Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-06-21 (Experimental Starting Date) to 2013-04-23 (Experimental Completion Date)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 421
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Waxy solid, slightly yellow if molten
Details on test material:
- Name of test material (as cited in study report): Bis-(2,6-diisopropylphenyl) carbodiimide
- Substance type: Benzeneamines
- Physical state: solid
- Analytical purity: 99.9%
- Lot/batch No.: 3110372
- Stability under test conditions: stability was determined analytically ("The analyses to validate stability were carried out under Study No. F1012107").Formulations were stable for a period of 7 days.
- Storage condition of test material: room temperature, light protection
- Other:
Molecular mass: 362.6 g/mol
Chemical structure: C25H34N2

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: HsdRCCHan:Wist
- Source: Harlan- Nederland, Kreuzelweg 53, 5960 AD Horst, The Nederlands
- Age at study initiation: about 9-10 weeks
- Weight at study initiation: males (259 g (240-273 g)); females (225 g (208-242 g))
- Fasting period before study: no
- Housing: At their arrival the animals were placed together, separated by sex, in some containers and transported into the animal room where they were put individually into Makrolon cages Type IIIh. After tattooing and during the experimental phase animals were kept singly in Makrolon cages Type IIIh on low-dust wood granules. When littering came near special nesting material. Cages, bedding and nesting material were changed weekly with new ones.
- Diet (e.g. ad libitum): ad libitum (fixed-formula standard diet [ssniff R/M-H, 10 mm (pellet), Germany]
- Water (e.g. ad libitum): ad libitum (tap water)
The results of food and water analyses were stored. The available data provided no indication of any effect on the study objective.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES: 57 days From: 2012-06-21 (Study Start Date (= Experimental Starting Date)) To: 2012-08-20 (Study Termination Date (=End of in-Life Phase))

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The formulations were prepared as needed taking into account the analytically determined stability. For the preparation of the formulations the content of test item was used for calculation.
The test item was administered as a solution in the vehicle. The formulations were stored at room temperature and light protection.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test material is well soluble in corn oil.
- Amount of vehicle (if gavage): 5 mL/kg bw/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses prior to the study:
Before the start of the study the suitability of the proposed formulations was confirmed by analysis of solutions containing the test substance in concentrations covering the concentrations to be used in this study. For determination of the stability analytical investigation was performed on samples stored for 15 days (14 mg/mL) and 7 days (0.2 mg/mL). The analyses to validate stability were carried out under Study No. F1012107.

Analyses during the in-life phase:
The test item concentrations (all doses including vehicle control formulation) were checked twice, at start and near termination.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: maximum 14 days.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: no

During the mating period the rats were co-housed overnight (afternoon up to nex morning). To determine the insemination date vaginal smears were taken from females in the morning following co-housing until the day when evidence of copulation was observed. The date, when a vaginal plug or sperms were found microscopically was taken as gestation day 0 for calculating the gestation length. Inseminated females were not further co-housed. Females, which exhibited marked weight gains although insemination had not been established, were not further co-housed. No duration of gestation could be determined in these animals. The vaginal smears were obtained using a flame-sterilized platinum loop and plated out on slides. The smears were stained for about one minute in May-Grünwald solution and were then microscopically examined.
Duration of treatment / exposure:
Males: at least 28 days
Females: mating period (maximum 14 days), gestation and lactation up to necropsy on day 4-6 p.p (altogether 57 days)
Frequency of treatment:
once daily (7 days each week)
Duration of test:
Altogether 57 days
Doses / concentrations
Remarks:
Doses / Concentrations:
1, 3 and 8 mg/kg bw (replaced by 5 mg/kg bw on day 11)
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose selection was based on results of a subacute (28 days) repeated dose toxicity study in Wistar rats (Hsd Cpb:WU), in which Bis-(2,6-diisopropylphenyl) carbodiimide was administered in cornoil once daily in nominal doses of 0 (vehicle control), 1, 4 and 16 mg/kg b.w. to 5 male and 5 female animals per dose group, as well as on data of an exploratory study in male rats.
Because of the findings in the genital tract, selection of dose levels was done without an additional dose-range-finding study as such a study was not expected to provide more conclusive information.
The NOAEL in the study (Report No.: AT06477) was 4 mg/kg b.w. in both sexes. At the high dose of 16 mg/kg b.w., 2 female animals died. In addition, clear-cut clinical symptoms were found. Both sexes showed a distinct reduction in body weight gain and a decrease in food consumption. Furthermore, changes in hematology and clinical chemistry were found. Target organs of toxicity were among others: heart, liver, lymphatic organs, kidney, as well as female genital tract with changes in ovaries and atrophy of oviducts, uterus and vagina.
Furthermore, a dose-range-finding study was performed earlier in male rats over 2 weeks, where 8 mg/kg b.w. were tolerated without symptoms, whereas 16 mg/kg b.w.resulted in toxicity including a reduction in testes weight.
Therefore, the dose levels of 1, 3 and 8 mg/kg b.w. were selected for this study.

