.1 g)
2nd application: mean value 40.6 g (SD  2.1 g)



ENVIRONMENTAL CONDITIONS
Housing: single
Cage Type: Makrolon Type II/III, with wire mesh top
(EHRET GmbH, 79302 Emmendingen, Germany)
Bedding: granulated soft wood bedding
(Rettenmaier & Söhne GmbH + Co. KG,
73494 Rosenberg, Germany)
Feed: pelleted standard diet, ad libitum
(Harlan Laboratories B.V.; Postbus 6174;
5960 AD Horst; The Netherlands)
Water: tap water, ad libitum,
(Gemeindewerke, 64380 Rossdorf, Germany)
Environment: temperature 22  3°C
relative humidity 45 - 80 %
artificial light 6.00 a.m. - 6.00 p.m.



IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Name: 30% dimethylsulfoxide / 70% polyethylene glycol 400
(30% DMSO / 70% PEG 400)
Supplier: VWR-Merck
64295 Darmstadt
Catalogue no.: DMSO: 1.02931.1000; PEG 400: 1.09726.0100
Route and Frequency of Administration: orally, twice
Volume Administered: 10 mL/kg b.w.
Details on exposure:
orally
Duration of treatment / exposure:
--
Frequency of treatment:
twice
Post exposure period:
--
Doses / concentrations
Remarks:
Doses / Concentrations:
250, 500, and 1000 mg/kg b.w..
Basis:

No. of animals per sex per dose:
2 males and 2 females (for each tested dose level) used in the pre experiments
35 males were used in the main experiment
Control animals:
yes
Positive control(s):
Name: CPA; Cyclophosphamide
Supplier: Fisher Scientific GmbH
61130 Nidderau, Germany
Catalogue no.: 203960010 (purity: > 98 %)
Dissolved in: sterile water
Dosing: 40 mg/kg b.w.
Route and frequency of administration: orally, once
Volume administered: 10 mL/kg b.w.

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). At least one slide was made from each bone marrow sample.
Evaluation criteria:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To describe a cytotoxic effect, the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
All animals per test group were evaluated as described.

The study was considered valid as the following criteria are met:
- at least 5 animals per group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.
- the positive control shows a statistically significant and biological relevant increase of micronucleated PCEs compared to the negative control.
Statistics:
nonparametric Mann-Whitney test

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
in range finding studies, the lowest dose not exhibiting toxic effects was evaluated. This is the highest dose applied in the main study
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY (Clinical Signs on Toxicity):

In the first pre-experiment 4 animals (2 males, 2 females) received once orally a single dose of 2000 mg/kg b.w. Novares LS 500 (CAS No 61788-44-1) formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 mL/kg b.w..
The animals treated with 2000 mg/kg b.w. showed signs indicative of toxicity as shown in the table:
toxic
reactions hours post-treatment
male / female
1 h 2-4 h 6 h 24 h 30 h 48 h
ruffled fur 2/0 2/2 2/2 1/- 1/1 1/1
In order to increase the exposure of the animals to the test item upon sponsor’s request the study design changed from single treatment to double treatment spaced by 24 hours.

In the second pre-experiment 4 animals (2 males, 2 females) received twice orally at 24 h intervals a dose of 2000 mg/kg b.w. Novares LS 500 (CAS No 61788-44-1) formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 mL/kg b.w..
The animals treated with 2000 mg/kg b.w. showed signs indicative of toxicity as shown in the table:
Number of males / females with finding
hours post 1. application hours post 2. application
1 h 2-4 h 6 h 24 h 1 h 2-4 h 6 h 24 h
- reduction
of spontaneous
activity 0/0 0/0 0/0 0/0 0/0 0/0 1/1 0/0
- abdominal
position 0/0 0/0 0/0 0/0 0/0 0/0 1/1 0/0
- ruffled fur 0/0 0/0 0/0 0/0 0/0 1/0 0/0 0/0
death 0/0 0/0 0/0 0/0 0/0 0/0 0/0 1/1


