N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May 2007 to 30 September 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

2001

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: M.A.F.F. in Japan 12 Nousan N° 8147 (2000)

Version / remarks:

M.A.F.F. in Japan 12 Nousan N° 8147 (2000)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The concentrations of the present study were based on the results of a subacute pilot feeding study in rats T9077717, performed at Bayer HealthCare AG, Wuppertal) and an oncogenicity rat feeding study (Laboratory Project ID 551-030) performed at International Research and Development Corporation Mattawan, Michigan, USA. In the subacute study MGK 264 was fed to Wistar rats in concentrations of 0, 625, 1250 and 10000 ppm for 4 weeks. In this study no effect on survival as well as appearance and behavior of the rats were obvious up to 10000 ppm.
At 10000 ppm males were reduced by 6% in terminal body weights. At 10000 ppm statistically significantly increased absolute (males: 26%; females 58%) and relative (males: 34%, females: 64%) liver weights were observed. At necropsy, enlargement and distinct lobulation of the liver was recorded in all high dose animals. In males, the mid dose group was also affected by a distinct lobulation (two males) and a swollen liver (one male).

Histopathologically the macroscopical findings correlated with a minimal to moderate hypertrophy and eosinophilic cytoplasmic change of the centrilobular hepatocytes, which was noticed in all high dose rats as well as in all mid dose males. These findings were considered as the morphological correlate of enzyme induction which is regarded as an adaptive response to the hepatocellular test substance metabolism. They are not considered as adverse effects.

Additionally, all high dose males and nearly all high dose females showed a minimal to moderate cholestasis of the intra-hepatic bile ducts with a minimal to slight pericholangiolar mononuclear cell infiltration. The affected bile ducts showed a prominent epithelium. In three males even a minimal to slight bile duct proliferation was observed. In rats with a slight or moderate cholestasis mild signs of degeneration of the bile duct epithelium were seen. As demonstrated by the ORO-stain, a minimal to moderate lobular fat accumulation was seen in males and females dosed at 625 ppm with an increase in severity.

In the oncogenicity study (0, 400, 2000 or 15000 ppm) 400 ppm MGK 264 were tolerated without toxic effects. At higher doses several signs of toxicity occurred including morphological liver changes comparable to those seen in the subacute study.

Thus, from these results 6400 ppm was considered to be be a maternal toxic dose, whereas 400 ppm as seen in the oncogenicity study was expected to be a NOAEL.

Therefore, this 2-generation reproduction study was dosed at dietary concentrations of 0, 400, 1600 and 6400 ppm.

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

EC Number:

204-029-1

EC Name:

N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

Cas Number:

113-48-4

Molecular formula:

C17H25NO2

IUPAC Name:

N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

impurity 1

Reference substance name:

(E)-1,4-Bis(2-ethylhexylamino)-2-butene-1,4-dione

Molecular formula:

C20H38N2O2

IUPAC Name:

(E)-1,4-Bis(2-ethylhexylamino)-2-butene-1,4-dione

impurity 2

Reference substance name:

1-(2-ethylhexyl)-1H-pyrrole-2,5-dione

Molecular formula:

C12H19NO2

IUPAC Name:

1-(2-ethylhexyl)-1H-pyrrole-2,5-dione

Test material form:

liquid

Details on test material:

Purity and characterisation analysis conducted on the following sample; Supplier: McLaughlin Gormley King ; Batch Number: AB9500

Specific details on test material used for the study:

MGK® 264
CAS No.: 000113-48-4
Batch No.: KP048TK
Content(s ): 94.2 %MGK 264 (December15, 2006 and December 11, 2007)

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

SPF-bred rats of the strain Wistar Crl:(Wl)WU BR supplied by the breeder Charles River GmbH, 97633 Sulzfeld, Germany.

Sex:

male/female

Details on test animals or test system and environmental conditions:

The environmental conditions in the animal room No. 245 Building 515 were standardized as follows:
Room temperature: 23 ± 2 °C
Air humidity: 55 ± 5%
Air exchange: ~ 10 passages per hour
Light/ Dark cycle: 12 hours rhythm

Occasional deviations from these specifications occurred, e.g. as a consequence of cleaning the animal room. They had no identifiable influence on the course of the study.

During light phase low-noise radio music program was played in the animal room.

During the acclimatization and experimental period animals were kept singly, except when co-housed for matings or with litters, under controlled environmental conditions in Makrolon ® cages Type IIIh. As bedding material low-dust wood granules (supplier: Ssniff Spezialdiaten Inc. 59494 Soest, Germany) and nesting material (day 20 p.c. to 14 p.p.) produced by S.P.P.S. in 25560 Frasne, France, were used. Cages and bedding were exchanged at least weekly with new ones. For environmental enrichment wooden blocks supplied by Tapvei OY, 73620 Kortteinen, Finland, were offered per cage during the whole in-life phase and renewed, if necessary.