- Rationale for animal assignment (if not random): by the random list
Prior to the start of the treatment phase male and female animals were assigned to the dose groups. For that purpose they were placed singly at their arrival in Type IIIh cages. Thereafter the animal weights were determined and recorded as well as the position of the animal on the shelf. Animals with extremely high or low body weights as well as surplus animals were removed. Based on evenly distributed chance numbers especially generated for this study, the animals were allocated to their final cage number specified by the random list. The cages were then placed one after the other in shelves in the order of increasing numbers. Control and test item treated animals were handled each in the same manner.
- Other:

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
The experimental animals were inspected twice a day for morbidity and mortality. General clinical examinations (in the home cage) were made daily (especially findings occurring during littering e.g. prolonged parturition) some time (about 30 to 60 minutes) after the administration of the last animal and recorded, if any.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all signs of illness or clinical reactions to treatment were recorded online during a detailed clinical observation at the following times:
F0-Males: Prior to the treatment and then weekly up to necropsy.
F0-Females: Prior to the treatment and then weekly during premating and mating period and, additionally, daily during gestation and lactation period.
This investigation included the evaluation of the general state of health, behavior, condition of the fur, and the orifices as well as excretory products.
If animals became ill, they were marked (with cage labels) or set apart, observed more frequently and sacrificed prematurely, if death seemed imminent.

BODY WEIGHT: Yes
- Time schedule for examinations: The individual body weights of all parental animals were determined just prior before the first treatment of animals and then daily thereafter. The individual body weights were used for calculation of the administration volume. The data of administration volume is held on file, but is not reported. Furthermore, body weights were recorded immediately before scheduled necropsies for calculation of relative organ weights

FOOD CONSUMPTION: yes
The food intake of F0 males was measured weekly during the premating period on a seven day basis. In F0 females, food intake was measured in the same way during the premating period. During gestation period determination of food intake was done on post-coital days 0-7, 7-14 and 14-20.
During lactation period determination of food intake was done on day 0-4 p.p.

SACRIFICE and GROSS NECROPSY
- Male animals: All surviving animals.
- Maternal animals: All surviving animals.
Dams were sacrificed on day 4 to 6 p.p. When pups were at least 4 days old dams were anesthetized with carbon dioxide and killed by exsanguination (at carotid artery) and examined for gross pathology. In F0 females the number of implantation sites was counted after staining the uterus with 10% ammoniumsulfide. In a very few cases discrepancies between the No. of implantation sites and the No. of pups delivered per female may have occurred. In the case that the number of pups born was higher than that of implantation sites, the pup number was taken for both parameters.
At necropsy also the number of corpora lutea in the right and left ovary was determined.
Females without pups were necropsied when not longer necessary for matings.
F0 males were killed as scheduled under carbon dioxide narcosis by exsanguination (at carotid artery) when they were administered 28 days to a minimum. They were necropsied and macroscopically examined in the same way as the females.

