Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2009 - January 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test substance name: PERKALINK 900
Chemical name : 1,3-bis(citraconimidomethyl) benzene
CAS-Reg. no.: 119462-56-5
Batch number: 812191/01
Purity: 92.3% (see CoA attached)
TNO dispense reference no.: 09002F
Appearance: off-white pastilles
Expiry date: 1 June 2010
Amount received: approx. 3 kg in 6 closed containers containing approx. 500 g
Date of receipt at TNO: 17 February 2009
Storage conditions: in a closed container at room temperature in the dark

Test animals

Species:
rat
Strain:
other: Wistar outbred Crl:WI(WU)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 5-6 weeks
- Weight at study initiation: (P) Males: 151.12-155.19 g; Females: 104.13-106.70 g; (F1) Males: 36.87-62.48 g; Females: 37.10-59.40 g
- Housing: Macrolon cages with wood shavings (Lignocel, Type 3/4) as bedding material and strips of paper (Enviro-dri) as environmental enrichment; 4/sex/group during premating period, mated females were housed individually
- Water: ad libitum; domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC)
- Diet: ad libitum; cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Witham, England)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
Experimental diets were prepared by mixing powdered Rat & Mouse No. 3 breeding diet, RM3 with the appropriate amounts of test substance. The
diets were mixed in a mechanical blender (Lödige, Paderborn, Germany). During the entire study, fresh batches of experimental diets were prepared approx. once every 2-5 weeks. The experimental diets were stored in a freezer (<-18°C). The feed in the feeders was replaced with fresh portions once a week, and filled up when necessary.

Homogeneity analyses were performed four times during the analytical part of the study.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Individually
- Sperm positive females that turned out to be non-pregnant were killed 26-28 days after copulation. Females that did not show evidence of copulation after the end of the 2-week mating period were also housed individually until sacrifice (approx. 2 weeks after the last day of the mating period)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Principle
The concentration of the substance in RM3 diet was determined by extraction of diet samples with acetonitrile. After shaking and centrifugation, an aliqout of the clear supernatant was diluted woth acetonitrile/methanol/MilliQ water 40/10/50 v/v% and the diluted extract was analysed using HPLC with UV detection at 218 nm. Quantification was obtained by comparison of the peak area of the substance in the sample extracts with those in calibration solutions containing known amounts of the test substance.

Validation criteria
Before analysis of study samples, the analytical method was validated by analysing three spiked samples per dose level, to conform to the following criteria:
- Linearity: the correlation coefficient of the calibration curves should be greater than or equal to 0.996;
- Recovery: the recovery of the test substance from test diet should be between 80% and 110% at each of the concentrations tested;
- Repeatability: the relative standard deviation in the percentage recovery of the three spiked diet samples per concentration level should be less than 10%.

Chromatography
The HPLC-UV conditions were as follow:
HPLC: Waters Alliance
Flow: 1 ml/min
Mobile phase: Acetonitrile/methanol/MilliQ water 40/10/50 v/v%
Gradient: Isocratic
Column: Phenomenex Luna C18, 5 µm, 250 x 4.6 mm
Column temperature: 25 ºC
Injection volume: 50 µl
Detection: DAD (218 nm)

