Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
only seven pregnant females in the high dose group
GLP compliance:
yes
Remarks:
(Mitsubishi Chemical Safety Institute Ltd.)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): 4,4'-Sulfonyldiphenol
- Physical state: white powder
- Analytical purity: 99.87 %
- Impurities (identity and concentrations): 2,4’-Dihydroxy diphenyl sulfone 0.13 wt%; Water 0.03 wt%
- Stability under test conditions: stability of the test substance in the administration solution of 1 and 200 mg/mL kept under dark and cold conditions was assured before start of administration.
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain as specified in the report: Crj:CD(SD)IGS
- Source: Tsukuba Breeding Center, Charles River Laboratories Japan
- Age at study initiation: 9 weeks
- Body weight ranges at study initiation: 329 to 374 g in males and from 206 to 251 g in females
- Housing: Each one male and female in mating period, a litter of pups in lactation period and one animal during gestation period
- Diet: Autoclave sterilized CRF-1, Oriental Yeast Co., Ltd, ad libitum
- Water: Tap water filtrated with 5 µm pore size filter followed by ultra-violet radiation, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous sodium carboxymethylcellulose solution with 0.1% Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was individually weighed for each dose and suspended using an agate mortar in 0.5% CMC-Na aqueous solution with 0.1% Tween 80 added. The administration solution was prepared once a week and kept in under dark and cold conditions to be used within 8 days after preparation. Before start of administration, the stability of the test substance in the administration solution of 1 and 200 mg/mL stored under cold and dark conditions was assured. In addition, the administration solutions for respective dose groups were analyzed at the time of first preparation to confirm that they were within +- 10% of the prescribed concentrations.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance is sparingly soluble in water.
- Amount of vehicle (if gavage): 5 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1 : 1
- Length of cohabitation: 7 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
The administration solutions for respective dose groups were analyzed at the time of first preparation to confirm that they were within +- 10% of the prescribed concentrations (data not available).
Duration of treatment / exposure:
males in total 45 days (including 14 days pre-mating period), females in total 40 to 46 days from mating through gestational period and parturition to day 3 of lactation. Animals without delivery were administered until day 25 after confirmation of copulation.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a preliminary test, the test substance was administered repeatedly for 14 days to each 3 male and female rats per group at doses of 0, 100, 300 and 1000 mg/kg bw., respectively. As a result, suppressed gain or decrease in body weight, decreased or low food intake and reduced weight of seminal vesicle were observed in males and females of the 300 and 1000 mg/kg bw groups. In addition, caecum abdominal distension was observed in the males of the 300 mg/kg bw group and in males and females of the 1000 mg/kg bw group. Furthermore, decreased weights of thymus and prostate gland were observed in males of the 1000 mg/kg group. Considering these results and the administration period in the main test, the high dose was determined as 300 mg/kg bw where clear toxicity is expected, and 3 doses in total including 60 and 10 mg/kg bw were set up at a common ratio of 5.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day (before and after administration)

BODY WEIGHT: Yes
- Time schedule for examinations: on the day 1, 3, 7, and 14 of test substance administration and once a week thereafter; additionally, females with successful copulation were weighed on day 0, 7, 14 and 20 of gestation period and on day 0 and 4 of lactation period. The body weight gain was calculated in males based on the body weight on day 1 and in females based on day 1 of test substance administration, and on day 0 of gestation as well as on day 0 of lactation, respectively, in pre-mating, gestation and lactation periods.

FOOD CONSUMPTION: Yes
- Mean food intake per animal per day was calculated for both males and females by measuring gross weight on the first day of gavage as well as on day 3, 7, and 14 of the treatment period, and for males once a week excluding mating period thereafter as well as females on day 0, 7, 14 and 20 of gestation period and day 0 and 4 of lactation period.

