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Toxicity to reproduction: other studies

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Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2003-03-25 to 2003-08-22 (experimental)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant study comparable to guideline with acceptable restrictions: 2 instead of 3 test groups with different doses of the test chemical.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD Protocol and Guidance for the Conduct of the Rodent Hershberger Assay; Phase 2 of the Validation of the Rodent Hershberger Assay
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: Hershberger Bioassay in Rats: A Short-term Screening Assay for (Anti)Androgenic Properties (OECD 441), Adopted: 7 September 2009
Deviations:
yes
Remarks:
(coefficient of variation was not determined; 2 instead of 3 test groups with different doses of the test chemical; no positive control)
GLP compliance:
yes (incl. certificate)
Type of method:
other: Hershberger Assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Further details on strain: CrIGIxBrIHan:Wl
- Source: Charles River, Sulzfeld, Germany
- Age at start of administration: 56 ± 1 days
- Weight at study initiation: no data
- Housing: individually in type DK III stainless steel wire mesh cages
- Diet: ground Kliba maintenance diet rat-mouse, meal, Provimi, Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2003-03-25 to 2003-04-25

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test substance was administered as a suspension in corn oil: the appropriate amount of test substance was weighed, depending on the dose group, subsequently suspended in corn oil using a high speed homogenizer, filled up to the desired volume and mixed using a magnetic stirrer. During the administration the test substance preparations were kept homogenous with a magnetic stirrer.
The test substance preparations were prepared at the beginning of the administration period and filled up in aliquots and kept cold in a refrigerator.

- As efficacy control for the test system Testosterone Propionate (CAS Nr.: 57-85-2, purity: 97 %, solid white powder) was administered as a solution in corn oil: the appropriate amount of Testosterone Propionate was weighed and filled up to the desired volume and mixed using a magnetic stirrer. Preparations were prepared at the beginning of the administration period in filled up in aliquots and kept cold in a refrigerator.

ADMINISTRATION VOLUME:
- Test substance preparations were administered to the animals at a volume of 5 mL/kg bw/day by gavage and 0.5 mL/kg bw/day subcutaneously, based upon the latest individual body weight determination.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical investigations of the test substance preparations were carried out at the Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF Aktiengesellschaft, Ludwigshafen, Germany. The recovery rates were in an acceptable range (100.6 - 101.8 % of the target concentration).
The stability of the test substance in corn oil was carried out from taking samples of the lowest concentration at the day of sample preparation and at the last day of using the test substance preparation.
Homogeneity analyses and concentration control analyses of the test substance preparations were carried out in samples of the highest and lowest concentration drawn from the bottom, middle and the top of the suspension at the start of the administration period. Additional concentration control analyses were performed with samples from the lowest concentration drawn from the middle of the suspension at the end of the administration period.
Duration of treatment / exposure:
10 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
300 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
6 males per group
Control animals:
other: yes, concurrent vehicle with and without Testosterone propionate (0.4 mg/kg bw) substitution
Details on study design:
- Animals were castrated 11 days before the first dosing.
The animals were anesthetized with Isoflo® (Essex GmbH, Munich, Germany). The scrotum as well as mesorchium was opened by a median incision and the testes were exteriorized. Blood vessels and seminal ducts were ligated. The testes and epididymides were removed. Finally, after confirming that no further bleeding occurred, the scrotum was closed with autoclips.

- Treatment (day 0 through 9)
The test substance was administered daily by gavage for about 10 days. Control animals received the vehicle, only. Testosteron Propionate (0.5 mL/kg bw) was administered daily by subcutaneous administration for 10 days.
At the end of the administration period the animals were sacrificed after a fasting period (withdrawal of food) for at least 16-20 hours.
Clinical examinations regards hormone status and pathology was performed.
Statistics:
- Clinical examinations (Food consumption, body weight, body weight change, food efficiency): Dunnett's test (two-sided) for the hypothesis of equal means;
- Hormones: Wilcoxon-test (two-sided) for equal medians;
- Pathology: non-parametric one-way analysis using Kruskal-Wallis Test (two-sided) and Wilcoxon Test;
- Markers for statistical significance: * p ≤ 0.05, ** p ≤ 0.01.

Results and discussion

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no anti-androgenic effects at highest tested dose

Any other information on results incl. tables

Clinical examination

Mortality

No animal died prematurely.

Clinical examinations

No substance-related findings were obtained.

Food consumption

Food consumption of group 3 was statistically significantly reduced on day 5. Since this finding has no influence on body weight data and moreover, it was a single occurrence and could not be observed on day 9, anymore, this observation was assessed as incidental in nature and without any biological relevance.

Food efficiency

No substance-related findings were obtained.

Water consumption

There were no overt deviations in volume between treated groups and controls.

Body weight data

No substance-related findings were obtained.