Duration of treatment / exposure:
Animals were exposed during the premating period of at least 10 weeks, during mating, gestation and lactation until sacrifice over two successive generations.
Frequency of treatment:
7 days/week
Details on study schedule:
After allocation to the treatment groups, the animals were fed diets containing the test substance from the start of the study until sacrifice. Each generation raised one litter. Vaginal smears were made 3 weeks prior to mating to evaluate length and normality of the estrus cycle.
After 10 weeks of treatment (premating period), each female was caged with a male from the same group until pregnancy occurs or 2 weeks elapsed. Vaginal smears were made daily to determine if sperm was present. The day of observation of sperm in the vaginal smear was considered to be day 0 of pregnancy. Upon evidence of copulation the females were caged individually for the birth and rearing of their pups until PN 21 or shortly thereafter, when pups were weaned and sacrificed. Sperm positive females that turned out to be non-pregnant were killed 26-28 days after copulation. Females that did not show evidence of copulation after the end of the 2-week mating period were also housed individually until sacrifice (approx. 2 weeks after the last day of the mating period). Dams were allowed to raise one litter.
Males were euthanized after mating at approx. 11-12 weeks of exposure.
On PN 4, litters of more than 8 pups were adjusted by eliminating extra pups by random selection to yield, as nearly as possible, 4 males and 4 females per litter. Pups euthanized at culling were examined externally for abnormalities and subsequently preserved in a neutral aqueous phosphate buffered 4% solution of formaldehyde.
On PN 21, the litters were weaned and 28 males and 28 females were selected at random from as many litters as possible in each group to rear the next generation. After selection of the pups for the next generation, of the remaining pups 1 male and 1 female F1-pup of each litter were subjected to a thorough necropsy and the tissues listed in section XXX were weighed and preserved. Pups not selected for the next generation or necropsy were examined externally and sacrificed.
The dams were sacrificed and subjected to a thorough necropsy and the tissues listed in section XXX were weighed, preserved and microscopically examined.
The animals selected from the F1-litters to rear the next generation, were treated at the same dose levels as their parents.
Vaginal opening and preputial separation were scored in the F1-generation.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
800, 1600 and 3200 mg/kg diet
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
57.4, 119.6 and 236.7 mg/kg bw/day
Basis:
actual ingested
males
Remarks:
Doses / Concentrations:
70.7, 139.2 and 268.5 mg/kg bw/day
Basis:
actual ingested
females
No. of animals per sex per dose:
28 (for both F0 and F1)
Control animals:
yes, plain diet
Details on study design:
The study was preceded by a dose-range finding study and a palatability study using the same species, strain and route of administration.

The dose-range finding study comprised 5 groups (one control and four test groups) of 4 male and 4 female rats. After allocation to the treatment groups, the animals were fed diets containing the test substance from the start of the study until sacrifice.
After a premating period of 2 weeks each female was caged with a male from the same group until pregnancy occurred. Vaginal smears were made daily to determine if sperm was present. The day of observation of sperm in the vaginal smear was considered to be day 0 of pregnancy. Upon evidence of copulation the females were caged individually for parturition and rearing of their pups until postnatal day 4 (PN 4) or shortly thereafter, when pups were sacrificed (see section 4.10.11) and dams were sacrificed for necropsy. The morning after birth was considered day 1 post partum. Consequently, for litters that were born during the day, but after the morning observation, that day was considered day 0 post partum.
Males were sacrificed after approximately the same period of exposure as the females; approx. 6 weeks.
The dose levels were selected by the Sponsor and were based on the results of a subacute toxicity study.

In general, it can be concluded that the substance administered in the diet up to levels of 3000 mg/kg (top-dose) was well tolerated. A decrease in body weight and food consumption was observed in the male animals of the 3000 mg/kg in the first period of administration (day 0 to 3 of the study). The test substance intake in the groups was 0, 22.2, 42.4, 88.1 and 177.0 mg/kg body weight for the control, low-, mid- high and top-dose groups, respectively for the male animals. The test substance intake over the entire study in the groups was 0, 29.4, 61.4, 116.1 and 220.7 mg/kg body weight for the control, low-, mid- high and top-dose groups, respectively for the female animals.
The palatability study with 3750 and 4500 mg/kg diet indicated that levels higher than 3000 mg/kg were palatable, but also some effects (weight loss during the first week) were observed at these concentrations.
Based on the results of the dose-range finding study and the palatability study the following dose levels were selected for the main study:
0, 800, 1600 and 3200 mg/kg diet for the control, low-, mid- and high-dose groups.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
General clinical observations
Each animal was observed daily in the morning hours by cage-side observations. On working days, all cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. On Saturdays, Sundays and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.

Body weight
Body weights of male and female rats were recorded at the start of administration of the test substance, and weekly thereafter during the premating period. Males were weighed once per week during the mating period until sacrifice. Females were weighed once per week during mating and mated females were weighed on days 0, 7, 14 and 21 during presumed gestation and on day 1, 4, 7, 14 and 21 of lactation. In addition, the animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.