OTHER:
- The starting day of administration was counted as day 0 of pre-mating period, the day of successful mating as day 0 of pregnancy and the day of parturition as day 0 of lactation, respectively.
- Gestation status was observed twice a day from day 21 to 25 of parturition.
Oestrous cyclicity (parental animals):
Vaginal smears of females were collected every day in the morning starting with the first day of treatment to the day of confirmed copulation and subjected to eosin-thionine staining (with Hemacolor multi-purpose quick staining kit) to inspect their estrus cycle and calculate the number of mean estrus cycle days until successful copulation.
Females whose estrus cycle was not in a range from 4 to 6 days were considered as those of irregular estrus cycle.
Sperm parameters (parental animals):
Parameters examined in male parental animals:
testis weight, epididymis weight
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Total number of offspring at birth (number of live offspring at birth, number of dead offspring at birth), sex, external abnormalities were checked on day 0 of lactation. Thereafter, general conditions, death etc. were observed every day until day 4 of lactation.
- Body weight: Live offspring was individually measured on day 0 and 4 of lactation. In addition, body weight gain was calculated based on the body weight on day 0 of lactation.
- Anogenital distance: Anogenital distance (AGD) of all the live offspring was measured.
Furthermore, relative distance divided by cube root of body weight on day 4 of lactation (AGD / ∛body weight) was calculated to compensate fluctuation of measured values due to body weight balance among individual offspring.
- Neonates were lactated for 4 days after birth (day 4 of lactation period) to observe every day lactation conditions including lactation, nest-building, eventual cannibalism and etc.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [day 45]
- Maternal animals: All surviving animals [day 4 of lactation]
- Animals without delivery were brought to necropsy on day 26 after confirmation of copulation.
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Ovaries and uterus of dams were removed on necropsy to check the number of corpora lutea and number of implantation sites.

HISTOPATHOLOGY / ORGAN WEIGHTS
- In all animals, organs and tissues from liver, thymus, testes, epididymis, seminal vesicle, prostate, uterus, ovaries, pituitary, vagina and gross abnormal lesions were fixed with 10% neutral phosphate buffer formalin solution to be stored.
- In addition, abnormalities were found in caecum of 60 and 300 mg/kg bw groups and hence, caecum was taken as reference from all the males and one female of the control groups, while their testes and epididymis were fixed with Bouin liquid.
- In the histopathological test, hematoxylin and eosin-stained samples were prepared based on usual technique from the said organs and tissues stored of all the males and females of the control groups and 300 mg/kg bw group and subjected to microscopic examination. As a result, changes in liver of males and females caused by the test substance were observed and hence, livers of all the males and females in 10 and 60 mg/kg bw groups were examined.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- After inspecting external appearance including oral cavity of all the live offspring on day 4 of lactation, they were euthanized similarly to their parent animals, followed by necropsy. After fixation in 10% neutral phosphate buffer formalin solution, animals were subjected to necropsy using a stereomicroscopy.
Statistics:
The measured data were subjected to Bartlett test for homogeneity of variances and they were subjected to one-way analysis of variances when variances were equal and to Kruskal-Wallis test when variances were not equal. In the case that significant differences were found among the groups, the data were subjected to multiple comparison according to Dunnett test. Some items were first subjected to Kruskal-Wallis test and also to multiple comparison of Dunnett type when significant differences were found among the groups. The data obtained in the histopathological test were subjected to x2 test for (a x b) or to Amitage x2 test when significant differences were found among the groups. Furthermore, Fischer’s exact test was performed. The significant level for each test was set as 5%. The target items for statistical analysis are as follows and the statistical analysis was not applied for general conditions and necropsy examination;
* Multiple comparison test: Body weight, body weight gain, food intake, organ weight, number of corpora lutea, number of implantation sites, number of offspring at birth, and anogenital distance
* Multiple comparison of Kruskal-Wallis and Dunnet type: Number of days required for copulation, number of estrus cycles which elapsed till successful copulation, mean number of days of estrus cycle, gestation period, implantation index, delivery index, live birth index, and viability index of offspring on day 4 after birth
* x2 test and Amitage x2 test: Histopathological test
Fischer’s exact test: Incidence index of females with irregular estrus cycle, copulation index, fertility index, gestation index, and sex (male/female)
Reproductive indices:
Copulation index (%): (Number of copulated animals / number of animals housed together) x 100
Fertility index (%): (Number of pregnant females / number of copulated animals) x 100
Gestation period: number of days from day 0 gestation to the day when the delivery was assured
Delivery index (%): (number of pregnant females delivered live offspring / number of pregnant females) x 100
Implantation index (%): (number of implantation sites / number of corpora lutea) x 100
Gestation index (%): (total number of offspring delivered / number of implantation sites) x 100
Offspring viability indices:
Live birth index (%): (Number of live offspring at birth / Total number of offspring at birth) x 100
Viability index (%) of offspring on day 4 after birth: (Number of live offspring on day 4 of lactation / number of offspring at birth) x 100