The parental animals were individually identified by cards on the ·cages specifying the test substance, the animal number, dose, sex and study number. The color of the cards for each dose group was different. In addition, the parental animals were identified by tattooed tail corresponding to the animal number on the cards. Pups were identified by foot tattoos.

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

Route: via diet
Frequency of Administration: continuously in the diet
Vehicle: basal diet - Provimi Kliba 3883.G4.S2S
Preparation of Diet Mixtures: at least once weekly
Storage of Diet Mixtures: room temperature
Diet Mixtures Stable for a Period of: 15 days

Details on mating procedure:

In general, the first adult F0 male was caged over night with the first F0 female from the same test group, whereas the fourth Fl female was mated with the first male of the corresponding dose group, and so on. Mating occurred at a maximum of 12 times during the three-week mating period until sperm cells were observed in vaginal smears taken the next morning. Inseminated females were not further cohoused with the below mentioned exception. Generally, if a male had to be necropsied prior to co-housing the next male was co-housed with two females.

Rematings
F0 and/or Fl females found sperm-positive after the first mating day but not shown to be pregnant (lack of weight gain within 14 days following insemination) were cohoused again over one week with the same male without checking insemination or measuring body weight and food intake during possible further pregnancy. According to our experience occurrence of sperms after the first co-housing day without a following pregnancy can happen, if an inexperienced male, co-housed with a female for the first time, inseminated the female outside the estrus.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A liquid chromatographic method for quantifying the test item in feed formulation was developed. For analytical investigations, representative samples from the test item formulation covering the test item concentration range used in the study, were taken. These samples were extracted with acetonitrile and after optional filtration quantified by reversed phase (C-18) high-performance liquid chromatography {HPLC) with UV detection DAD, diode array detector; wavelength: 210 nm). Standard solutions of the authentic test item were used for calibration. Linearity, Precision, Specificity, Robustness and Accuracy of the analytical method were evaluated apart from this GLP-study and fulfil the predefined acceptance criteria. Additional system suitability tests in terms of specificity, precision and linearity indicated that qualified analytical procedures were followed during the study.

Duration of treatment / exposure:

The purpose of this two-generation reproduction study was to evaluate possible effects of MGK 264 on the entire reproduction process in Wistar rats. MGK 264 was administered to groups of 25 male and 25 female rats at concentrations of 0 (control), 400, 1600 and 6400 ppm in their diet. During the premating period the test substance intake was: F0: 32.9, 137.3 and 565.6 mg/kg (males); 51.2, 212.4 and 851.9 mg/kg (females).
Fl: 39.9, 180.0 and 733.5 mg/kg (males); 53.0, 238.5 and 1051.2 mg/kg (females).

Parental F0 animals received the test substance for a period of about 10 weeks and were then allowed to mate over a period of up to three weeks. F 1 offspring were nursed up to an age of four weeks. Some of them were selected for further treatment and for breeding a F2a or F2b generation. Generally, the test substance was administered to each animal up to necropsy.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Remarks:

Control Group

Doses / concentrations 2

Dose / conc.:

400 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

1 600 ppm (nominal)

Doses / concentrations 4

Dose / conc.:

6 400 ppm (nominal)

No. of animals per sex per dose:

25

Details on study design:

The F0 animals were pretreated with the test substance for about 10 weeks up to the cohabitation period. Within the weeks 8 - l 0 of the premating period, examinations on estrus cycle were performed. Then the mating period of 3 weeks followed.

After a gestation period of about 22 days litters were born and the dams were allowed to rear them. If necessary, four days after birth the FI litters were reduced ( = culled) to eight pups. If possible, four male and four female pups remained per litter. Pups found in a moribund state on day 4 were excluded from lactation. This was done to investigate possible malformations and to prevent cannibalism during the further rearing period. The remaining FI pups were reared to an age of four weeks and then necropsied when not used as weanlings for further treatment. F0 females were killed and necropsied when about 28 days old Fl pups had been weaned. F0 males were killed after the mating period partly in the course of spermatological investigation.

Twenty-five male and 25 female Fl rats per group were selected for further treatment and to breed the F2 generation. This was done by randomly selecting one male and one female as far as possible from each litter. The weaned Fl offspring was treated further with the substance for at least 10 weeks (premating period started on the day when the last Fl rat had been appointed to its group) and then co-housed for mating. During the last three weeks of this period estrus cycle determinations were performed. The procedures during the mating, gestation and lactation period of Fl rats were the same as described for F0 rats.

