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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
Study period:
December 2015 - January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals, number 420 "Acute Oral Toxicity - Fixed Dose Procedure"
Version / remarks:
(adopted: December 17, 2001)
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals, number 423 "Acute Oral Toxicity - Acute Toxic Class Method"
Version / remarks:
(adopted: December 17, 2001)
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Test material

Constituent 1
Chemical structure
Reference substance name:
p-vinylphenol
EC Number:
220-103-6
EC Name:
p-vinylphenol
Cas Number:
2628-17-3
Molecular formula:
C8H8O
IUPAC Name:
4-vinylphenol
Test material form:
liquid
Details on test material:
- Name as cited in the report: 4-Vinylphenol
- name: p-Vinylphenol
- CAS: 2628-17-3
- Batch No.: STBF6743V
- Storage Conditions: room temperature
- Expiration date: June 2020
- Description: yellow liquid
- Purity: 9.7% in propylene glycol, FG

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
ANIMALS
Species: Mouse (mus musculus)
Strain: NMRI, young healthy adult
Source: Charles River, 97633 Sulzfeld, Germany
Number of animals: 5 of each sex per dose group
Initial age at start of acclimatisation: 6 -12 weeks
Age at start of treatment: Minimum 7 weeks
The animals were derived from a controlled full barrier maintained breeding system (spf). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes and underwent an adequate acclimatisation period after arrival. The animals were randomly distributed to test groups.

HUSBANDRY
The animals were barrier maintained (semi-barrier) in an air conditioned room. The experiment was conducted under standard laboratory conditions.
Housing: 5 animals of identical sex per cage
Cage type: IVC cage (Polysulphone), Type 11 L
Bedding: Altromin sawfiber bedding (Bateh: 02102150820)
Feed: Free access to Altromin 1324 (Bateh: 0631) maintenance diet for rats and mice
Air change: At least 10 x per hour
Water: Free access to tap water, sulphur acidified to pH value of approx. 2.8 (drinking water, municipal residue control, micro-biologically controlled at frequent intervals)
Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich.
The animals were randomly distributed into the test groups. The animals of each test group and sex were housed in separate cages that were individually marked (study number, sex, control / test group). The animals of each cage were individually marked for identification by tai! and ear drawing.

ENVIRONMENT
Temperature 22 ± 3 °C
Relative humidity 55 ± 10%
Artificial light 6:00 - 18:00

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Propylene gycol and aqua ad iniectabilia
Details on exposure:
PREPARATION OF THE TEST ITEM
The test item was available as solution in propylene glycol with a content of 4-Vinylphenol of 9.7%. To obtain a non-toxic dose of propylene glycol the test item was further diluted in Aqua ad iniectabilia.
For all dose groups the test item was administered as split dose with a time period of 2 h between both applications. The volume administered ip for each application was 10 mL/kg bw.
Duration of treatment / exposure:
Single administration of a split dose with a time period of 2 h between applications.
Frequency of treatment:
The animals received the test item twice as split dose by the intraperitoneal route with a time period of 2 h between applications.
Post exposure period:
44 h and 68 h
Doses / concentrationsopen allclose all
Dose / conc.:
19.4 mg/kg bw (total dose)
Remarks:
0.2 MTD
Dose / conc.:
48.5 mg/kg bw (total dose)
Remarks:
0.5 MTD
Dose / conc.:
97 mg/kg bw (total dose)
Remarks:
1 MTD
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- Name: CPA (cyclophosphamide)
- CAS No.: 50-18-0
- Supplier: Sigma
- Catalogue No.: C0768
- Batch No.: SLBG4216V
- Dissolved in: physiological saline
- Dosing: 40 mg/kg bw
- Route and frequency of administration: ip, single
- Volume administered: 10 mL/kg bw

The solution was aliquoted and stored at <=-15°C. On day of administration the solution was freshly thawed. The stability of CPA at room temperature is quite good (3.5% is hydrolysed per day in aqueous solution). It is acceptable that the positive control can be administered by a route different from the test agent and sampled at only a single time. The sampling time for the positive control is 44 h after treatment.