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- Salivation was observed immediately before or immediately after gavage in 7 males of the 300 mg/kg bw group from day 22 after start of administration to day 54 of administration and in one female from day 17 after start of administration to day 15 of gestation, but all of them recovered in approx. 30 minutes after administration.
- No abnormalities were found in any males and females of the 10 and 60 mg/kg bw groups.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Body weight gain was significantly suppressed from day 3 (males, day 3: 85%, day 7: 62%, day 14: 40%, day 21: 32%, day 28: 23%, day 35: 21%) or day 7 (females, before mating: day 7: 75%, day 14: 39%, during gestation period: day 7: 20%, day 14: 22%, day 20: 23%) after start of administration and decrease in food consumption was observed in males and females of the 300 mg/kg bw group. Furthermore, significant difference from the control group was found in body weight in females on day 4 of lactation in 60 mg/kg bw group and low food intake were found on day 4 of lactation in 10 and 60 mg/kg bw groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- In the estrus cycle test, extended cycles were observed in 300 mg/kg bw group and 5/12 animals showed a continued diestrus for 6 to 10 days, but all the cases succeeded in copulation and no significant differences from the control group were observed in terms of copulation index, number of days required for copulation and number of estrus cycles which elapsed till successful copulation. However, 4 out of the animals showing a continued diestrus in 300 mg/kg bw group did not conceive and revealed a declined fertility index (58.3%).
The animals of the 10 and 60 mg/kg bw groups showed no significant differences from the control group in every inspection item and index.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- No changes attributable to the compound were observed in parameters such as mating index, delivery index, gestation index, number of corpora lutea, gestation length, parturition state, or lactation behaviour.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- Relative weights of liver (12%) and pituitary gland (19%) were increased and absolute weight of seminal vesicles (14%) were decreased in males of 300 mg/kg bw group (significantly different from the control group).
No significant differences from the control group were found in males and females of the 10 and 60 mg/kg bw groups.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- Distension of the caecum was observed at each one male and female of the 60 mg/kg bw group and all males (12/12) and 4/12 females of the 300 mg/kg bw group.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- Changes to be attributable to the test substance were observed in caecum and liver of males and females of all test groups. Mucosal epithelium diffusion in caecum was observed in 1/12 females of the 60 mg/kg bw group and 11/12 males and 4/12 females of the 300 mg/kg bw group, and hyperplasia and diffusion of absorptive epithelium were significant, and single cell necrosis of absorptive epithelium was found in 5/12 males and 1/12 females of the 300 mg/kg bw group. No clear change was observed in one male of the 60 mg/kg bw group in which distension of the caecum was found at necropsy.
- In liver, hypertrophy of centrolobular hepatocytes was observed in 5/12 males and 3/12 females without pregnancy of the 300 mg/kg bw group, and extramedullary hematopoiesis was observed in males and females of each group including the control group.
- Furthermore, inflammatory cell infiltration, micro-granuloma, focal necrosis and single cell necrosis in liver, thymus atrophy, pyelectasis, sperm-granuloma in epididymis, and lymphocyte infiltration in prostate were occasionally observed in each group including the control group, but no clear tendency to increasing abnormalities in 300 mg/kg bw group was found and thus, the effects were assumed not to be test substance related.
In addition, no histological abnormalities were found in the pituitary gland exhibiting an enhanced relative weight and in the seminal vesicle with a decreased absolute weight in males.
- No abnormalities were found in genitals of infertile males and females.