As the study results concerning viability and lactation, received from first mating of F1 rats, had to be clarified, it was decided to perform a second mating to produce F2b litters using the same mating scheme.

One week after the last F2a litter was weaned, mating procedures and activities during gestation and lactation were repeated as done for the production of F2a pups except cycle determinations, additional mating procedures and measurement of the ano-genital distance. Concerning implantation sites of F1 females the occurrence or missing of implantations instead of the number was recorded on data sheets but not entered into the TASC system.

All litter and pup parameters already done for the F2a pups, were evaluated for F2b pups. At weaning F2b pups were killed as described for F2a pups with necropsy and organ measurements.

F1 males were sacrificed after 2nd mating procedures.

The F1 dams were killed as scheduled after their F2b litters had been weaned at about day 28 p.p. as described for F0 dams.

Positive control:

N/A

Examinations

Parental animals: Observations and examinations:

Inspections on mortality and morbidity of the animals were done twice daily (once daily on weekends and public holidays). Clinical findings were recorded individually daily.

All signs of illness or clinical reactions to treatment (especially during littering) were recorded daily (cage-side observation). All further clinical symptoms were also recorded. This investigation included the observation of the general state of health, behavior, condition of the fur, and the orifices as well as excretory products. Any further findings e.g. prolonged parturition were recorded as well. A detailed clinical examination ·was done prior to the first administration of the test substance in the diet and then weekly as a rule and during the gestation and lactation periods as follows:
- Gestation on day 0 (=day of a sperm positive smear or vaginal plug) 7, 14 and 20.
- Lactation on day 0 (day of birth), 4, 7,-14, 21 and 28.
Body weights were recorded directly prior to the first administration and thereafter weekly up to necropsy (except males and females not pregnant) and during the gestation and lactation periods as follows:
During gestation on day p.c. 0, 7, 14 and 20.
During lactation on day p.p. 0, 4, 7, 14, 21 and 28.
On the day of scheduled necropsy.
The individual food consumption was measured (by weighing the quantity of food provided and back-weighing the amount, which remained unconsumed) as follows:
-Males: Weekly from week 1 up to necropsy (except during mating periods).
- Females: Weekly from week 1 up to mating.
During gestation day p.c. 0-7; 7-14; 14-20.
During lactation day p.p. 0-4; 4-7.
From these food intake data the intake of the test substance was calculated per animal and per kg body weight per day, week or for a given period.
The algorithms used for all these calculations are described in the Annex (Chapters 8.2 and 8.3) of the final report (attached).
During the mating periods vaginal smears were taken in the morning after rats had been co-housed over night to determine time to insemination and gestation length. The vaginal smears were obtained using a flame-sterilized platinum loop and plated out on slides. Smears were stained for about 1 minute with MAY GRUNWALD 'S Eosine-methylene blue solution modified for microscopy.
The date on which sperms or a vaginal plug were found microscopically was taken as gestation Day 0 for calculating the gestation length. Females which exhibited marked weight gains although insemination had not been established were not further co-housed. No duration of gestation could be determined for these animals.

Oestrous cyclicity (parental animals):

Estrus cycle length determination was done by evaluation of vaginal smears (see above) received daily over 19 consecutive days about 3 weeks prior to the mating period. The smears were examined microscopically for large serrated cells indicating that estrus had occurred. These data were used to determine the estrus cycle length and whether females were cycling properly.

Sperm parameters (parental animals):

Spermatological investigation was performed in all surviving F0 and Fl males of the 0 and 6400 ppm group on the day of necropsy as routine.

Just after testes and epididymides had been dissected sperm samples were collected from the right cauda epididymis and suspended in HAM's F 10 tissue culture medium (37°C). A sample of about 20 μI of the culture medium was placed on a 37°C warmed and siliconated slide and then covered with a warmed cover glass. Then sperm motility was recorded on 100 spermatozoa during minute 1 and minute 5 after preparation of the sample using semi-dark field microscope. The microscope stage was warmed up at 37°C. Spermatozoa showing a forward swimming movement were given a positive score.
Furthermore, the difference between motility recorded during the first minute and that measured during the fifth minute was calculated. Data of these analyses were recorded off-line and video taped.

Spermatozoa morphology was evaluated in a formalin citrate fixed and Eosin G stained sperm sample collected from the right cauda epididymis. Morphological changes of the head, upper and middle tail were evaluated on 200 spermatozoa using the microscope as mentioned above. Data of these analyses were recorded off-line and video taped.

Determination of spermatozoa density was performed in a suspension (0.9% NaCl) of minced cauda epididymis tissue by counting of spermatozoa in a hemocytometer and calculation of spermatozoa density per mg epididymis. The samples used for counting had been heated (> 70°C) shortly. Each sample was counted in 5 tertiary squares of two secondary squares.