Examinations

Tissues and cell types examined:
Blood cells / erythrocytes
Details of tissue and slide preparation:
see "Any other information on materials and methods"
Evaluation criteria:
There are several criteria for determining a positive result:
- dose-related increase in the number of micronucleated cells and/or
- biologically relevant increase in the number of micronucleated cells for at least one of the dose groups.
According to the OECO guideline, the biological relevance as well as the statistical significance of the results are the criteria for the interpretation.
A test item is considered to be negative if there is no biologically relevant and/or statistically significant increase in the number of micronucleated cells at any dose level.
Statistics:
For the statistics the nonparametric Mann-Whitney test was used.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
other: vehicle served as negative control
Positive controls validity:
valid

Any other information on results incl. tables

PRE-EXPERIMENT

The test item was available as solution in propylene glycol with a content of 4-Vinylphenol of 9.7%.

To rule out that propylene glycol per se causes systemic toxicity, the highest non-toxic dose level of propylene glycol was determined first. One male mouse received a single dose of 10 mL/kg bw pure propylene glycol ip and showed strong signs of systemic toxicity such as reduction of spontaneous activity, prone position, ataxia, bradykinesia, constricted abdomen and abnormal breathing. Based on animal welfare aspects the mouse was euthanized one hour after application. The dose was reduced to 2 mL/kg bw and applied ip to one male animal. The mouse showed strong systemic toxicity such as reduction of spontaneous activity, prone position, ataxia, bradykinesia and hunched posture and was euthanized two hours after application based on animal welfare aspects. The dose was reduced to 1 mL/kg bw and applied ip to one male animal. The mouse showed clear signs of systemic toxicity such as reduction of spontaneous activity, prone position, ataxia, bradykinesia and constricted abdomen and was euthanized 30 minutes after application based on animal welfare aspects. The dose was further reduced to 0.5 mL/kg bw and applied ip to one male animal. This animal did not show any clinical signs of systemic toxicity during a time period of two hours after application. Thus the animal received a second dose of 0.5 mL/kg bw propylene glycol ip two hours after the first application and did not show any symptoms during a time period of 72 h.

Based on these findings a cumulative intraperitoneal dose of propylene glycol of 1 mL/kg bw applied as split dose of two times 0.5 mL/kg bw with a time period of 2 h between applications was

determined as the highest non-toxic dose of propylene glycol.

To obtain a non-toxic dose of propylene glycol the test item was further diluted in Aqua ad iniectabilia at a ratio of 1:20 (w/v) leading to a concentration of the 4-Vinylphenol/propylene glycol

solution of 50 mg/mL corresponding to 4.85 mg/mL 4-Vinylphenol. The test item was administered two times at a dose of 500 mg/kg bw 4-Vinylphenol/propylene glycol solution corresponding to 48.5 mg/kg bw 4-Vinylphenol with a time period of 2 h between applications leading to a cumulative dose of 97 mg/kg bw 4-Vinylphenol.

The selection of the highest dose was conducted in accordance with the following current international guidelines for assessment of acute toxicity (OECD 420, OECD 423), particularly with respect to selection of dose spacing and animal welfare aspects.

In the pre-experiment a cumulative dose of 97 mg/kg bw of 4-Vinylphenol administered as split dose of two applications with 48.5 mg/kg bw each was evaluated. Three male and three female mice received a cumulative dose of 97 mg/kg bw ip and showed toxicity such as reduction of spontaneous activity, prone position, bradykinesia, constricted abdomen, ataxia, hunched posture, piloerection and half eyelid / eye closure.

Due to the results obtained in the pre-experiment 97 mg/kg bw 4-Vinylphenol was chosen as maximum tolerated dose (1 MTD) in the main experiment.

