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Endocrine disrupter mammalian screening – in vivo (level 3)

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Administrative data

Endpoint:
endocrine disrupter mammalian screening – in vivo
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:
2015
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 441 (Hershberger Bioassay in Rats): A Short-term Screening Assay for (Anti)Androgenic Properties
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-methylenedi-2,6-xylenol
EC Number:
226-378-9
EC Name:
4,4'-methylenedi-2,6-xylenol
Cas Number:
5384-21-4
Molecular formula:
C17H20O2
IUPAC Name:
4-[(4-hydroxy-3,5-dimethylphenyl)methyl]-2,6-dimethylphenol
Test material form:
solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on species / strain selection:
Standard as per guidelines
Sex:
male
State:
castrated male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd.
- Females (if applicable) nulliparous and non-pregnant: NA
- Age at study initiation: 50 days old
- Weight at study initiation: Phase 1: 196.5 to 262.3 g. Phase 2: 188.4 to 268.6 g.
- Fasting period before study: No
- Housing: The rats were housed 3 animals per cage in suspended plastic cages with solid floors and wood flake bedding.
- Diet (e.g. ad libitum): standard certified laboratory rodent diet RM1(E)SQC), ad libitum.
- Water (e.g. ad libitum): Free access to tap water
- Acclimation period: At least 5 days

DETAILS OF FOOD AND WATER QUALITY: Tap water, diet, wood flakes and chewblocks are regularly analysed and no contaminants were known to be present which could in the opinion of the Study Director interfere with or affect the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2°C
- Humidity (%): 55 +/- 15%
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Standard route of test substance administration
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was formulated in corn oil and the test formulations were prepared on two occasions for
each phase of dosing (1 day before the first day of dosing and on Day 4 of dosing) and stored at 2-8 °C until use. In both Phase 1 and Phase 2, the testosterone propionate was prepared fresh on each day of dosing. The required amount of test material was ground in a mortar and pestle and made up to volume with the required amount of vehicle (corn oil) in order to achieve the required concentration of 0.8 mg/mL. Homogeneity was maintained using magnetic stirring prior to, and during dosing and the formulations appeared homogenous at the time of dosing. In Phase 2, the flutamide was prepared fresh on each day of dosing. The required amount of test material was ground in a mortar and pestle and made up to volume with the required amount of vehicle (corn oil) in order to achieve the required concentration of 0.6 mg/mL. Homogeneity was maintained using magnetic stirring prior to, and during dosing and the formulations appeared homogenous at the time of dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil is a standard vehicle for regulatory testing.
- Concentration in vehicle: Test for androgenic activity; 5 mL/kg for the oral administration of the test substance and 0.5 mL/kg for the subcutaneous administration of the positive control substance (TP). Test for anti-androgenic activity; 5 mL/kg for the oral administration of the test substance or flutamide and 0.5 mL/kg for the subcutaneous administration of the positive control substance (TP).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test article at concentrations from 10 mg/mL to 250 mg/mL was at least 8 days at room temperature and refrigerated conditions.
The homogeneity of test formulations at 20 and 200 mg/mL was tested as part of this study. Test formulations were mixed by 20-fold inversion and magnetic stirring for a minimum of 30 min. Single samples (nominally 1 mL) were taken for assay from the top, middle and bottom of the formulation. Formulations were considered homogeneous if results had a coefficient of variation within 5%. Overall mean was expected to be within +10/-15% of nominal value. For the analysis of dose formulations, four formulation samples (nominally 1 mL accurately weighed) were collected from the middle of each dose formulation. Two formulation samples of
each test item formulation used, including vehicle were analysed. The remaining samples were retained refrigerated at 2-8 °C as contingency samples and were discarded following the confirmation of the results.

The homogeneity of test formulations at 20 and 200 mg/mL was confirmed for this study. The mean concentration of test substance in the test formulations analysed for both Phase 1 and Phase 2 of the study were within applied limits of +10%/-15% of the nominal concentrations, confirming accurate formulation. Absence of test substance was verified in the vehicle formulations (Groups 1 and 6).
Duration of treatment / exposure:
10 consecutive days for both phases
Frequency of treatment:
Phase 1:
The corn oil vehicle or test substance were administered by oral gavage for 10 consecutive days using a dose volume of 5 mL/kg/day. The testosterone propionate (Group 5 – Positive control for androgenic activity), was administered by subcutaneous injection for 10 consecutive days at a dose volume of 0.5 mL/kg/day. Subcutaneous doses were administered to the dorsoscapular and
lumbar regions via a sterile needle and syringe, with the dose sites having been shaved prior to dosing and the dose site rotated each day. Animals in all groups were weighed on a daily basis during the dosing period and the dosage level (i.e. volume) was adjusted daily based on the concurrent daily measures of body weight. The animals were dosed in the same manner and time sequence at approximately 24 h intervals, with the exception that on the last day of dosing animals were dosed in the planned necropsy order. The volume of dose and time that it was administered were recorded on each day.