Determination of spermatid head density was performed in a suspension (0.9% NaCl plus 200 μl Triton x-100) of homogenized testis tissue by counting of spermatid heads in a hemocytometer. Each sample was counted in 5 tertiary squares of two secondary squares.

Litter observations:

Number of live pups;
Pup weight
External alterations (=clinical and necropsy observation)
Number of dead pups
Ano-genital measurements X (only F2)
Sex of each pup (M/F) x
Developmental milestones (F1 Post weanlings)

Postmortem examinations (parental animals):

All parental rats to be necropsied as scheduled were killed by exsanguination under C02 narcosis.
Males and non pregnant females: When no longer needed for matings.
Females with pups: At weaning of 28 days old pups or up to 2 days later.
All parental rats to be necropsied unscheduled were also killed by exsanguination
under C02 narcosis.

Macroscopical post-Mortem Examination
Macroscopical investigation was done on all rats. Animals were subjected to detailed post-mortem examinations as follows. Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Implantation sites of FO females were counted after staining the uterus with 10% aqueous ammonium sulfide. In cases where implausible discrepancies existed between the number of observed implantation sites and the number of pups delivered (more pups than implantation sites) the number of pups was used as number of implantation sites. In case of autolytic rats or rats killed pre-scheduled the number of pups was taken as the number of implantation sites.
Concerning implantation sites of FI females the occurrence or missing of implantations instead of the number was recorded after staining the uterus with 10% aqueous ammonium sulfide.
Vaginal smears were performed and evaluated at scheduled necropsy as precaution but not reported herein.
Rats found dead or which had to be killed in moribund condition were necropsied as early as possible in the same way.

Determinations of MGK 264 in Tissues
At terminal necropsy samples of the liver, peri-renal fat and sceletal muscle (right femur) were taken from the first ten Fl males sacrificed scheduled. The samples were frozen at about 20°C and delivered to Dr. Krebber (Bayer CropScience AG BCS-D-ROCS, 040789 Monheim) for analytical determination of MGK 264. Raw data of these determinations will be stored there.

Preservation of Tissues
The following tissues of adult FO and Fl rats were generally fixed in I 0% formalin with the exception of ovaries, the left and two thirds of the right kidney and testes, which were fixed in Davidson's solution.

Organ weights
Organs were weighed at necropsy, see table 5-4 in full report for details.
Histopathological Evaluations
The organs were microscopically examined at least in the control and high dose groups. See table 5-4 in full report for details.
This included also all FO/Fl rats from all dose groups, which died intercurrently (as far as possible) and those, which were killed moribund. Organs altered possibly by the treatment and any gross and/or microscopically changed lesions were examined in all rats. Staging of ovarian follicles was done in Fl females (control and high concentration) only. All reproductive organs of pairs without pups were investigated (except those of the male No. 240 of female No. 337).

Postmortem examinations (offspring):

Sacrifice of Pups
In case of sacrifices done prior to weaning pups were killed under CO2 narcosis.
Weanlings on day 28-30 p.p. were killed by cervical dislocation under CO2 narcosis.

Macroscopical post-mortem examination
All pups culled for standardization of litter size, found dead or had to be killed moribund during lactation were necropsied and investigated macroscopically 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 (life birth) or not.

All F1 weanlings not selected for further treatment were killed when they were at least 4 weeks old and examined macroscopically.

All F2a and F2b weanlings were killed after a 4 week lactation period and examined macroscopically.

Preservation of Tissues
Grossly abnormal tissues if any were fixed in all pups/weanlings. In F1, F2a and F2b weanlings brain, spleen, thymus and uterus of one male and one female out of the firstly necropsied 5 litters per group were fixed in 10% formalin.

Organ Weights
The brain, spleen, thymus and uterus of one male and one female per F1 /F2a/F2b litter were trimmed and weighed as soon as possible after dissection. The ratio of organ weights to body weights was calculated. Therefore, all these weanlings were weighed at day of necropsy.

Statistics:

Statistics
Statistical evaluation was performed on an Alpha 800 5/500 computer (TASCsystem (see below)) using the following methods:
a) Analysis of Variance (ANOVA) and in case of significant results Dunnett's test as post hoc test for:
• Body weights and body weight gains of the male and female animals
• Food consumption of the male and female animals
• Number of implantation sites per female
• Number of viable pups per female
• Organ weights at necropsy
• Number of estruses
• Time to insemination
• Life birth, viability and lactation rate
• Determination of estrous length

b) 2 by 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, fertility, gestation and rearing rate

c) Kruskall-Wallis test and in case of significant differences Dunnett's test for:
• Number of prenatal loss per litter
The sperm and spermatid count data were not evaluated statistically, because no meaningful differences occurred between the high dose and control groups.
Generally, differences between the control group and groups treated with the test substance groups were considered as statistically significant when p ≤ 0.05.
Significant differences from the control are indicated with* for p ≤0.05 and **for p ≤0.01
Follicle counts were evaluated statistically using the Wilcoxon Mann-Whitney U test (details see Pathology Report).