OTHER FINDINGS (PARENTAL ANIMALS)
- No change in the number of corpora lutea was observed in the 300 mg/kg bw group but the number of implantation sites tended to decrease, showing a significant decrease in the implantation index (64.89%) from the control group (95.80%). In addition, the total number of offspring (9.1) in the 300 mg/kg bw group was not significantly different but it was lower than the control group (14.3).
- Furthermore, gestation index in each group including the control group was 100%, and no significant differences from the control group were observed in terms of gestation period and delivery index.
- No abnormalities was observed in lactation behaviour of each maternal animal including the control group.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
for parental toxicity
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
for reproductive toxicity
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: estrous cycles, fertility index, number of implantations, implantation index, number of live births and number of live offspring

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
- Total number of offspring at birth, number of live offspring at birth and number of live offspring on day 4 of lactation were decreased in the 300 mg/kg bw group at systemic parental toxicity.
- In addition, no significant difference in live birth index and viability index on day 4 was observed between the test substance administered group and the control group, and no abnormalities in external appearance and general conditions were found in offspring of each group including the control group.
- No changes attributable to the compound were observed in parameters including the sex ratio, the live birth index, and viability index on day 4.

BODY WEIGHT (OFFSPRING)
- No significant differences in male/female body weight and body weight gain were observed between the control group and the treatment groups.

SEXUAL MATURATION (OFFSPRING)
- No significant differences in male/female anogenital distance were observed between the control group and the treatment groups.

GROSS PATHOLOGY (OFFSPRING)
- No abnormalities in each group including the control group were observed in dead offspring on day 0 to 4 of lactation and live offspring on day 4 of lactation.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: total number of offspring

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1 Body weight of rats during gavage of the test substance (Body weight gain in brackets)

Dose

[mg/kg bw]

 

Day of treatment

 

Males

0

3

7

14

21

28

35

42

 

 

 

No.

Mean body weight [g ± SD]

 

 

 

0

12

351.9 ± 14.3

373.8 ± 15.5

(21.9 ± 4.8)

395.9 ± 16.8

(44.0 ± 7.9)

435.9 ± 24.4

(84.0 ± 18.6)

457.8 ± 27.1

(105.9 ± 22.1)

482.1 ± 31.0

(130.2 ± 25.5)

499.8 ± 33.9

(147.9 ± 29.3)

511.8 ± 33.1

(159.9 ± 28.4)

 

 

10

12

354.3 ± 12.5

373.5 ± 14.9

(19.2 ± 5.0)

397.4 ± 16.9

(43.1 ± 6.6)

435.9 ± 20.8

(81.6 ± 11.9)

458.7 ± 27.4

(104.3 ± 18.9)

482.7 ± 27.3

(128.3 ± 18.0)

500.4 ± 31.4

(146.1 ± 23.1)

514.5 ± 36.4

(160.2 ± 28.2)

 

 

60

12

352.5 ± 10.6

373.7 ± 11.7

(21.2 ± 6.8)

396.8 ± 13.1

(44.3 ± 8.9)

437.9 ± 19.9

(85.4 ± 15.9)

458.2 ± 24.4

(105.7 ± 20.1)

486.2 ± 28.7

(133.7 ± 22.9)

507.3 ± 31.1

(154.8 ± 24.8)

523.5 ± 34.0

(171.0 ± 28.1)

 

 

300

12

354.2 ± 11.1

357.5 ± 14.4*

(3.3 ± 12.7**)

370.8 ± 23.4**

(16.6 ± 20.8**)

404.5 ± 30.6**

(50.3 ± 26.7**)

426.6 ± 32.4*

(72.4 ± 28.3**)

454.2 ± 37.6

(100.0 ± 32.9*)

471.3 ± 45.4

(117.1 ± 40.5*)

486.0 ± 54.3

(131.8 ± 49.2)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Premating period

Day of gestation

Day of lactation

 

Females

0

3

7

14

0

7

14

20

0

4

 

No.