Food consumption
Food consumption was measured per cage by weighing the feeders. The results are expressed in g per animal per day and g per kg body weight per day. Food consumption of male rats was measured twice weekly, except during the mating period. Food consumption of female rats was measured twice weekly during the premating period. Food consumption of mated females was recorded weekly during pregnancy and on day 1, 4, 7, 14 and 21 of lactation.

Intake of the test substance
The intake of the test substance per kg body weight per day was calculated from the nominal dietary concentration of the test substance, the food consumption and the mean body weight measured at the beginning and the end of the pertaining period.
Estrous cyclicity (parental animals):
Vaginal smears to evaluate the estrus cycle length and normality were made daily for about 3 weeks prior to mating. Smears were made and stained of all females, but only the smears of the control group and the high-dose group were evaluated. No treatment-related changes were observed in the high-dose group; therefore the evaluation of vaginal smears was not extended to the intermediate-dose groups.
Sperm parameters (parental animals):
Epididymal sperm motility, count and morphology
At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis of all males of all groups. For this purpose, the cauda epididymis was dissected, weighed, and thereafter minced in M199 medium containing 0.5% bovine serum albumin. Sperm motility and, after sonification and DNA staining, the cauda epididymal sperm reserves (sperm count) were measured for all males of all groups, using the Hamilton Thorne Integrated Visual Optical System (IVOS). In addition, a smear of the sperm solution was prepared and stained for all males, but only the smears of the control and the high-concentration group males were examined for morphology as no treatment-related changes were observed in the high-dose group.

Testicular sperm count
At necropsy, the left testis of all males of all groups were placed on dry ice and subsequently stored in a freezer (<-70°C) for later determination of the number of homogenization-resistant spermatids. The testes to be analysed were thawed just before further processing. Following removal of the tunica albuginea, the testicular parenchyma were weighed, minced and homogenized in Saline Triton X-100 solution. Following DNA-staining, the homogenization-resistant sperm heads were enumerated using the IVOS. The daily sperm production was calculated. The evaluation of homogenization-resistant spermatids was performed in the control group and high-concentration group as no treatment-related changes were observed in the high-dose group.
Litter observations:
Parturition and litter evaluation
At the end of the gestation period, females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded. To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.

Litter size, sexes and weight
The total litter size and numbers of each sex as well as the number of stillbirths, live and dead pups and grossly malformed pups were evaluated on days 1, 4, 7, 14, and 21 of lactation. The pups were individually weighed days 1, 4, 7, 14, and 21 of lactation. Mean pup weight and pup weight change were calculated per sex and per both sexes combined.

Weaning and selection of pups
At weaning on PN 21 the study director provided a list for the selection of pups for necropsy (F1- and F2-pups) and the next generation (F1-pups).

All stillborn pups, pups found dead and pups that are terminated in a moribund condition during the study were examined macroscopically for structural abnormalities and pathological changes. Gross necropsy was also performed on pups of dams that died during lactation (these pups were sacrificed at the time of the dam’s death), and on pups showing external abnormalities at weaning. Organs and tissues showing macroscopic abnormalities were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde and examined microscopically.
Postmortem examinations (parental animals):
Gross necropsy and histology of parental animals
All surviving male and female parent rats were euthanized by exsanguination from the abdominal aorta under CO2/O2 anaesthesia and then examined grossly for pathological changes. A necropsy was also performed on animals that died intercurrently (if not precluded by autolysis) or that were killed because they were moribund.

Male animals were sacrificed after mating at approx. 11-12 weeks after the start of the administration of the test substance.
Female animals were sacrificed:
- females with a litter, at or shortly after day 21 of lactation
- females with no viable pups, shortly after the death of the last pup.
- non-pregnant females, 21-26 days after copulation
- non-mated females, approx. 3-4 weeks after the end of the mating period

Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde except for the testes which was preserved in Bouin's fixative:
- adrenals
- brain
- epididymides (left cauda which was used for sperm analysis)
- kidneys
- liver
- ovaries
- pituitary gland
- prostate
- seminal vesicles and coagulating glands
- spleen
- testes (right testis was preserved in Bouin’s fixative, the left one was used for sperm analysis)
- thyroid
- uterus (after counting of the implantation sites)
- vagina
- organs and tissues showing macroscopic abnormalities