Signs of toxicity in the pre-experiment (97 mg/kg bw)

 Signs

Time post-application / sex (3 male and 3 female mice)

 

1. Application

 30 min

 1 h  2 h

 2. Application

30 min

 1 h  2 h  3 h  4 h 24 h 48 h 72 h
   m  f  m  f  m  f  m  f  m  f  m  f  m  f  m  f
 Red. spont. activity  3  3
 Prone position
   Constricted abdomen
   Bradykinesia
 Ataxia 0 0 0 0 0 2 3
Hunched posture 
  Piloerection
  Half eyelid closure
 Eye closure

MAIN EXPERIMENT

Toxicity

All animals treated with the highest dose (1 MTD) showed strong toxic effects. Male animals showed symptoms until 44 h after the second application. Female animals showed toxic symptoms until 24 h after the second application. Mice treated with 48.5 mg/kg bw (0.5 MTD) showed the same signs of toxicity as displayed for the 1 MTD dose group animals execpt for ataxia,

hunched posture and eye closure. These signs of toxicity were in total less intensely developed (mild/moderate) than in the 1 MTD group animals. 24 h after the second application no toxic

symptoms were observed anymore in the male and female 0.5 MTD animals. The animals treated with 19.4 mg/kg bw (0.2 MTD) showed mild toxic effects after the treatment with the test item. 1 h after the second applieation no toxic symptoms were observed anymore in this group.

The weight variation of the animals did not exceed ± 20% of the mean weight of each sex, as recommended in OECD guideline 474.

Signs of toxicity in the main experiment (97 mg/kg bw)

 Signs

Time post-application / sex (5 male and 5 female mice)

 

1. Application

 30 min

 1 h  2 h

 2. Application

30 min

 1 h  2 h  3 h  4 h 24 h 48 h 72 h
   m  f  m  f  m  f  m  f  m  f  m  f  m  f  m  f
 Red. spont. activity 5

5

 Prone position

   Constricted abdomen

   Bradykinesia

 Ataxia

0

5

5

0

0

5

5

Hunched posture 

  Half eyelid closure

 Eye closure

Relative PCE

The relative PCE (rel. PCE = proportion of polychromatic (immature) erythrocytes among total erythrocytes) was determined for each animal. The relative PCE is the supportive end point to assess

cytotoxicity, which helps to demonstrate a target cell exposure with the test item.

The negative controls (44 h, 68 h) were within the historical control limits of the negative control (1.12% - 4.46% for males, 0.88% - 3.16% for females). The mean values noted for the 44 h negative control were 2.27% (male mice) and 1.50% (female mice). The mean values detected for the 68 h negative control were 3.05% (male mice) and 2.02% (female mice).

The animal group treated with 0.2 MTD showed mean values of the relative PCE of 1.91 % (male mice) and 2.38% (female mice). The mean value observed in the male group was decreased and the value noted in the female group was statistically significantly increased as compared to the concurrent negative control. However, the mean values were within the historical control limits of the negative control.

The dose groups which were treated with 0.5 MTD showed mean values of the relative PCE of 2.30% (male mice) and 1.68% (female mice). The values observed in both groups were slightly increased compared to the concurrent negative control, but these differences were not statistically significant. Moreover, the me an values were within the historical control limits of the negative control.

The animals who received 1 MTD (44 h sampling) showed mean values of 1.81% (male mice) and 0.98% (female mice). The values observed in both groups were decreased compared to the concurrent negative control, but these differences were not statistically significant. Moreover, the mean values were within the historical control limits of the negative control.

The animal group which was treated with 1 MTD (68 h sampling) showed mean values of the relative PCE of 1.12% (male mice) and 0.93% (female mice). The values observed in both groups

were statistically significantly decreased compared to the concurrent negative control.

The decrease and/or increase of PCE va lues in treated animals compared to control animals is a hint of a target cell exposure of the test item.

Micronucleated polychromatic erythrocytes

For all dose groups, including positive and negative controls, 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes.

The negative controls (44 hand 68 h) evaluated were within the historical control limits of the negative control (0.14 - 0.32% for males, 0.13% - 0.31 % for females). The mean values of micronuclei observed for the negative control (44 h) were 0.21 % (male mice) and 0.23% (female mice). The mean values of the 68 h negative control were 0.18% (male mice) and 0.21 % (female mice).