HISTOPATHOLOGY / ORGAN WEIGHTS
The weights of the heart, kidneys (both), liver, lungs, testes (both), epididymides and ovaries with oviducts were recorded during the scheduled necropsies. The organ weights were specified in both absolute and relative terms. The relative organ weights were calculated by normalization to 100 g body weight determined immediately prior to necropsy of the pertinent animal. Overview on organs evaluated microscopically is presented in Table 2 (in "Any other information on materials and methods incl. tables").
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No (OECD 421 study)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]
The numbers of live and dead pups as well as the sex of the pups (including those of dead pups, if possible) were determined shortly after birth (on postpartum day 0) and on day 4 p.p. At these time points individual body weights and clinical signs were recorded as well. Note was taken of any apparent malformations.

Necropsies of Pups:
Unscheduled Necropsies
Pups that were found dead at birth, that died during lactation as well as those killed (with carbon dioxide) in moribund condition were macroscopically inspected after opening the body cavities, with particular attention on the organs of reproduction except for cases of autolysis or cannibalism. This included also visible skeletal abnormalities as far as possible.
A lung flotation in water was performed during the necropsy of pups found dead on the day of the first litter inspection to determine whether pups had breathed at birth or not.
Scheduled Necropsies
When pups were 4-6 days old they were killed under carbon dioxide anesthesia and were examined for macroscopical alterations as described above.
Statistics:
Statistical evaluation was performed on an Alpha 800 5/500 computer [TASC-system (see above)] using the following methods:
a) Analysis of Variance (ANOVA) and in case of significant results Dunnett test as post hoc test for:
Body weights and body weight gains
Food consumption
Number of implantation sites per female
Number of viable pups
Prenatal loss per litter
Organ weights at necropsy
Time to insemination
Live birth and viability rate
Sex ratio
Duration of gestation
Pup weights

b) 2*N CHi2 test; in case of significant differences Fisher's exact test with Bonferroni correction for:
- Number of viable pups per group based on the number of implantations
- Insemination gestation and fertility index

c) CHi2 test and Fisher’s Exact test:
- Stillborn as well as died, missing and/or cannibalized pups.
The results for the groups, which had received the test substance, were compared with those for the control group. Generally, differences between the control group and groups treated with the test substance groups were considered as statistically significant when p ≤ 0.05.
In these types of statistical processing of measurement values a large number of comparisons are made, which may also lead to false-positive statements. On account of this problem for the evaluation not only the statistical significance but also the biological and toxicological relevance is considered.
For statistical evaluations of histopathological findings, if any, the Path Data program was used.
Indices:
Reproductive indices:
Insemination index (%) = (No. of sperm positive females*/ No. of females co-housed with a male) x 100
Fertility index (%) = (No. of pregnant females***/ No. of sperm positive females*) x 100
Gestation index (%) = (No. of females with live pups/ No. of pregnant females***) x 100
* Including pregnant females that were not sperm positive
*** Animals with implantation sites

Offspring viability indices:
Live birth index (%)** = (No. of live pups at birth/ total No. of pups born) x 100
Viability index (%)** = (No. of live pups on day 4/ No. of live pups born) x 100
** Index calculation from litter means
Historical control data:
The state of health of the strain is routinely tested for the most important specific pathogens on a random basis. The results of these tests are held on file in the test facility. Data on physiology and spontaneous changes in Wistar rats are also available.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: deaths, clinical signs, reduced body weight and food intake, findings at necropsy.

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

One female (No. 91) of the high dose group (8 mg/kg b.w.) died on study day 8 and one female (No. 90) of the high dose group (8 mg/kg b.w.) was killed moribund on study day 11 after two days without treatment.

Males: No toxicologically relevant clinical findings were observed at daily cage-side or detailed clinical observations in all dose groups.