Mean body weight [g ± SD]

 

 

 

0

12/11/11

229.1 ± 9.5

236.0 ± 11.4

(6.9 ± 7.7)

244.9 ± 12.3

(15.8 ± 6.6)

264.2 ± 11.9

(35.1 ± 6.9)

272.8 ± 13.5

 

315.8 ± 14.6

(43.0 ± 5.4)

352.6 ± 14.9

(79.8 ± 5.5)

436.5 ± 22.4

(163.6 ± 14.4)

325.5 ± 22.0

360.1 ± 17.1

(34.5 ± 13.9)

10

12/11/11

228.4 ± 11.6

237.3 ± 13.1

(8.8 ± 6.1)

245.8 ± 13.9

(17.4 ± 6.4)

263.8 ± 17.2

(35.4 ± 9.8)

277.1 ± 19.6

 

320.7 ± 18.4

(43.6 ± 5.8)

358.5 ± 21.8

(81.4 ± 10.9)

433.1 ± 36.7

(156.0 ± 30.1)

327.5 ± 31.2

354.1 ± 20.9

(26.5 ± 17.2)

60

12/12/12

228.2 ± 9.2

233.5 ± 13.2

(5.3 ± 9.4)

241.6 ± 12.8

(13.4 ± 7.8)

262.3 ± 16.3

(34.1 ± 10.9)

266.8 ± 12.9

 

304.9 ± 16.0

(38.2 ± 6.9)

338.7 ± 18.2

(71.9 ± 10.3)

418.6 ± 21.9

(151.8 ± 13.2)

314.3 ± 25.2

333.0 ± 24.2*

(18.7 ± 16.2)

300

12/7/7

230.3 ± 12.5

228.8 ± 15.7

(-1.5 ± 10.5)

234.3 ± 15.9

(4.0 ± 10.4**)

251.7 ± 16.1

(21.4 ± 10.0**)

264.4 ± 16.9

 

298.9 ± 19.6

(34.4 ± 6.5*)

326.9 ± 19.9*

(62.4 ± 7.2**)

390.4 ± 29.2**

(126.0 ± 17.9 **)

316.0 ± 15.0

338.6 ± 20.0

(22.6 ± 8.8)

Significantly different from control group (*: p<0.05; **: p<0.01)

Table 2 Reproductive performance

Dose

[mg/kg bw]

Number of animals

Mean oestrus cycle (± SD)

Incidence of females with irregular oestrus cycle

Copulation index (%)

Fertility index (%)

0

12

4.08 ± 0.21

0/12

100.0

91.7

10

12

4.01 ± 0.11

0/12

100.0

91.7

60

12

4.14 ± 0.34

1/12

100.0

100.0

300

12

5.57 ± 1.81**

5/12*

100.0

58.3

Significantly different from control group (*: p<0.05; **: p<0.01)

 

Table 3 Delivery data

Dose

[mg/kg bw]

Number of animals

Number of corpora lutea

Number of implantation sites

(Mean ± SD)

Total number of offspring

(Mean ± SD)

Implantation index (%)

Delivery

index (%)

0

11

16.6 ± 2.0

15.9 ± 1.9

14.3 ± 2.1

95.80 ± 5.75

90.03 ± 10.08

10

11

15.9 ± 3.6

13.3 ± 5.3

12.5 ± 5.0

80.84 ± 26.01

94.60 ± 8.19

60

12

17.3 ± 2.1

14.8 ± 2.0

13.5 ± 1.8

86.15 ± 7.76

91.22 ± 7.71

300

7

15.7 ± 2.8

10.7 ± 5.5

9.1 ± 4.2

64.89 ± 26.57**

89.75 ± 12.11

Significantly different from control group (*: p<0.05; **: p<0.01)

 

Table 4 Litter size (Sex ratio in brackets)

Dose

[mg/kg bw]

Number of animals

Total number of offspring at birth

(Mean ± SD)

Number of live offspring at birth

(Mean ± SD)

Number of live offspring on day 4

(Mean ± SD)

males

females

total

males

females

total

total

0

11

6.6 ± 1.3

7.6 ± 2.7

14.3 ± 2.1

(73/84)

6.5 ± 1.3

7.6 ± 2.7

14.2 ± 2.1

(72/84)

14.1 ± 2.2

(72/83)

10

11

7.3 ± 3.3

5.2 ± 3.3

12.5 ± 5.0

(80/57*)

7.3 ± 3.3

5.2 ± 3.3

12.5 ± 5.0

(80/57*)

12.4 ± 5.2

(79/57*)

60

12

6.8 ± 1.2

6.8 ± 1.7

13.5 ± 1.8

(81/81)

6.7 ± 1.2

6.8 ± 1.7

13.4 ± 1.7

(80/81)

13.3 ± 1.6

(80/80)