Clinical pathology

Hormones

Control groups

-compared to the values of castrated animals which received 0.4 mg/kg bw/day of testosterone propionate as reference (group 1) androgen testosterone levels were significantly decreased in the serum of castrated male rats (group 0);luteinizing hormone concentrations were significantly increased.

- No significant difference in serum dihydrotestosterone concentrations was observed.

Treatment groups

- No treatment-related effects on testosterone, dihydrotestosterone and luteinizing hormone concentrations were found in the serum of castrated males after co-administration of 1,000 mg/kg bw/day of the test item and 0.4 mg/kg bw/day of testosterone propionate for 10 days when compared with the hormone levels of animals given testosterone propionate as reference androgen, only.

- a slight, but statistically significant increase in testosterone in the serum of rats whichreceived the test item is considered to be an incidental finding.

Pathology

Organ weights

The following substance-related, statistically significantly changes in mean organ weight were observed for rats treated withthe test item (when compared with control group 1):

- increased absolute / relative liver weight (group 2 [17.4% / 20.8%], group 3 [23.0% / 27.3%])

- increased relative prostate ventral fresh weight (group 2 [15.6%], group 3 [33.3%])

The significantly increased absolute and relative liver weights exhibited a dose-response relationship and were therefore assessed as a substance-related effect.All other mean relative weight parameters did not show significant differences.The increased prostate weights were considered incidental.

 

In the rats that did not receive testosterone (group 0), the absolute and relative weights of the following organs were significantly changed (when compared with control group 1):

- seminal vesicle [-92.7%]

- bulbo-urethal gland [-81.8%]

- prostate ventral fresh [-86.7%]

- prostate ventral fixed [-88.2%]

- glans penis [-44.8%]

- adrenal glands [+21.4%]

- Musc. levator ani with Musc. Bulbocavernosus [-61.0%]

The increased weight of adrenal glands was considered incidental.

As expected, in the males of group 0 the absolute and relative weights of the accessory sex organs were significantly reduced when compared to animals treated with testosterone propionate, only. Histologically, prostate, seminal vesicle and the bulbo-urethral gland were immature.

After treatment with the test item, the absolute and relative weights of the accessory sex organs were not significantly reduced. The histology of prostate, seminal vesicle and the bulbo-urethral gland was comparable to the control group. Additionally, the maximum coefficient of variation perfromance criteria was not exceeded (see table 2).

Gross lesions

All gross lesions occurred singly.

Histopathology

The accessory sex organs (ventral prostate, seminal vesicle and bulbo-urethral gland) were immature in males of dose group 0 (vehicle control). The administration of testosterone propionate (dose group 1) led to the maturation of the accessory sex organs. In the treatment group (dose group 3) the histology of prostate, seminal vesicle and the bulbo-urethral gland was comparable to dose group 1.There were no histopathological findings.

Table 1: Hershberger assay: summary of results

Treatment

Vehicle control

(corn oil)

TestosteronePropionate

Test substance

Group

Group 0

Group 1

Group 2

Group 3

Dose [mg/kg bw/d]

0

0.4

300

1000

Number of animals

6

6

6

6

Clinical findings

none

none

none

none

Body weight day 0 [g]

Mean

(± S.D.)

 

199.4

(± 7.7)

 

206.2

(± 7.3)

 

202.8

(± 6.6)

 

202.6

(± 8.2)

Body weight, day 7 [g]

Mean

(± S.D.)

 

222.0

(± 9.9)

 

233.7

(± 10.7)

 

228.2

(± 14.6)

 

225.5

(± 10.0)

Hormones

 

 

 

 

Testosterone [nmoL/L]

Mean

(± S.D.)

 

0.53(**)W

(± 0.32)

 

3.72

(± 0.77)

 

n.d.

 

5.35(*)W

(± 1.46)

Dihydrotestosterone [pmoL/L]

Mean

(± S.D.)

 

361.38

(± 66.76)

 

325.13

(± 25.02)

 

n.d.

 

356.15

(± 78.51)

Luteinizing hormone [µg/L]

Mean

(± S.D.)

 

14.33(**)W

(± 2.58)

 

2.41

(± 1.78)

 

n.d.

 

2.90

(± 3.84)

Absolute weights

 

 

 

 

Terminal body weight [g]

Mean

(± S.D.)

 

206.517

(± 11.346)

 

218.367

(± 7.232)

 

213.133

(± 9.278)

 

211.0

(± 6.287)

Liver [g]

Mean

(± S.D.)

 

5.907

(± 0.382)

 

6.558

(± 0.644)

 

7.702 (*)KW

(± 1.178)

 

8.068 (**)KW

(± 0.594)

Kidneys [g]

Mean

(± S.D.)

 

1511.533(*)

(± 122.656)

 

1783.683

(± 318.88)

 

1857.583

(± 461.392)

 

1681.667

(± 137.173)

Target Sex Accessory Tissues

 

 

 

 

Seminal vesicle [mg]

Mean

(± S.D.)