Females during premating and mating: In-cage observations revealed piloerection, a sqatting position and accelerated breathing after dosing in females of the high dose group. No remarkable clinical observations were noticed in treated females up to 3 mg/kg b.w.. In high dosed females sunken flanks, a hunched back, a high-stepping gait, an increased respiratory rate, piloerection and paleness could be observed.

Females during gestation: No remarkable clinical observations were noticed up to 3 mg/kg b.w.. (As none of the high-dosed females (8/5mg/kg b.w.) was pregnant, no clinical observation during gestation could be observed here.)

Females during lactation: No remarkable clinical observations were noticed in treated females up to 1 mg/kg b.w.. One female of the 3 mg/kg b.w. dose group exhibited paleness and piloerection postpartum. None of the high-dosed females littered.

Females without litter: Up to a dose of 3 mg/kg b.w. no remarkable clinical findings could be observed. The females of the high dose group presented piloerection at daily clinical observation and cage-side observation after treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

A statistically significant reduction of body weight (-8.6%) could be observed in males of the highest dose group (only during days 1-8 (8 mg/kg bw))(Table 1). In high-dosed females a statistically significant decrease (-10.6%) of body weight could be observed on study day 8 during the premating phase (8 mg/kg bw) (Table 2). After a drug-holiday of two days (study day 9 and 10) and continuation at a reduced dose of 5 mg/kg b.w. on study day 11, the high-dosed females recovered. Body weight was not affected anymore during the second week of premating.
During the first week of gestation a statistically significant reduction of body weight gain (-28.8%) could be observed in females of the 3 mg/kg b.w.. No effects on body weight gain could be observed during the second and third week of gestation up to 3 mg/kg b.w.. Pregnancy of high-dosed females1) could not be detected.
Body weights were not affected in the lactation period up to 3 mg/kg b.w. (no female of the high dose group littered).

In males and females food intake was not affected by the compound at the doses of 1 and 3 mg/kg b.w. during the premating period. At the high dose1), a statistically significant decrease in food intake per animal per day (males -9.9%, females -37.4%) could be observed in the first week. Statistically significant reduced food intake related to body weight (g/kg b.w./day) could be observed only in females of the high dose group1) (-38.1%) in the first week. After a two days drug holiday and the reduction of dose from 8 to 5 mg/kg b.w. on study day 11, food intake was comparable in all groups during the second week of the premating period (see Table 3).
No pregnancy could be observed in the high dose group.
Food intake in g/animal/day and g/kg/body weight/day was reduced in females of the 3 mg/kg b.w. dose group during the first week of gestation. No effect on food consumption was seen during the second and third week of gestation and during the lactation period (see Table 4).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

At the dose levels of 1 mg/kg b.w. and 3 mg/kg b.w., no treatment-related findings concerning fertility, gestation indices as well as the mean duration of gestation and number of females with live born pups could be observed (see Table 7).
Insemination was slightly reduced in high-dosed rats and additionally fertility and gestation were severely affected. No gestation could be observed in this group, which has to be attributed clearly to the treatment with the test article.
Mating performance was not affected. An overview on the mating performance of the F0 animals is given in Table 8. Only those females were included, in which the mating date could be determined.
As shown in Table 9 no toxicological relevant effects on the number of implantation sites, total No. of pups at birth and prenatal losses could be seen at 1 mg/kg b.w. No implantation sites and no pups were seen in high-dosed females. At 3 mg/kg prenatal losses were slightly increased (statistically not significant) compared to control animals.


ORGAN WEIGHTS (PARENTAL ANIMALS)

No relevant effect on absolute or relative organ weights was found at any dose level in males and in females up to 3 mg/kg b.w. (no organ weight determined in high-dosed females).
A slight statistically significant decrease in absolute epididymides weight in high-dosed males could be observed.
Additionally a slight statistically significant increase in relative liver weight of high-dosed females was seen. The same trend, but not being statistically significant could be observed in absolute liver weights of high-dosed females (see Table 6).