Phase 2: The vehicle, test substance or flutamide were administered by oral administration for 10 consecutive days using a dose volume of 5 mL/kg/day. Testosterone propionate was also administered by subcutaneous administration at a dose volume of 0.5 mL/kg/day, immediately after each oral administration. The testosterone propionate was administered to the dorsoscapular and lumbar
regions via a sterile needle and syringe, with the dose sites having been shaved prior to dosing and the dose site rotated each day. Animals in all groups were weighed on a daily basis during the dosing period and the dosage level (i.e. volume) was adjusted daily based on the concurrent daily measures of body weight. The animals were dosed in the same manner and time sequence at approximately 24 h intervals, with the exception that on the last day of dosing animals were dosed in the planned necropsy order. The volume of dose and time that it was administered were recorded on each day.
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 castrated males
Control animals:
yes, concurrent vehicle
Positive control:
The positive (androgenic) control for Phase 1 was testosterone propionate.

The positive (antiandrogenic) control for Phase 2 was flutamide

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the dosing period, general clinical observations were made immediately after dosing and at the end of each treatment group, 1-2 h after dosing and again at the end of the working day.


BODY WEIGHT: Yes
- Time schedule for examinations: daily

SPECIFIC ENDPOINTS
- Weight of seminal vesicles: Yes
- Weight of ventral prostate: Yes
- Weight of levator ani-bulbocavernosus muscle: Yes
- Weight of Cowper's glands: Yes
- Weight of glans penis: Yes

Optional investigations:
- Determination of liver, paired kidney, and paired adrenal weights: No
- Hormones determined: serum luteinising hormone, follicular stimulating hormone, testosterone, serum T4 and T3 levels: No
Sacrifice and pathology:
GROSS PATHOLOGY: Approximately 24 h after the last administration of the test substance, the rats were euthanized by cervical dislocation. The necropsy order was randomized across groups to avoid progression directly up or down dose groups that could affect the data. The five androgen-dependant tissues (SV, VP, LABC, COW and GP) were excised from all animals, carefully trimmed of excess adhering tissue and fat, and their fresh (unfixed) weights recorded to the nearest 0.1 mg. Each tissue was handled with particular care to avoid the loss of
fluids and to avoid desiccation. No fluid loss occurred during the dissection procedure. No pathological changes/visible lesions were noted during necropsy.

HISTOPATHOLOGY: No
Statistics:
See 'any other information on materials and methods incl. tables'.

Results and discussion

Endocrine disrupting potential:
ambiguous
Maximum tolerated dose level exceeded:
no
Remarks:
The highest dose is 1000 mg/kg/day (limit dose).

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No effects in both phases of the study.
Mortality:
no mortality observed
Description (incidence):
No mortality observed on both phases of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effects on body weight were observed in both phases of the study.
Food consumption and compound intake (if feeding study):
not examined
Water consumption and compound intake (if drinking water study):
not examined
Clinical biochemistry findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Phase 1: Test substance administered orally at doses of 100, 300 and 1000 mg/kg/day for 10 consecutive days showed no statistically significant changes in the weights of the seminal vesicles, ventral prostate, levator ani-bulbocavernosus, paired Cowper’s glands or glans penis compared with vehicle-treated control animals. Based on these findings there was no evidence of androgenic activity of the substance in this study.

Phase 2: Test substance administered by gavage at doses of 100 and 300 mg/kg/day for 10 consecutive days in animals concurrently treated with subcutaneous testosterone propionate (0.4 mg/kg/day) showed no significant reductions in the seminal vesicles, ventral prostate, levator ani-bulbocavernosus,
paired Cowper’s glands or glans penis, compared with the vehicle/testosterone propionate treated control animals. Test substance administered orally at the limit dose of 1000 mg/kg/day for 10 consecutive days in animals concurrently treated with subcutaneous testosterone propionate (0.4 mg/kg/day) produced statistically significant (p <0.05, based on means adjusted to terminal bodyweights) decreases in the weights of seminal vesicles and levator ani-bulbocavernosus muscle without
displaying any degree of reduced growth in any of the remaining tissues compared with vehicle treated control animals.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Details on results:
Phase 1: Although four of the ten possible CVs (those for levator ani-bulbocavernosus and glans penis in both groups) in the control and high dose treatment groups exceed the maximum designated values for the agonist (refer any other information on materials inclc methods), none of the target tissues was considered
of less statistically significant importance because this criteria was exceeded, i.e. p values for
these parameters did not fall between 0.05 and 0.10.
Phase 2: None of the CVs exceeded the maximum values designated for the antagonist study
(refer to section 3.5).
These results confirm the statistical validity of this study.