Reproductive indices:

See attached background information for equations

Offspring viability indices:

See attached background information for equations

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no remarkable changes in clinical appearance and behavior of parental rats during daily in-cage or detailed clinical observations up to 6400 ppm (F0 and Fl in both sexes). The relatively high incidence of 6400 ppm F0 females exhibiting a bloody muzzle is considered incidental, because such findings were not evident in males and F1 females.
The following clinical symptoms not shown in the following tables were recorded during daily cage-side observations and are considered incidental:
Breathing sounds and increased salivation in one 0 ppm F 1 female on day 12 of gestation after the 1st mating.
Blood in bedding material in one 400 ppm Fl female on day 25 p.c. after the 1st mating.

Mortality:

no mortality observed

Description (incidence):

No unscheduled deaths occurred. Therefore, no treatment-related increase in mortality was seen in males and females up to 6400 ppm.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Selected body weight and food consumption (g/kg body weight/day) data received during the premating periods are summarized in Table 6-3 of the final report (attached). A summary table of all study findings has also been attached for reference.
As shown there up to the end of the premating periods no adverse effect on body weights and body weight gain was seen up to 1600 ppm in F0 and F1 rats. In F0 females as well as in Fl males and females receiving 6400 ppm body weights (≤0.05) and body weight gain (p>0.05) were slightly reduced (up to 6%). The daily food intake per body weight was unchanged up to 1600 ppm and slightly increased at 6400 ppm in F1 rats.

Selected group mean body weights and food consumption values for pregnant or nursing dams are summarized in Table 6-4 of the final report (attached). Following these data no adverse changes in body weight development were seen in pregnant or lactating F0 or F1 females up to 1600 ppm. At 6400 ppm statistically significant body weight depression (6-10%) was observed in both generations. The daily food intake per body weight was slightly enhanced at 1600 and/or 6400 ppm in F0 and F1 females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The daily food intake per bodyweight was unchanged up to 1600 ppm and slightly increased at 6400ppm in F0 & F1 rats. Accordingly at this concentration a slightly higher test substance intake was calculated than expected from the theoretical dose factor.

Test substance intake
Based on food consumption and body weight data, the doses expressed as mean daily mg test substance/kg body weight during the premating period are presented in Table 6-5 of the final report (attached). The values for the F0 and Fl generation are considered to be representative for the test substance intake for the entire study. During the premating periods the test substance intake per kg body weight increased roughly in the dose groups as expected from the theoretical dose factor up to 1600 ppm in F0 and F 1 rats. 6400 ppm rats ingested some times slightly more test substance as expected from the dose factor, because of a slight increase in food intake per body weights. This was also true for F0 females during gestation and lactation period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The appearance and behavior of the parental animals were not toxicologically relevantly influenced due to the test substance up to 6400 ppm.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A summary table of all study findings has also been attached for reference.
Microscopical examination:
The following treatment-related organ changes were noted:
Liver:
- hypertrophy and/or eosinophilic cytoplasmic change of centrilobular hepatocytes in males at 1600 ppm and above and in females at 400 ppm and above in both generations
- periportal hypertrophy in females at 1600 ppm and above (F0 generation)
- deposition of a brownish pigment in/around the bile ducts with prominent bile duct epithelium, periductular cellular infiltration and/or degeneration of bile duct epithelium at 6400 ppm in both sexes and generations
- presence of hydropic foci in males at 6400 ppm (both generations)
- increased amount of periportal centrilobular fat storage in F0/F1 males beginning at 1600 ppm and in F0 and F1 females at 6400 ppm.
Kidneys:
- increased pigment storage of a brownish pigment at 1600 ppm and above in both sexes (F0 and/or F1 generation)
- increased severity in basophilic cortical tubules in 6400 ppm F0 males
- increased severity in hyaline droplets in males at 6400 ppm (F0 generation)
Thyroid glands:
- follicular cell hypertrophy and colloidal alteration in males at 1600 ppm and/or 6400 ppm in both generations
- follicular cell hypertrophy in F0 and F1 females at 6400 ppm
Female genital tract:
- altered luteinization of the corpora lutea of F0 and F1 females at 6400 ppm

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Results from the evaluation of vaginal smears performed at the end of the premating periods indicate that there were no toxicologically relevant effects on the estrus cycle up to 6400 ppm in F0 and F1 rats (see Table 6-7 of the final report (attached)).