GROSS PATHOLOGY (PARENTAL ANIMALS)

Necropsy did not reveal any test item related findings at the dose of 1 mg/kg b.w. and 3 mg/kg b.w..
One high-dosed female1) (No. 91) died on study day 8 and another female (No. 90) of the highest dose group1) was killed moribund after a two days drug holiday on study day 11.
The following other findings were observed, which all were regarded as chance finding, due to the lack of dose-dependency or due to the absence of correlating histopathological findings:
In the control group one male rat presented a dark-red thymus.
At 1 mg/kg b.w. a deformation in spleen (male No. 13), a distinct liver lobulation (male No. 20), a smaller right testes (male No. 22), and an enlarged left testes (male No. 24) could be observed.
At 3 mg/kg b.w. a dilatation of right pelvis (male No.25), a nodule on the kidney (male No. 27), and a rough surface of spleen (male No.28) could be observed.
One male of the high-dose group revealed a smaller parostate gland (No. 41) and another male revealed dilatation of pelvis (No. 42).
One female of the control group (No. 53) presented a brownish-red nodule on the liver.
At 1 mg/kg b.w. a brown red mass was observed in the right horn of a uterus (No. 71).
At 3 mg/kg b.w. one female presented a smaller left kidney and an enlarger right kidney. Furthermore a reddish cyst was observed on the ovaries. This animal (No. 79) did not present any implantation sites or corpora lutea. Another female of the 3 mg/kg b.w. group presented several dark-red masses in the left horn of the uterus (No.80).
At the high-dose group of 8 mg/kg b.w. female No 90 which was killed moribund, presented a changed surface of kidneys, a smaller thymus and a red, elastic lump at the mucosa of the gastro-esophageal vestibule.
Female No 91 that died on study day 8 presented an enlarged adrenal gland, a slightly collapsed, dark-redded lung and aqueous liquid in the chest cavity.
No relevant changes in number of corpora lutea or the number of animals with implantation sites or without implantation sites could be observed up to 3 mg/kg b.w. At 5 mg/kg b.w. neither corpora lutea nor implantations sites were seen (Table 5).

HISTOPATHOLOGY (PARENTAL ANIMALS)

One female of group 04 was found dead on study day 8. This animal had a slight myocardial degeneration, an alveolar edema in the lungs, slight multifocal bile duct hyperplasia, and signs of moderate tubular degeneration and regeneration in the kidneys. Another animal of this group had to be euthanized due to a marked ulcer in the forestomach. Therefore, after a short treatment free period, dosing was reduced to 5 mg/kg b.w./d beginning with study day 11, and no further mortalities occurred during the course of the study. During terminal necropsy, no implantation sites and no corpora lutea were recorded in the ovaries in any of the animals from the high dose group (04). This has to be attributed clearly to the treatment with test article. In contrast, the distribution of the number of implantation sites and corpora lutea was comparable among the controls and the animals treated with 1 or 3 mg/kg b.w./d body weight Stabaxol. This became evident also from the histopathological investigation of the ovaries (uterus not investigated). No treatment related findings were seen in other organs investigated in this study.

Effect levels (maternal animals)

Dose descriptor:
NOEL
Effect level:
1 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING)
The total numbers of pups born, stillborn pups, the live birth index, percentage of males born, the litter size at birth and the viability index were not affected by treatment (see Table 10).

CLINICAL SIGNS (OFFSPRING)
No clinical signs with a dose-dependent distribution up to 3 mg/kg b.w. were observed in F1 pups during the five days of lactation.
The following findings were considered as spontaneous findings: Two pups of the mid dose group were cold to touch, with one of these pups beeing pale.

BODY WEIGHT (OFFSPRING)
No toxicological relevant findings concearning pup weights at birth and on day 4 p.p. were noticed (see Table 11).


