Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 July 2017 - 04September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
in vivo required for non-EU registration

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
3 November 2015
Type of assay:
other: in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Appearance: white to off white powder or flakes
Storage conditions: at room temperature protected from light
Specific details on test material used for the study:
Stability at higher temperatures: maximum temperature: 60°C for a maximum duration of 1 hour

Test animals

Species:
mouse
Strain:
NMRI
Details on species / strain selection:
Species and strain are recommended by international guidelines (e.g. OECD, EC).
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany.
- Age at study initiation: 6 weeks
- Weight at study initiation: 35.4 ± 2.0 g (within the range of 33-38 g)
- Assigned to test groups randomly: yes
- Fasting period before study: 3 - 4 hours prior to dosing until approximately 1 hour after administration of the test item.
- Housing: group housing in cages (maximum 5 animals per sex per cage) containing sterilized sawdust as bedding material.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: ≥5 days

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18.0-24.0 (actual: 21.5 - 22.4)
- Humidity (%): 40-70 (actual: 46-73)
- Air changes (per hr): ≥10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 31 July 2017 To: 25 August 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: polyethylene glycol 400
- Justification for choice of vehicle: the test item was stable in PEG400 for at least 5 hours at room temperature under normal laboratory conditions and 8 days in the refrigerator over the concentration range 1 to 200 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Specific gravity of vehicle: 1.125 g/mL
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: dosing solutions were prepared on the day of administration. No correction was made for the purity/composition of the test item. Before use the test item was crushed and ground in a mortar with pestle to improve the consistency. Test item concentrations were treated with ultra-sonic waves and heated up to a maximum temperature of 59.0°C until it had completely dissolved. Test item concentrations were dosed within 4 hours after preparation.

Duration of treatment / exposure:
Two treatments were performed, administered at a 24-hour interval.
Frequency of treatment:
Treatment was performed in 2 dosings with 2-3 hours in between.
Post exposure period:
The sampling time was 48 hours after first dosing.
Doses / concentrations
Dose / conc.:
2 000 mg/kg bw (total dose)
No. of animals per sex per dose:
5 male animals per sampling time for the treatment group, positive control group and vehicle control group each (15 animals in total). Based on the results of the dose-range finding test that did not indicate differences in toxicity between sexes, the main test was performed with only male animals.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide dissolved in physiological saline

- Justification for choice of positive control: recommended in international guidelines (e.g. OECD, EC)
- Route of administration: oral gavage
- Doses / concentrations: single dose of 40 mg/kg bw

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes from the bone marrow were examined for micronuclei.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: in the dose-range finding test performed with 3 male and 3 female animals, no toxicity was observed up to 2000 mg/kg bw. Therefore, the main test was performed as a limit test with a dose level of 2000 mg/kg bw (maximum recommended dose in accordance with regulatory guidelines).

TREATMENT AND SAMPLING TIMES: Two treatments were performed, administered at a 24-hour interval. The test item is administered as a split dose, i.e., two treatments on the same day separated by no more than 2-3 hours, to facilitate administering a large volume necessary due to limited solubility of the test item.
Bone marrow was sampled 48 hours after the first dosing.

DETAILS OF SLIDE PREPARATION: Femurs were flushed with approximately 2 ml of fetal calf serum. The cell suspension was collected and centrifuged at 216 g for 5 min. The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. At least two slides were prepared per animal.
The slides were automatically stained using the "Wright-stain-procedure" in a HEMA-tek slide stainer (based on Giemsa).

METHOD OF ANALYSIS:
At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%).
The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

OTHER: Animals were checked for mortality at least twice a day. The toxic signs were recorded at least once a day. The animals were weighed just prior to dosing.
Evaluation criteria:
A test item is considered positive in the micronucleus test if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control;
b) The increase is dose related when evaluated with a trend test;
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control;
b) There is no concentration-related increase when evaluated with a trend test;
c) All results are within the 95% control limits of the negative historical control data range.

ACCEPTABILITY CRITERIA:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes. The positive control data will be analyzed by the Students t test (one-sided, p < 0.05) in case of homogeneous variances or by the Welch t test in case of inhomogeneous variances (one-sided, p < 0.05).
Statistics:
The positive control data were analyzed by the Students t test (one-sided, p < 0.05) to determined statistical significance.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: three males and three females were treated with 2000 mg/kg bw
- Clinical signs of toxicity in test animals: no
- Rationale for exposure: according to guidelines (e.g. OECD, EC)
- Other: observation period after dosing was 3 days

RESULTS OF DEFINITIVE STUDY (see table 1)
- Induction of micronuclei: no increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of Ethylene glycol dibenzoate treated animals compared to the vehicle treated animals.
- Ratio of PCE/NCE: no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group
- Appropriateness of dose levels and route: since no toxicity was observed in the dose-range finding test and no differences between the sexes were observed, it was justifiied to use 2000 mg/kg bw as only dose level.
- Statistical analysis: since a statistical effect was only observed in the treatment group treated with the positive control, statistical analysis was applied to this treatment group only.
- All animals tested showed no treatment related clinical signs of toxicity or mortality.

ACCEPTABILITY OF ASSAY:
- The concurrent vehicle control data were within the 95% control limits of the distribution of the historical negative control database (see table 1-3).
- The positive control induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes (p<0.001). The number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database (i. e. 29.8 +/- 7.9)

Any other information on results incl. tables

Table 1 Mean Number of Micronucleated Polychromatic Erythrocytes and Ratio of Polychromatic/Normochromatic Erythrocytes

Group

Treatment

Dose

(mg/kg body weight)

Number of micronucleated polychromatic erythrocytes

Ratio polychromatic/ normochromatic erythrocytes

 

 

 

(mean ± S.D.)(a,b)

(mean ± S.D.)(a,c)

 

 

 

 

 

 

 

 

 

A

Vehicle Control

0

1.0

±

0.7

1.04

±

0.07

B

Test Item

2000

1.0

±

1.0

0.99

±

0.11

C

CP

20

29.8

±

7.9(d)

0.80

±

0.22

Vehicle control = PEG400

CP = Cyclophosphamide.

(a)Five animals per treatment group. ; (b)At least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.; (c)The ratio was determined from at least the first 1000 erythrocytes counted.; (d)Significantly different from corresponding control group (Students t test, Welch t test, P < 0.001).

Table 2 Historical data for the vehicle controls (males)

Mean number of micronucleated polychromatic erythrocytes per 2000 cells

1.34

SD

1.10

n

230

Upper control limit

(95% control limits)

3.76

Lower control limit

(95% control limits)

-1.09

SD = Standard deviation; n = Number of observations

Distribution historical negative control data from experiments performed between January 2012 and October 2017.

Table 3 Historical data for the positive controls (males)

Mean number of micronucleated polychromatic erythrocytes per 2000 cells

24.80

SD

9.45

n

224

Upper control limit

(95% control limits)

47.18

Lower control limit

(95% control limits)

2.42

SD = Standard deviation; n = Number of observations

Distribution historical negative control data from experiments performed between January 2012 and October 2017.

Applicant's summary and conclusion

Conclusions:
In a bone marrow micronucleus test with male mice, performed according to OECD guideline 474 and GLP principles, it is concluded that Ethylene glycol dibenzoate is not clastogenic or aneugenic up to and including a dose level of 2000 mg/kg bw.
Executive summary:

In a bone marrow micronucleus test with male mice, performed according to OECD guideline 474 and GLP principles, it is concluded that Ethylene glycol dibenzoate is not clastogenic or aneugenic up to and including a dose level of 2000 mg/kg bw.