The mean values of micronuclei observed after treatment with 0.2 MTD were 0.31% (male mice) and 0.24% (female mice). The value observed in the male group was increased compared to the concurrent negative control, but this increase was not statistically significant. Moreover, the value was within the historical control limits of the negative control. The value observed in the female group was within the range of the concurrent negative control and within the historical control limits of the negative control.

The mean values noted for the 0.5 MTD dose group were 0.24% (male mice) and 0.24% (female mice). The values observed in the male and female group were within the range of the concurrent negative control and within the historical control limits of the negative control.

The dose group treated with 1 MTD (44 h sampling) showed mean values of 0.31 % (male mice) and 0.18% (female mice). The value observed in the male group was increased compared to the concurrent negative control, but this increase was not statistically significant. Moreover, the value was within the historical control limits of the negative control. The value observed in the female group was within the range of the concurrent negative control and within the historical control limits of the negative control.

The mean values observed for the 1 MTD (68 h sampling) were 0.23% (male mice) and 0.15% (female mice). The values observed in the male and female group were within the range of the concurrent negative control and within the historical control limits of the negative control.

No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the study and under the experimental conditions reported, the test item 4-Vinylphenol did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse.
Therefore, the test item 4-Vinylphenol is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the Mammalian Erythrocyte Micronucleus Test.
Executive summary:

This study was performed to investigate the potential of 4-Vinylphenol to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse, which is the endpoint of this test

to assess genotoxicity.

The test item was available as solution in propylene glycol with a content of 4-Vinylphenol of 9.7%. To obtain a non-toxic dose of propylene glycol the test item was further diluted in Aqua ad iniectabilia. For all dose groups the test item was administered as split dose with a time period of 2 h between both applications. The volume administered ip for each application was 10 mL/kg bw. Peripheral blood samples were collected for micronuclei analysis 44 h and 68 h after the second application of the test item.

A pre-experiment was performed as dose range finding study based on the OECD guideline 474 and other relevant documents (OECD 420, OECD 423). Based on the outcome of the dose range finding study, a dose of 97 mg/kg bw was selected as maximum tolerated dose (MTD).

In the main experiment three dose levels were used covering a range from the maximum tolerated dose to little or no toxicity. The following dose groups were selected based on the toxicity observed

in the pre-experiment:

 Doses  Concentration [mg/kg bw]
 1 MTD  97
 0.5 MTD  48.5
 0.2 MTD  19.4

The animals treated with doses of 0.2 MTD and 0.5 MTD showed mild and moderate signs of systemic toxicity, respectively. The animals treated with a dose of 1 MTD showed strong signs of systemic toxicity such as reduction of spontaneous activity, prone position, constricted abdomen, bradykinesia, ataxia, hunched posture and half eyelid / eye closure.

For all dose groups, including positive and negative controls, 10000 polychromatic erythrocytes per animal were scored for incidence of micronucleated immature erythrocytes. The negative controls

(44 h, 68 h) were within the historical control limits of the negative control. The mean values noted for the dose groups which were treated with the test item (44 h, 68 h) were within the range of the

concurrent negative control except for the values of the 0.2 MTD and 1 MTD (44 h) male dose groups that were increased compared to the concurrent negative control. However, these increases

were not statistically significant and the values were within the historical control limits of the negative control. Thus the observed increases were considered as not biologically relevant.

No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.

The nonparametric Mann-Whitney Test was performed to verify the results. No statistically significant increases (p< 0.05) of cells with micronuclei were noted in the dose groups of the test item evaluated. Additionally, the X2 Test for trend was performed to test whether there is a dose related increase in the micronucleated cells frequency of the dose groups of the 44 h sampling time.

No statistically significant increase in the frequency of micronucleated cells was observed.

Cyclophosphamide (40 mg/kg bw) administered ip was used as positive control, which induced a statistically significant increase in the micronucleus frequency. This demonstrates the validity of the

assay.