Microscopic examination was performed on these organs of all rats of the control and high-dose groups. Treatment-related abnormalities were observed in the ovaries, uterus and vagina. In consultation with the sponsor it was decided to extend the examination of these organs to the intermediate groups.
In addition, reproductive organs of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined.
Postmortem examinations (offspring):
At weaning 1 male and 1 female pup per litter were subjected to a thorough necropsy. Pups were euthanized by exsanguination from the abdominal aorta under CO2/O2 anaesthesia and then examined grossly for pathological changes. Special attention was paid to the organs of the reproductive system. The following organs were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde for possible future examination:
- brain
- spleen
- thymus
- organs and tissues showing macroscopic abnormalities
Statistics:
The results were analyzed using the methods mentioned below. Other statistical tests may be performed when considered appropriate. P< 0.05 was considered as a level of significance.
- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, organ weights and food consumption data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.
- Fisher's exact probability test were used to evaluate the number of mated and pregnant females and females with live pups.
- Number of implantation sites, live and dead pups were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney U test.
- Estrus cyclicity were evaluated by Fisher’s exact test (number of acyclic animals and number of animals with prolonged estrus period), ANOVA followed by Dunnetts multiple comparison tests (number of cycles per animal) and Kruskal-Wallis non parametric ANOVA followed by Mann-Whitney U test (length of the longest cycle).
- Sperm parameters were evaluated by ANOVA followed by Dunnetts multiple comparison tests (epididymal and testicular sperm count and numerical sperm motility parameters) or by Kruskal-Wallis non parametric ANOVA followed by Mann-Whitney U test (motility parameters expressed as a percentage and sperm morphology).
- Histopathological changes were evaluated by Fisher's exact probability test.
Reproductive indices:
For each mating the following data were presented for each group:
- number of females placed with males
- number of males mated with females
- number of successful copulations (= number of females mated)
- number of males that became sire
- number of pregnant females as demonstrated by the presence of implantation sites observed at necropsy.
- number of females surviving delivery
- number of females with liveborn and (all) stillborn pups
- number of pups delivered (live- and stillborn)
- number of live pups at day n
- number of pups lost between days
- number of litters lost entirely at day n
- number of male pups at day n
- number of implantation sites
- number of lost implantations
- litter size at day n

The following parameters were calculated:
- pre-coital time = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
- mating index= (number of females mated/number of females placed with males) x 100
- male fertility index = (number of males that became sire/number of males placed with females) x 100
- female fertility index = (number of pregnant females/number of females placed with males) x 100
- female fecundity index = (number of pregnant females/number of females mated) x 100
- gestation index = (number of females with live pups/number of females pregnant) x 100
- live birth index = (number of pups born alive/number of pups born) x 100
- pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100
- sex ratio day n = (number of live male pups on day n/ number of live pups on day n) x 100
- number of lost implantations = number of implantations sites - number of pups born alive
- post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
F0-generation:
No mortalities were observed. The clinical signs observed in the animals during the premating, mating, gestation and lactation period were only seen in one or a few animals and were normal for animals of this strain and age.
F1-generation:
One female of the mid-dose group was killed moribund on GD 21. This animal showed poor health, weakness, cold, piloerection and vaginal discharge (other than red) on GD 21. Because mortality was not observed in animals of the high-dose group, this finding was considered an isolated finding.
In 2, 3, 7 and 7 female animals of the control, low, mid- or high-dose group, respectively a thread was observed in the vagina during the premating period. This observation was scored as “vagina: hymen partially present” after the vagina was open; the increase of this finding was not statistically significant.
No other remarkable clinical signs were observed in the animals during the premating, mating, gestation and lactation period; signs were only seen in one or a few animals and were normal for animals of this strain and age.