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Results of sperm analyses are summarized in Table 6-8 of the final report (attached). There no toxicologically relevant effects on sperm parameters ( epididymal sperm count, sperm motility and morphology and testicular spermatids counts) were detected in F0 and FI rats up to 6400 ppm.

Reproductive performance:

no effects observed

Description (incidence and severity):

Results of reproductive performance for the parental animals are summarized in Table 6-9 of the final report (attached). There no adverse changes attributable to the test substance were detected on indices for insemination, gestation, fertility and rearing, on gestation length as well as on number of litters born and duration of co-housing period up to insemination.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

400 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Critical effects observed:

no

Lowest effective dose / conc.:

1 600 ppm

System:

hepatobiliary

Organ:

liver

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical observations on pups revealed no remarkable increase in clinical symptoms up to 6400 ppm in Fl pups.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was a relatively low F1 pup viability index at 0 ppm (control group), whereas the highest viability index and consequently lowest number of unscheduled pup deaths were seen at 6400 ppm.

The litter data of F1 rats showed no toxicologically relevant changes up to 6400 ppm.
Litter data of F1 pups were not indicative for a test substance-related effects on pup survival up to 6400 ppm.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Data on pup weights are presented in Table 6-18 of the final report (attached).
Up to Day 7 p.p. there no meaningful decrease in F1 pup weights occurred up to 1600 ppm. On Day 14, 21 and/or 28 pup weights were found to be decreased (partly statistically significantly) in all male and female pup generations at 6400 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The daily food intake per bodyweight was unchanged up to 1600 ppm and slightly increased at 6400ppm in F0 & F1 rats. Accordingly at this concentration a slightly higher test substance intake was calculated than expected from the theoretical dose factor.

Test substance intake
Based on food consumption and body weight data, the doses expressed as mean daily mg test substance/kg body weight during the premating period are presented in Table 6-5 of the final report (attached). The values for the F0 and Fl generation are considered to be representative for the test substance intake for the entire study. During the premating periods the test substance intake per kg body weight increased roughly in the dose groups as expected from the theoretical dose factor up to 1600 ppm in F0 and F1 rats. 6400 ppm rats ingested some times slightly more test substance as expected from the dose factor, because of a slight increase in food intake per body weights.

Food efficiency:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

The mean age at vaginal opening was unchanged in F1 females up to 1600 ppm and that at balano-preputial separation was unchanged in F1 males up to 6400 ppm.
The body weight of males at the time of balano-preputial separation lay within the range of historical controls.
At 6400 ppm vaginal opening was statistically significantly delayed, whereas the body weight was unchanged at this time point indicating the delay of the vaginal opening is most probably secondary to body weight reduction rather than a specific effect on sexual maturation.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were no statistically significant or dose-dependent changes in absolute and relative pup organ weights at 400 ppm.
There was a dose-dependent (partly statistically significant) increase in absolute and relative liver weights in males beginning at 400 ppm and in females beginning at 1600 ppm (p≤0.01 at 6400 ppm). This finding was considered an adaptive response to a changed liver function and not considered as adverse.

Absolute and relative adrenal weights decreased (p≤0.01) at 6400 ppm in Fl males.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic examination:
In F1 females liver enlargement was noticed at 6400ppm.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Microscopical examination:
The following treatment-related organ changes were noted:
Liver:
- hypertrophy and/or eosinophilic cytoplasmic change of centrilobular hepatocytes in males at 1600 ppm and above and in females at 400 ppm and above in F1 generation
- deposition of a brownish pigment in/around the bile ducts with prominent bile duct epithelium, periductular cellular infiltration and/or degeneration of bile duct epithelium at 6400 ppm in both sexes of F1 generation
- presence of hydropic foci in males at 6400 ppm F1 generation
- increased amount of periportal centrilobular fat storage in F1 males beginning at 1600 ppm and in F1 females at 6400 ppm.
Kidneys:
- increased pigment storage of a brownish pigment at 1600 ppm and above in both sexes F1 generation
Thyroid glands:
- follicular cell hypertrophy and colloidal alteration in males at 1600 ppm and/or 6400 ppm in F1 generation
- follicular cell hypertrophy in F1 females at 6400 ppm
Female genital tract:
- altered luteinization of the corpora lutea of F1 females at 6400 ppm
- increased number of F1 females being in met-/diestrus cycle at 6400 ppm

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

400 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Concerning F2a pups there was a higher incidence of pups being relatively small at 6400 ppm. None of the remaining clinical signs were dose-dependently (no milk spot, thin pups) increased up to 6400 ppm.