GROSS PATHOLOGY (OFFSPRING)
No macroscopical alterations with a remarkable incidence or dose-dependency were observed at pup necropsies.
The litter of dam No. 82 dosed at 3 mg/kg b.w. consisted of two pups. One pup died on day 0 and revealed no milk in stomach; the other pup died on day 1 and was found in an autolytic state.

Effect levels (fetuses)

Remarks on result:
other: Explanation
Remarks:
No toxicologically relevant clinical signs were observed in F1 pups.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Analysis of Test Item in the Vehicle

The analytical content checks of the test item in the vehicle, which were performed twice during the study in all concentrations, showed that all dose formulations agreed to the target values within defined limits.

Table 1.Body Weights and Body Weight Gain in F0 Males

Dose in mg/kg b.w.

0

1

3

81)

Day

Body weight

 1

260.9

259.4

257.9

255.8

 8

290.3

293.2

285.3

275.3**

Dose in mg/kg b.w.

0

1

3

5

15

319.5

321.3

310.7

301.2**

22

333.5

337.3

326.5

311.5**

29

353.8

355.3

345.2

327.9**

36

371.3

376.6

360.8

339.2**

Difference to control

 

+1.4%

-2.8%

-8.6%

 

Body weight gain

1-36

110.4

117.2

102.9

 83.4

Difference to control

 

 +6.2%

 -6.8%

-24.5%

Table 2.Body Weights and Body Weight Gain in F0 Females

Dose in mg/kg b.w.

Day 1 premating

Day 8 premating

Day 15 premating

 

Gestation Day p.c.

Lactation Day p.p.

0

7

14

20

0

4

 

Body weights in g

0

224.8

237.4

246.2

 

247.4

277.3

307.6

373.3

288.2

297.9

1

225.8

238.7

246.3

 

250.7

276.7

306.8

372.8

302.3

306.3

3

223.7

236.8

239.3

 

243.1

264.4

293.4

357.8

279.8

289.5

51)

225.6

212.2**

236.6

 

 

 

 

 

 

 

 

Body weight gain in g

 

 

 

Day 1-15

 

 

Gestation Day p.c.

Lactation Day p.p.

 

 

 

Premating

 

 

0-7

7-14

14-20

0-4

0

 

 

21.4

 

 

29.9

30.2

65.8

9.7

1

 

 

20.5

 

 

26.0

30.2

66.0

4.0

3

 

 

15.6

 

 

21.3**

29.0

64.4

9.8

51)

 

 

11.0

 

 

 

 

 

 

Table 3.Food Intake during the Premating Period

 

Mean Food Consumption during the Premating Period

Dose

 

Week 1

Week 2

Week 1

Week 2

mg/kg b.w.

Sex

g / animal / day

g / kg body weight / day

0

m

22.3

21.0

85.4

72.1

1

m

22.3

20.8

85.9

70.9

3

m

21.3

19.7

82.7

69.2

51)

m

20.1*

20.4

78.8

74.2

0

f

16.3

15.9

72.7

67.1

1

f

16.0

15.8

71.1

66.2

3

f

15.8

14.8

70.6

62.6

51)

f

10.2**

14.3

45.0**

67.1

Table 4.Food Intake in Females during Gestation or Lactation Period

 

Mean Food Consumption during Gestation or Lactation Period

Dose

Gestation

Gestation

Gestation

Lactation

mg/kg b.w.

Day 0-7

Day 7-14

Day 14-20

Day 0-4

 

g / animal / day

0

18.6

21.4

23.3

26.9

1

17.6

20.2

24.4

26.0

3

16.1**

19.4

23.2

30.1

51)

-

-

-

-

 

g / kg body weight / day

0

75.3

77.0

75.8

94.0

1

70.5

72.9

79.4

86.0

3

66.4*

73.2

79.1

107.5

51)

-

-

-

-

1) day 1-8 of study 8 mg/kg b.w., day 9-10 of study not dosed, day 11 of study onwards 5 mg/kg b.w.