In the third pre-experiment 4 animals (2 males, 2 females) received twice orally at 24 h intervals a dose of 1500 mg/kg b.w. Novares LS 500 (CAS No 61788-44-1) formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 mL/kg b.w..
The animals treated with 1500 mg/kg b.w. showed signs indicative of toxicity as shown in the table:
Number of males / females with finding
hours post 1. application hours post 2. application
1 h 2-4 h 6 h 24 h 1 h 2-4 h 6 h* 24 h
- reduction of
spontaneous
activity 0/0 0/0 0/0 0/0 1/2 2/2 2/2 -/-
- abdominal
position 0/0 0/0 0/0 0/0 0/0 0/0 2/2 -/-
- eyelid
closure 0/0 0/0 0/0 0/0 0/2 0/2 2/2 -/-
- ruffled fur 2/0 2/2 0/0 0/0 2/2 2/2 2/2 -/-
- tumble 0/0 0/0 0/0 0/0 0/0 0/0 1/1 -/-
- apathy 0/0 0/0 0/0 0/0 0/0 0/0 2/2 -/-
- convulsion 0/0 0/0 0/0 0/0 0/0 0/0 1/0 -/-
- death 0/0 0/0 0/0 0/0 0/0 0/0 2/2 -/-
*: Due to the severity of the clinical signs observed at that time point it was decided to euthanize all of the animals.
-/-: no observation possible.
In the fourth pre-experiment 4 animals (2 males, 2 females) received twice orally at 24 h intervals a dose of 1000 mg/kg b.w. Novares LS 500 (CAS No 61788-44-1) formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 mL/kg b.w..
The animals treated with 1000 mg/kg b.w. showed signs indicative of toxicity as shown in the table:
Number of males / females with finding
hours post 1. application hours post 2. application
1 h 2-4 h 6 h 24 h 1 h 2-4 h 6 h 24 h
ruffled fur 1/0 0/0 0/0 0/0 2/0 0/0 0/0 0/0


In the fifth pre-experiment 4 animals (2 males, 2 females) received twice orally at 24 h intervals a dose of 1250 mg/kg b.w. Novares LS 500 (CAS No 61788-44-1) formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 mL/kg b.w..
The animals treated with 1250 mg/kg b.w. showed signs indicative of toxicity as shown in the table:
Number of males / females with finding
hours post 1. application hours post 2. application
1 h 2-4 h 6 h 24 h 1 h 2-4 h 6 h 24 h
- reduction of
spontaneous
activity 0/0 0/0 0/0 0/0 1/2 1/1 1/1 0/-
- abdominal
position 0/0 0/0 0/0 0/0 0/0 0/1 0/1 0/-
- eyelid
closure 0/0 0/0 0/0 0/0 0/0 0/1 0/1 0/-
- ruffled fur 0/0 0/0 0/0 0/0 1/2 0/1 1/1 0/-
- tremor 0/0 0/0 0/0 0/0 0/0 0/0 0/1 0/-
- death 0/0 0/0 0/0 0/0 0/0 1/1 0/0 0/1
-/-: no observation possible.

On the basis of these data 1000 mg/kg b.w. was considered the maximum tolerated dose by using a double application spaced by 24 hours.
No sex specific differences were observed for clinical signs. In agreement with the sponsor the main study was performed using males only.


RESULTS OF DEFINITIVE STUDY:

In the main experiment the treated animals of the high (1000 mg/kg b.w.), mid (500 mg/kg b.w.) and low (250 mg/kg b.w.) dose of the test item, as well as the animals treated with the vehicle control (30% DMSO / 70% PEG 400) did not show any evidence of toxicity.

Any other information on results incl. tables

Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE/ total erythrocytes
scoring 48 hours after treatment

Table1: vehicle

animal no.

sex

test group

dose mg/kg b.w.

micronucleated cells per
2000 PCEs per animal

PCE per 2000 erythrocytes

    1

m

30% DMSO / 70% PEG 400

0

8

        1145

    2

m

 

2

        1276

    3

m

 

 

2

        1048

    4

m

 

 

3

        1150

    5

m

 

 

1

        1185

    6

m

 

 

2

        1063

    7

m

 

 

0

        1257

 

sum

18

        8124

 

mean

2.6

        1161

 

percent cells with micronuclei

0.129

 

Table2: test item

animal no.

sex

test group

dose mg/kg b.w.

micronucleated cells per
2000 PCEs per animal

PCE per 2000 erythrocytes

8

m

Novares LS 500

(CAS No 61788-44-1)

250

1

        1078

9

m

 

0

        1098

10

m

 

3

        1088

11

m

 

 

2

        1135

12

m

 

 