 

18.667(**)

(± 5.726)

 

271.017

(± 49.476)

 

294.183

(± 39.369)

 

319.283

(± 79.871)

Bulbo-urethral gland [mg]

Mean

(± S.D.)

 

4.2(**)

(± 0.865)

 

24.05

(± 2.671)

 

26.117

(± 4.872)

 

23.517

(± 6.677)

Prostata ventral fresh [mg]

Mean

(± S.D.)

 

11.933(**)

(± 4.536)

 

97.517

(± 10.634)

 

110.05

(± 13.539)

 

125.6

(± 22.789)

Prostata ventral fixed [mg]

Mean

(± S.D.)

 

12.017(**)

(± 4.02)

 

112.15

(± 12.574)

 

123.017

(± 14.497)

 

139.117

(± 27.694)

Glans penis [mg]

Mean

(± S.D.)

 

32.617(**)

(± 7.947)

 

63.867

(± 5.908)

 

61.5

(± 5.055)

 

63.617

(± 10.427)

levator ani-bulbocavernosus muscle [mg]

Mean

(± S.D.)

 

125.017(**)

(± 23.198)

 

336.783

(± 55.578)

 

331.983

(± 44.589)

 

340.35

(± 37.854)

Relative weights

 

 

 

 

Liver [%]

Mean

(± S.D.)

 

2.859

(± 0.075)

 

3.002

(± 0.257)

 

3.627(*)KW

(± 0.647)

 

3.821(**)KW

(± 0.205)

Kidneys [%]

Mean

(± S.D.)

 

0.732

(± 0.046)

 

0.816

(± 0.137)

 

0.877

(± 0.245)

 

0.797

(± 0.061)

Target Sex Accessory Tissues

 

 

 

 

Seminal vesicle [%]

Mean

(± S.D.)

 

0.009(**)

(± 0.003)

 

0.124

(± 0.024)

 

0.138

(± 0.014)

 

0.151

(± 0.038)

Bulbo-urethral gland [%]

Mean

(± S.D.)

 

0.002(**)

(± 0.0)

 

0.011

(± 0.001)

 

0.012

(± 0.002)

 

0.011

(± 0.003)

Prostata ventral fresh [%]

Mean

(± S.D.)

 

0.006(**)

(± 0.002)

 

0.045

(± 0.004)

 

0.052(*)

(± 0.005)

 

0.06(*)

(± 0.011)

Prostata ventral fixed [%]

Mean

(± S.D.)

 

0.006(**)

(±0.002)

 

0.051

(± 0.005)

 

0.058

(± 0.005)

 

0.066

(± 0.014)

Glans penis [%]

Mean

(± S.D.)

 

0.016(**)

(±0.003)

 

0.029

(± 0.003)

 

0.029

(± 0.002)

 

0.03

(± 0.005)

levator ani-bulbocavernosus muscle [%]

Mean

(± S.D.)

 

0.06(**)

(±0.009)

 

0.154

(± 0.023)

 

0.156

(± 0.018)

 

0.161

(± 0.015)

W = Wilcoxon-Test, Two-sided, K = Kruskal-Wallis-Test; (*) p ≤ 0.05; (**) p ≤ 0.01; n.d. = not determined.

Table 2: Coefficient of variation for target sex accessory tissues

Coefficient of variation (%)

Treatment

TestosteronePropionate

(negative control)

Test substance

Seminal vesicle

19.4

25.2

Prostata ventral fresh

8.9

18.3

Prostata ventral fixed

9.8

21.2

levator ani-bulbocavernosus muscle

14.9

9.3

Bulbo-urethral gland

(Cowpers gland)

9.1

27.3

Glans penis

10.3

16.7

 

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, regarding clinical examinations, hormone investigations as well as pathological evaluations, no indication of an anti-androgen efficacy of the test item oxybenzone dosed at 300 and 1000 mg/kg bw was determined.
Executive summary:

The Rodent Hershberger Assay was performed in accordance with OECD Protocol and Guidance, Phase 2 of the test validation. To determine a possible anti-androgen efficacy, the test substance was administered to groups of 6 castrated but Testosterone propionate (0.4 mg/kg bw/d) treated/substituted male Wistar rats (which were subdivided in three subsets) by gavage for 10 days at dose levels of 300 (group 2) and 1,000 (group 3) mg/kg bw/day. A vehicle control group was treated with corn oil by gavage (group 0). Another control group was administered subcutaneously 0.4 mg/kg bw/day Testosterone propionate (group 1) to test the androgenic efficacy.

Clinical examinations revealed no substance-related findings in both treatment groups (300 and 1,000 mg/kg bw/day). Concerning hormone levels, co-administration of 1,000 mg/kg bw/day of the test substance and 0.4 mg/kg bw/day of testosterone propionate did not affect testosterone, dihydrotestosterone and luteinizing hormone in the serum of castrated male rats when compared with castrated animals given 0.4 mg/kg bw/day of testosterone propionate as reference androgen, only.