In the F2b generation the number of pups showing no milk spot and/or pups being thin was higher in treated pups than in corresponding controls. Because there was no dose dependence (no milk spot) or the control incidence was relatively low (thin pups), if compared with that of 0 ppm F2a pups a test substance specific effect is not assumed at least up to 1600 ppm. Futhermore, there was no dose dependence, if the number of litters affected is considered. The fact that there were more relatively small F2a pups and more thin F2b pups at 6400 ppm might be due to the test substance as pup weights were also decreased at this dose.

Generally, in generation studies clinical pup findings such as lacking milk spot, to be relatively small and/or thin are seen with a wide variation as seen in this study and in studies performed roughly at the same time in the same facility.

Taken together clinical appearance of pups in the present study was not considered to be toxicologically relevantly affected up to 1600 ppm in any generation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Up to Day 7 p.p. there was no meaningful decrease in F2a/b pup weights up to 1600 ppm. At 6400 ppm a marginal but reprotoxicologically relevant weight depression was seen in F2b pups.
On Day 14, 21 and/or 28 pup weights were found to be decreased (partly statistically significantly) in all male and female pup generations at 6400 ppm. There was also a marginal body weight depression (p>0.05) in F2a males at 1600 ppm (means were at the lower limit of the range of historical controls), which cannot be completely excluded to be test substance-related, because a dose dependence existed. However, this finding does not indicate a reprotoxicological effect as at this age pups ingested the test substance by their own via diet.

The partly marginally lower weights of F2a/F2b weanlings at 400 ppm are covered by historical contol values (males and females) or were not changed dose dependently (females). Therefore, they do not reflect a test substance effect.

Taken together marginal effects on pup weights cannot be excluded at 1600 ppm (not reprotoxicologically relevant) and reprotoxicologically relevant pup weight depression is considered at 6400 ppm.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Changes in absolute and relative brain, spleen, thymus or uterus weights were recorded in 6400 ppm and to a lesser extent in 1600 ppm pups. All of these were considered to be associated with effects on body weight and therefore not a primary effect of MGK 264. No organ weight effects were seen in the 400 ppm pups.

.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no remarkable changes at necropsy.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Measurements of Ano-Genital Distance in F2a Pups
Detailed data on ano-genital measurements are given in the Annex in the final report (attached). There was no effect on this parameter up to 6400 ppm.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

1 600 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

6 400 ppm (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions described for this study, the parental systemic NOAEL is 400 ppm (33 mg/kg bw/day in males, 51 mg/kg bw/day in females), based on adverse effects on the kidney and liver beginning at 1600 ppm.

The NOAEL for reproductive parameters is 1600 ppm (127 mg/kg bw/day). At 6400 ppm, the maternally toxic dose level, a pup weight depression was noted in most generations (in F2b pups already before Day 14 p.p.) indicating a reprotoxic effect.

Executive summary:

MGK 264 was examined for possible effects on reproduction in a two-generation reproduction study in Wistar rats. MGK 264 was administered to groups of 25 male and 25 female rats at concentrations of 0, 400, 1600 or 6400 ppm in their diet.

Effects, which occurred in this study, are summarized in the attachment ‘2-generation reproduction toxicity study summary of study findings’.

The test substance was stable and homogeneously distributed in the diet. Analytical content checks revealed that the test substance mixtures in the diet were properly performed.

There was no change in survival up to 6400 ppm in parental males and females.

The appearance and behaviour of the parental animals were not toxicologically relevantly influenced due to the test substance up to 6400 ppm.

No adverse effects on body weights were evident up to 1600 ppm. At 6400 ppm body weight depression was seen in F0 and F1 females and F1 males.

The food intake per kg body weight was increased at 6400 ppm in F0 and/or F1 rats. Accordingly, at this concentration a slightly higher test substance intake was calculated than expected from the theoretical dose factor.

Sperm analyses revealed no substance-related effects at 6400 ppm (in F0 and F1).

There was a dose-dependent increase in absolute and relative liver weights of F0 and F1 parental rats partly beginning at 400 ppm. In 6400 ppm females this finding correlated with liver enlargement seen at necropsies. Beginning at 400 ppm (females) or beginning at 1600 ppm (males) an increase in hypertrophy and/or eosinophilic cytoplasmic change of centrilobular hepatocytes was noted in both generations. Periportal hypertrophy increased in F0 females beginning at 1600 ppm. These findings are regarded as an adaptive response to a changed liver function and not considered as adverse.

At 6400 ppm additional liver changes such as deposition of a brownish pigment in/around the bile ducts with prominent bile duct epithelium, periductular cellular infiltration and/or degeneration of bile duct epithelium was seen in both sexes and generations. At 6400 ppm hydropic foci were seen in the liver of F0 and F1 males. An increase in the amount of periportal/centrilobular fat storage was seen in F0/F1 males beginning at 1600 ppm. Periportal fat storage was also observed in F0 and F1 females at 6400 ppm. Thus, adverse effects on the liver were assumed in males beginning at 1600 ppm and females at 6400 ppm.