4

        1269

13

m

 

 

0

        1152

14

m

 

 

1

        1255

 

sum

11

        8075

 

mean

1.6

        1154

 

percent cells with micronuclei

0.079

 

Table3: test item

animal no.

sex

test group

dose mg/kg b.w.

micronucleated cells per
2000 PCEs per animal

PCE per 2000 erythrocytes

  15

m

Novares LS 500

(CAS No 61788-44-1)

500

2

        1082

  16

m

 

4

        1149

  17

m

 

0

        1135

  18

m

 

 

2

        1275

  19

m

 

 

2

        1149

  20

m

 

 

1

        1324

  21

m

 

 

0

        1217

 

sum

11

        8331

 

mean

1.6

        1190

 

percent cells with micronuclei

0.079

 

Table4: test item

animal no.

sex

test group

dose mg/kg b.w.

micronucleated cells per
2000 PCEs per animal

PCE per 2000 erythrocytes

  22

m

Novares LS 500

(CAS No 61788-44-1)

1000

1

        1350

  23

m

 

2

        1210

  24

m

 

2

        1350

  25

m

 

 

0

        1245

  26

m

 

 

0

        1174

  27

m

 

 

2

        1214

  28

m

 

 

4

        1139

 

sum

11

        8682

 

mean

1.6

        1240

 

percent cells with micronuclei

0.079

 

Table5: positive control

animal no.

sex

test group

dose mg/kg b.w.

micronucleated cells per
2000 PCEs per animal

PCE per 2000 erythrocytes

  29

m

Cyclophosphamide

40

56

        1248

  30

m

 

 

45

        1144

  31

m

 

 

39

        1257

  32

m

 

 

57

        1040

  33

m

 

 

51

        1137

  34

m

 

 

36

        1158

  35

m

 

 

34

        1190

 

sum

318

        8174

 

mean

45.4

        1168

 

percent cells with micronuclei

2.271

 

Summary of Micronucleus Test Results

test group

dose mg/kg b.w.

application before preparation (h)

PCEs with micronuclei (%)

range

PCE per 2000 erythocytes

vehicle

          0

    48

0.129

 0 -8

         1161

test item

      250

    48

0.079

 0 -4

         1154

test item

      500

    48

0.079

 0 -4

         1190

test item

    1000

    48

0.079

 0 -4

         1240

positive control

        40

    24

2.271

34 -57

         1168

Biometry

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Vehicle control versus test group

Significance

p

 250 mg Novares LS 500 (CAS No 61788-44-1)/kg b.w.

-

n.t.

 500 mg Novares LS 500 (CAS No 61788-44-1)/kg b.w.

-

n.t.

1000 mg Novares LS 500 (CAS No 61788-44-1)/kg b.w.

-

n.t.

   40 mg CPA/kg b.w.

+

0.0003

       -     =    not significant
        +    =    significant
       n.t. =    not tested

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.
Executive summary:

This study was performed to investigate the potential of Novares LS 500 (CAS No 61788-44-1) to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

Novares LS 500 was administered orally twice at an interval of 24 hours. The test item was formulated in 30% dimethylsulfoxide / 70% polyethylene glycol 400, which was also used as vehicle control; the dose volume was 10 mL/kg b.w.. A positive control group received cyclophosphamide once at 40 mg/kg b.w.. Forty eight hours after the first administration of the test item (24 h after the last treatment) the bone marrow cells were collected for micronuclei analysis.

Seven males per test group were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

The following dose levels of the test item were investigated: 250, 500, and 1000 mg/kg b.w..

The highest dose was estimated by a pre-experiment to be suitable. As observed in pre-experimental assessments, at higher dose levels, animals died.

The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that Novares LS 500 (CAS No 61788-44-1) did not have any cytotoxic properties in the bone marrow when given orally in this experiment. It is considered that the clinical signs and deaths expressed at doses higher than 1000 mg/kg bw, used in this experiment indicated that Novares LS 500 was absorbed and that the bone marrow would have been exposed to the test item.

In comparison to the corresponding vehicle controls, there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with Novares LS 500 (CAS No 61788-44-1) were below to the value of the vehicle control group. Additionally all values were within the historical vehicle control data base.

Cyclophosphamide administered once orally at 40 mg/kg b.w. was used as positive control, which showed a statistically significant increase of induced micronucleus frequency.