* = p0.05    ** = p0.01

Table 5. Implantation sites

Dose in mg/kg b.w.

0

1

3

5

Implantation sites

9

8

10

0**

No implantation sites

3

4

2

10

Table 6. Organ weights

Dose in mg/kg b.w.

Sex

Body Weight (g)

Heart

Kidneys

Liver

Lung and Trachea

Epididy-mides

Testes

Ovaries

 

 

 

 

absolute (mg)

 

 

0

m

377.8

1156

2500

14262

1846

1398

3759

-

1

m

384.3

1179

2672

15815

1808

1400

3837

-

3

m

367.6

1229

3101

13185

1852

1398

3768

-

5

m

345.8**

1155

2500

13748

1703

1287*

3502

-

 

 

 

 

relative (mg / 100 g body weight)

 

 

0

m

377.8

0.3064

0.6629

3.7709

0.4887

0.3715

0.9983

-

1

m

384.3

0.3067

0.6951

4.1055

0.4710

0.3650

0.9988

-

3

m

367.6

0.3360

0.8678

3.5864

0.5062

0.3805

1.0281

-

5

m

345.8**

0.3334

0.7228

3.9706

0.4916

0.3728

1.0143

-

 

 

 

 

absolute (mg)

 

 

0

f

297.9

1015

1970

13063

1410

-

-

141

1

f

308.7

1052

1899

14294

1693

-

-

163

3

f

289.5

1090

1983

14734

1442

-

-

149

5

f

-

-

-

-

-

-

-

-

 

 

 

 

relative (mg / 100 g body weight)

 

 

0

f

297.9

0.3409

0.6624

4.3950

0.4735

-

-

0.0475

1

f

308.7

0.3418

0.6169

4.6307

0.5427

-

-

0.0526

3

f

289.5

0.3774

0.6857

5.0969*

0.5000

-

-

0.0516

5

f

-

-

-

-

-

-

-

-

Table 7.Data Concerning Insemination, Fertility and Gestation (F0)

Dose

mg/kgb.w.

0

1

3

5

Insemination index

%

100.0

100.0

100.0

90.0

Fertility index

%

75.0

66.7

83.3

0.0

Gestation index

%

100.0

87.5

90.0

-

Gestation length

Days

22.56

22.50

22.63

-

Co-housed females

n

12

12

12

10

Litters with liveborn pups

n

9

7

9

0

Table 8. F0 Mating Performance

 

Mean Time to Insemination

Dose mg/kg b.w.

0

1

3

5

Mating Days until Day 0 p.p.

3.0

4.8

3.6

2.9

Table9. Evaluation of Implantation Sites in F0 Females

Dose

No. of Implantation Sites

Total No. of Pups at Birth

Prenatal Loss

mg/kg b.w.

per Litter

Total

Means per Litter

0

12.56

113

106

0.78

1

11.71

82

76

0.86

3

12.89

116

98

2.0

5

-

-

0

-

Applicant's summary and conclusion

Conclusions:
Overall, a steep dose response curve was observed and the following no-observed-(adverse)-effect-levels were determined:
F0 Rats: NOAEL: 3 mg/kg bw.
Reproduction/Developmental Toxicity: NOEL : 1 mg/kg bw., due to the slight increase of prenatal losses at 3 mg/kg b.w..
Executive summary:

Bis-(2,6-diisopropylphenyl) carbodiimide was administered daily via gavage in corn oil as vehicle to 12 male and 12 female Wistar rats per dose group, in doses of 0, 1, 3 or 8 mg/kg body weight. Dosing of the highest dose group treated with 8 mg/kg bw was stopped on study day 8, as toxicity in this dose was too pronounced for the purpose of the study. Animals of both sexes were not treated on study day 9 and 10. In the morning of study day 11 dosing was continued at a reduced dose of 5 mg/kg bw.

Treatment started 2 weeks prior to mating and continued during the mating period of up to 2 weeks. Males were dosed further up to necropsy for a total period of at least 4 weeks and females were dosed during gestation and lactation up to their necropsy on day 4-6 p.p.