300

7

4.1 ± 1.7*

5.0 ± 2.7

9.1 ± 4.2

(29/35)

4.1 ± 1.7*

5.0 ± 2.7

9.1 ± 4.2

(29/35)

9.1 ± 4.2

(29/35)

Significantly different from control group (*: p<0.05; **: p<0.01)

Applicant's summary and conclusion

Conclusions:
Based on the test results, the NOAEL for parental and reproductive toxicity was considered to be 60 mg/kg bw/day.
Executive summary:

In the repeated dose oral toxicity study according to GLP and OECD 421 (Reproduction/Developmental Toxicity Screening Test), rats (12/sex/dose) were administered a repeated oral dose (gavage) of the test item (analytical purity: 99.87%) at dose levels of 10, 60 and 300 mg/kg body weight. The test item was suspended in vehicle (0.5% aqueous sodium carboxymethylcellulose solution with 0.1% Tween 80) and administered at a volume of 5 mL/kg bw.

The animals were observed daily. Body weight measurements and determination of reproductive parameters as well as pathological examinations were performed in parental animals. Viability, body weight, sexual maturation were examined and after sacrifice on day 4 of lactation offspring was subjected to gross pathology.

Salivation was observed in the high dose group immediately following test substance application. Reduced food intake and body weight gain from day 3 after start of administration to the time of necropsy were observed in males and females of the high dose group. At necropsy, abdominal distension of the caecum was observed in males and females of the mid and high dose group, mostly with mucoepithelial cell necrosis, which might be a reaction of the absorptive epithelium to physical stimulus. Similar results were found in the 28 -day repeated dose oral toxicity study in rats, thereof indicating that the effects might be similar changes caused by the test substance. Furthermore, increased relative liver weight observed in the males of the high dose group as well as centrolobular hypertrophy of hepatocytes found in both the males and females, may indicate enzyme induction, which is often observed after administration of chemical substances causing induction of drug-metabolizing liver enzymes. Hypertrophy of hepatocytes was only found in non-pregnant females, and it is assumed that hepatocytes of delivering females are enlarged due to lactation and therefore conceal the test substance induced effects.

With respect to cecum effects in rats the relevance for humans is unclear. Significant anatomical and functional differences of the cecum exists for rodents and humans. In rodents this anatomical structure is large and in comparison to humans has a significant function in digestion. Nevertheless these disturbances in the content and structure of the gastrointestinal tract in rats may have a significant impact e.g. on body weight development.

No abnormalities were observed in parental animals of the high dose group with regard to copulation index, parturition index, delivery index, number of corpora lutea, gestation period, parturition status and lactation behaviour, but extended oestrus cycle, increased number of animals showing irregular oestrus cycle, tendency to decreased number of implantation sites, and significantly decreased implantation index were observed. Most of the females exhibiting continued dioestrus were not fertilized and tendency to decreased fertility index was noticed. Since no effects on delivery index and live birth index were observed in the high dose group, the decrease in fertility index and total number of offspring at birth may partly be attributable to the increased pre-implantation loss. No histopathological abnormalities were found in pituitary glands in males of the high dose group, exhibting increased relative weight and in seminal vesicles with decreased absolute weight. Moreover, no abnormalities possibly causing infertility were found in histological examinations of genitals of both males and females and thus, the reasons for decreased reproductive performance in paternal animals remained unclear.

No effects were observed with respect to growth of the offspring after birth, as no abnormalities were found in sex ratio (male/female), body weight, viability index of pups on day 4 after birth, anogenital distance, general conditions, external appearance and necropsy findings.

Since the relevance of rat cecum effects is questionable for humans and no other adverse efects occurred at 60 mg/kg bw, the dose is considered to be a NOAEL for parental toxicity.

Based on the distension in caecum and histological changes in caecum observed in males and females of the mid and high dose group, the no-observed adverse effect level regarding repeated oral administration of the test substance is10 mg/kg bw/day for both, males and females.

Based on extended oestrus cycle, increased number of animals showing continued dioestrus, decreased fertility index, number of implantation sites, implantation index, and total number of live offspring at birth observed in the high dose group, the no-observed adverse effect level for reproductive development is 60 mg/kg bw/day for parental animals and pups.