 

Kidney histopathology revealed an increased storage of brownish pigment beginning at 1600 ppm in both sexes of the F0 and/or F1 generation, an increase in severity of basophilic cortical tubules in 6400 ppm F0 males and an increase in severity of hyaline droplets in males at 6400 ppm (F0 generation).

 In thyroid glands, follicular cell hypertrophy and colloidal alteration increased in males beginning partly at 1600 ppm in both generations, whereas follicular cell hypertrophy increased in F0 and F1 females at 6400 ppm. These alterations are considered to be secondary to the changes in liver function mentioned above.

The absolute and relative weights of the ovaries/oviducts were decreased at 6400 ppm in F0 females.

At 6400 ppm altered luteinization of the corpora lutea (F0 and F1 females) and/or an increased number of F1 females being in met-/diestrus cycle were observed. These findings indicate a test substance-induced alteration of the ovarial function at 6400 ppm.

 The absolute and relative weights of seminal vesicles/coagulation glands were increased at 6400 ppm in F0 males without a morphological correlate.

The absolute brain weights were decreased in 6400 ppm F0 females, whereas the relative weights were inconspicuous. Because there was no morphological correlate to this finding and because brain weights in F1 females and males were comparable to control values a toxic effect on the brain is not considered.

Absolute and relative adrenal weights decreased without a morphological correlate in F1 males at 6400 ppm.

Absolute spleen weights were reduced without a morphological correlate in 6400 ppm F1 females due to reduced body weights.

The parameters of the reproductive performance such as insemination, fertility, gestation and rearing indices as well as gestation length and the number of litters born were not influenced by the test substance up to 6400 ppm.

The litter data such as live birth index, prenatal loss, number of born pups and sex distribution were not affected by the test substance at levels of up to 6400 ppm.

Though the total number of F1 pups born was not adversely changed up to 6400 ppm the mean litter size of F1 pups was marginally reduced at 6400 ppm due to marginally reduced number of implantation sites. It can not be excluded that the reduced number of implantation sites is caused by altered ovarian function.

The viability and lactation indices were not test substance-relatedly reduced up to 6400ppm.

Concerning clinical findings of pups there were no toxicologically relevantly changes up to 1600 ppm in any generation. At 6400 ppm thin or small pups were more often seen than at 0 ppm.

The pup weights were unaffected at 400 ppm. At 1600 ppm depressed F2 pup weights occurred from Day 14 p.p. onwards indicating a not reprotoxicological effect, because pups of this age are beginning to ingest test substance by food.

At 6400 ppm, the maternally toxic dose level, a pup weight depression was noted in most generations (in F2b pups already before Day 14 p.p.) indicating a reprotoxic effect.

At 6400 ppm vaginal opening was delayed most probably secondary to body weight reduction rather than being a specific effect on sexual maturation.

Ano-genital measurements revealed no test substance-related effect up to 6400 ppm.

At pup or weanling necropsies no remarkable macroscopical findings were noted in the F1 or F2 generation up to 6400 ppm.

Partly beginning at 1600 ppm relative brain weights increased in male and/or female weanlings of all generations. In some pup generations a decrease was found in corresponding absolute brain weights. Partly beginning at 1600 ppm absolute spleen weights decreased in all weanling generations, whereas corresponding relative weights were mostly not affected. Partly beginning at 1600 ppm the absolute thymus weights decreased in male F1 and F2a/F2b weanlings, whereas corresponding relative weights were not changed.

The absolute uterus weights decreased in F1 weanlings at 6400 ppm and in F2a weanlings at 1600 and 6400 ppm, whereas corresponding relative weights were not changed. The lower body weights are assumed· to be the reason for the changes in the weights of brain, spleen, thymus and uterus.

Determinations of MGK 264 in the peri-renal fat, liver and sceletal muscle revealed the highest content in the peri-renal fat tissue.

Thus, under the conditions described the parental systemic NOAEL is 400 ppm (33 mg/kg bw/day in males, 51 mg/kg bw/day in females), based on adverse effects on the kidney and liver beginning at 1600 ppm.

 

The NOAEL for reproductive parameters is 1600 ppm (127 mg/kg bw/day). At 6400 ppm, the maternally toxic dose level, a pup weight depression was noted in most generations (in F2b pups already before Day 14 p.p.) indicating a reprotoxic effect.

The offspring NOAEL is at 400 ppm (29 mg/kg bw/day) due to the decrease in pup weights from Day 14 p.p. onwards due to direct intake of the test substance.