BODY WEIGHT AND BODY WEIGHT CHANGE
Males of the F0-generation:
Mean body weight of the high-dose group was statistically significantly decreased when compared to the control group from week 1 until sacrifice. Mean body weight change of this group was statistically significantly decreased between weeks 0-1, 2-3, 4-7 and 9-10. Mean body weight of the mid-dose group was statistically significantly decreased in weeks 1, 2, 5, and 9. Body weight change of the animals of this group was statistically significantly decreased between weeks 0-1, 4-5 and 9-10. Mean body weights and body weight changes of animals of the low-dose group were comparable to the control group; only an incidental difference was observed in the body weight change in week 4-5.
Males of the F1-generation:
Mean body weights of the animals of the mid- and high-dose groups were statistically significantly decreased when compared to the control group; the high-dose group during the entire period and the mid-dose group in weeks 0-6. Body weight change of animals of the high-dose group was statistically significantly decreased when compared to the control group between weeks 0-7 (except in week 4-5) and body weight changes of the mid-dose group only in week 1-2. Body weight change of the low-dose group was statistically significantly increased in weeks 3-4 and 10-11.

Premating period:
Females of the F0-generation:
Mean body weight of the high-dose group was statistically significantly decreased when compared to the control group in weeks 1-3. Mean body weight change of this group was statistically significantly decreased between weeks 0-2. Body weight change of animals of the mid-dose group was statistically significantly decreased between weeks 0-1 and 2-3 and increased between week 9-10. Body weight change of animals of the low-dose group was statistically significantly increased between week 1-2.
Females of the F1-generation:
Mean body weight of the high-dose group was statistically significantly decreased when compared to the control group from week 0 (shortly after weaning) until mating onwards. Mean body weight change of this group was statistically significantly decreased between weeks 0-2 and statistically significantly increased between weeks 3-5, 6-7 and 8-9. Mean body weight of the animals of the mid-dose group was statistically significantly decreased in weeks 0-4. Body weight change was not decreased; only an incidental statistically significant increase was observed in week 6-7.
Gestation period:
F0-generation:
Mean body weights of the pregnant animals of the dosed groups were comparable to the control group. Mean body weight change of the mid-dose group was statistically significantly increased when compared to the control group between GD 14-21.
F1-generation:
Mean body weights of the pregnant animals of the high-dose group were statistically significantly decreased when compared to the control group on GD 0, 7 and 14. No differences in body weight change were observed between the dosed groups and the control group.
Lactation period:
F0-generation:
Mean body weights of the dams of the high-dose group were statistically significantly decreased when compared to the control group on day 7, 14 and 21 of lactation (LD). Mean body weight changes of the mid- and high-dose groups were statistically significantly decreased between LD 7-14 and statistically significantly increased between LD 14-21; the dams of the high-dose group lost weight between LD 7-14.
F1-generation:
Mean body weights of the dams of the high-dose group were statistically significantly decreased when compared to the control group during the entire lactation period (LD 1-21). Mean body weight change of the high-dose group was statistically significantly decreased between LD 7-14 when compared to the control group. The dams of the high-dose group lost weight between LD 7-14; the dams of the control group gained weight during this period. The body weight changes between LD 14-21 of dams of the mid- and high-dose groups were statistically significantly different from the control group; these dams lost less weight (mid-dose) or gained weight (high-dose) when compared to the dams of the control group.

FOOD CONSUMPTION
Males of the F0-generation:
Mean food consumption (expressed as g/animal/day) was statistically significantly decreased in the mid-dose group in week 0-1, weeks 4-7 and 9-10 and in the high-dose group during the entire premating period and after mating until sacrifice. Mean food consumption calculated as g/kg bw/day was statistically significantly decreased in the first week of the study in the high-dose group.
Males of the F1-generation:
Mean food consumption (expressed as g/animal/day) was statistically significantly decreased in the mid-dose group between weeks 1 and 3 and in the high-dose group during the entire premating period and after mating until sacrifice. Mean food consumption calculated as g/kg bw/day was statistically significantly increased between weeks 3 and 5 and weeks 6 and 11 in the high-dose group.