Effect levels

Remarks on result:
other: Effect levels not appropriate for these study types.

Any other information on results incl. tables

Effects of oral administration of the test substance on the accessory sex organ weights in castrated rats –absolute values (Phase 1 – Androgen activity)

Organ (mg)

Dose (mg/kg/day)

Vehicle control

100

300

1000

TP (0.4)

N

Mean

SD

CV

N

Mean

SD

CV

N

Mean

SD

CV

N

Mean

SD

CV

N

Mean

SD

CV

Seminal vesicles

6

62.9

8.24

13

6

74.6

16.71

22

6

66.5

7.58

11

6

61.9

11.4

18

6

452.9

109.81

23

Ventral prostate

6

44

13.33

30

6

31.0

14.49

47

6

37.7

6.94

18

6

33.9

8.04

24

6

140.8

35.07

25

LABC

6

174.3

60.68

35

6

213.9

27.11

13

6

218.2

45.20

21

6

1467.

62.94

43

6

451.4

96.22

21

Cowper’s gland

6

7.3

2.12

29

6

10.3

3.71

36

6

8.5

5.03

59

6

6.1

2.5

42

6

35.7

7.80

22

Glans penis

6

58.2

13.48

23

6

56.2

9.96

18

6

52.3

20.89

40

6

60.3

22.10

37

6

82.8

15.20

18

 

Organ

Dose (mg/kg/day)

100

300

1000

TP (0.4)

N

P Value

N

P Value

N

P Value

N

P Value

Seminal vesicles

6

0.134

6

0.579

6

0.813

6

0.000**

Ventral prostate

6

0.042

6

0.523

6

0.217

6

0.000**

LABC

6

0.285

6

0.237

6

0.452

6

0.000**

Cowper’s gland

6

0.275

6

0.641

6

0.676

6

0.000**

Glans penis

6

0.842

6

0.550

6

0.829

6

0.0019*

 

Effects of oral administration of test substance on the accessory sex organ weights in castrated rats –absolute values (Phase 2 – Anti-androgen activity)

Organ (mg)

Dose (mg/kg/day)

Vehicle control + TP (0.4)

100 + TP (0.4)

300 + TP (0.4)

1000 + TP (0.4)

Flutamide (3) + TP (0.4)

N

Mean

SD

CV

N

Mean

SD

CV

N

Mean

SD

CV

N

Mean

SD

CV

N

Mean

SD

CV

Seminal vesicles

6

447.2

55.08

12

6

373

90.79

24

6

397.8

69.45

17

6

350.7

60.96

17

6

110.6

27.22

25

Ventral prostate

6

176.4

8.69

5

6

162.9

70.75

43

6

147.8

52.34

35

6

153.9

25.09

16

6

46.9

13.07

28

LABC

6

580.4

71.49

12

6

521.8

61.45

12

6

514.4

104.56

20

6

492.1

99.57

20

6

247.6

58.77

24

Cowper’s gland

6

35.8

8.18

23

6

36.8

11.21

30

6

36.3

7.71

21

6

37.2

9.66

26

6

13.8

7.17

52

Glans penis

6

81.8

9.87

12

6

82.4

5.53

7

6

85.7

9.25

11

6

80

10.43

13

6

61.7

11.03

18

 

Organ

Dose (mg/kg/day)

100 + TP (0.4)

300 + TP (0.4)

1000 + TP (0.4)

Flutamide (3) + TP (0.4)

N

P Value

N

P Value

N

P Value

N

P Value

Seminal vesicles

6

0.056

6

0.195

6

0.015*

6

0.000**

Ventral prostate

6

>0.05

6

>0.05

6

>0.05

6

<0.01**

LABC

6

0.163

6

0.173

6

0.072

6

0.000**

Cowper’s gland

6

0.847

6

0.921

6

0.788

6

0.000**

Glans penis

6

0.913

6

0.484

6

0.748

6

0.001**

* Significantly different from controls at P<0.05

* Significantly different from controls at P<0.01

Applicant's summary and conclusion

Conclusions:
In phase 1 the test substance administered orally by gavage for 10 consecutive days to castrated male rats showed no statistically significant changes in mean organ weights in any of the five androgen-dependent tissues compared with vehicle-treated control animals. Based on these finding, the test material is considered to have no androgenic effects.

In conclusion for phase 2, the test substance, administered orally by gavage for 10 consecutive days in amimals treated with subcutaneous TP (0.4 mg/kg/day) showed reduced growth in two of five androgen sensitive tissues (seminal vesicles and levator anibulbocavernosus muscles) and only at the limit dose of 1000 mg/kg/day without displaying any degree of reduced growth in any of the remaining tissues.

As the test substance did not reduce growth in all target tissues relative to TP treatment alone, the study results do not meet the OECD criteria for a positive androgen antagonist response. Therefore, the response to the test substance is considered equivocal.