Investigations were performed on general tolerance of the test compound by the parental animals as well as on effects on reproduction including early postnatal development of F1 pups. The animals were regularly observed and weighed, food intake and reproduction parameters were determined. Selected organs were weighed and organs were subjected to macroscopical and histopathological investigations.

The test substance was stable in the vehicle for the duration of use. Formulations given to the rats were prepared appropriately.

Survival of both sexes was not affected in the groups treated with 1 or 3 mg/kg bw. At the high dose of 8 mg/kg bw one female (No. 91) died on study day 8 and one female (No. 90) was killed moribund on study day 11 after a two days drug holiday. The high dose was reduced to 5 mg/kg bw after the two days of drug holiday. With that dose there was no obvious clinical toxicity and no further mortality.

Clinical observations did not reveal any relevant adverse effects in males up to the high dose and in females up to the mid dose of 3 mg/kg bw An impaired general condition was observed in females starting at 8 (5) mg/kg bw. During premating and mating in-cage observations the females of this dose group revealed piloerection, a squatting position and accelerated breathing after dosing. Furthermore, sunken flanks, a hunched back, high-stepping gait, an increased respiratory rate, piloerection and paleness were noted. The females of the high dose group without litter presented piloerection at daily clinical observation and cage-side observation after treatment. None of the high-dosed females was pregnant or littered.

Body weight was reduced in males of the highest dose group. High-dosed females which revealed a decrease in bw on day 8 of premating, recovered after the two days of drug holiday and a reduction to 5 mg/kg bw. The reduction of food intake observed in high-dosed females and males in the first week of premating was comparable in all groups during the second week of the premating period. Food intake was also reduced in females of the 3 mg/kg bw dose group during first week of gestation, but no effect on food consumption was seen during the second and third week of gestation and during the lactation period, anymore. (No pregnancy could be observed in the high dose group).

Necropsy revealed an enlarged adrenal gland, a slightly collapsed, dark-redded lung and aqueous liquid in the chest cavity of the high-dose female No. 91, which died on study day 8.

High-dosed female No. 90, which was killed moribund after a two days drug holiday on study day 11, presented a changed surface of kidneys, a smaller thymus and a red, elastic lump at the mucosa of the gastro-esophageal vestibule at necropsy.

Histopathology of No. 91 revealed a slight myocardial degeneration, an alveolar edema in the lungs, slight multifocal bile duct hyperplasia, and signs of moderate tubular degeneration and regeneration in the kidneys and No. 90 presented a marked ulcer in the forestomach.

During terminal necropsy, no implantation sites and no corpora lutea were recorded in the ovaries in any of the animals from the high dose group. This has to be attributed clearly to the treatment with test article. In contrast, the distribution of the number of implantation sites and corpora lutea was comparable among the controls and the animals treated with 1 or 3 mg/kg bw/day body weight test substance. No further histopathological, treatment-related and toxicologically relevant findings could be observed up to the highest dose group.

The insemination index was slightly reduced in high-dosed rats. Additionally fertility and gestation were severely affected in the high dose. No gestation could be observed in this group, which has to be attributed clearly to the treatment with the test article. No implantation sites and no pups were seen in high-dosed females. At 3 mg/kg bw a slight, but statistically not significant increase of prenatal losses was observed.

No toxicologically relevant clinical signs were observed in F1 pups during lactation. One pup of dam No. 82 of the 3 mg/kg bw dose group died on day 0 and revealed no milk in it´s stomach; the other pup of this dam died on day 1 and was found in an autolytic state. 

Overall, a steep dose response curve was observed and the following no-observed-(adverse)-effect-levels were determined: NOAEL of 3 mg/kg bw for F0 rats (general toxicity) and NOEL of 1 mg/kg bw for reproduction and developmental toxicity, due to the slight increase of prenatal losses at 3 mg/kg bw.