Premating period:
Females of the F0-generation:
Mean food consumption (expressed as g/animal/day) was statistically significantly decreased in the mid- and high-dose group during the first week of the study. Mean food consumption calculated as g/kg bw/day was statistically significantly decreased in the first week of the study in the high-dose group.
Females of the F1-generation:
Mean food consumption (expressed as g/animal/day) was statistically significantly decreased in the mid-dose group in week 1 to 2 and in the high-dose group between weeks 0-6. No statistically significant difference was observed in mean food consumption calculated as g/kg bw/day between the groups.
Gestation period:
F0-generation:
Mean food consumption of the pregnant females (expressed as g/animal/day and g/kg body weight/day) was statistically significantly decreased in the high-dose group between GD 14-21.
F1-generation:
Mean food consumption of the pregnant females (expressed as g body weight/day) was statistically significantly increased in the high-dose group between GD 0-7. No other differences in food consumption were observed between the groups.
Lactation period:
F0-generation:
Mean food consumption of the dams was statistically significantly decreased in the high-dose group between LD 1-4 and 7-21 (expressed as g/animal/day) and between LD 7-21 (expressed as g/kg body weight/day).
F1-generation:
Mean food consumption (expressed as g/animal/day) of the dams was statistically significantly decreased in the mid-dose group between LD 14-21 and in the dams of the high-dose group between LD 1 and 21. Mean food consumption (expressed as g/kg body weight/day) of the dams of all dosed groups was statistically significantly decreased when compared to the control between LD 7-21.

ESTRUS CYCLE
F0-generation:
No statistically significant differences were observed in the estrus cycle between the exposed and the control groups.
F1-generation:
No statistically significant differences were observed in the estrus cycle between the exposed and the control groups.

FERTILITY AND REPRODUCTIVE PERFORMANCE
F0-generation:
All females of the dosed and control groups were found sperm positive except one female of the low-dose group. 26, 26, 27, 28 female animals of the control, low-, mid- and high-dose group, respectively, delivered a live litter. No treatment-related differences were observed in pre-coital time, mating index, female fecundity index and male and female fertility index, duration of gestation and post-implantation loss. All dams survived the delivery and there were no dams with all stillborn pups in any of the groups.
F1-generation:
All females of the dosed and control groups were found sperm positive except one female of the high-dose group. 28, 26, 26, 27 female animals of the control, low-, mid- and high-dose group, respectively, delivered a live litter. One animal of the mid-dose group was killed moribund on GD 21. One animal of the mid-dose group was pregnant (1 implantation site); no live pups were observed. The duration of the gestation period was statistically significantly decreased in the low- and high-dose groups. The duration of the gestation period was at least 21 days for all dams; except for 1 dam of the low-dose group the duration of gestation was 20 days. The duration of gestation was within the historical control range and not considered to be an adverse effect. No treatment-related differences were observed in pre-coital time, mating index, female fecundity index and male and female fertility index and post-implantation loss.

SPERM ANALYSIS
Epididymal sperm motility
F0-generation:
Sperm motility in the dosed and the control groups was comparable.
F1-generation:
Sperm motility in the dosed and the control groups was comparable.

Epididymal sperm count
F0-generation:
Epididymal sperm counts in the dosed and the control groups were comparable.
F1-generation:
Epididymal sperm counts in the dosed and the control groups were comparable.

Sperm morphology
F0-generation:
Only a few abnormal sperm were observed in the high-dose and the control groups.
F1-generation:
Only a few abnormal sperm were observed in the high-dose and the control groups.

Testicular resistant sperm
F0-generation:
Homogenisation resistant sperm counts were comparable in the high-dose and the control groups.
F1-generation:
The testicular parenchym weight of the high-dose group was statistically significantly decreased when compared to the control; this is related to the decreased absolute testis weight of this group. Homogenisation resistant sperm counts were comparable in the high-dose and the control groups.

NECROPSY OBSERVATIONS OF PARENTAL ANIMALS
Organ weights
The statistically significant decrease in absolute and relative weights of the ovaries and uterus of the females of the high-dose group of the F0- and the F1-generation were considered related to treatment. The other effects observed on absolute and relative organ weights were inconsistent and or considered to be related to the effects on body weight.

Macroscopic observations
F0-generation:
Gross examination at necropsy revealed no exposure-related findings.
F1-generation:
One animal of the mid-dose group was killed moribund on GD 21. This animal lost approximately 14 g body weight between GD 14-21 (all other pregnant animals gained weight) and showed poor health, weakness, cold, piloerection and vaginal discharge (other than red) on GD 21. The uterus of this animal contained 1 early resorption and 9 live but small for age pups; at necropsy the head of one pup was outside the vagina. The other changes occurred in only a few animals and are common gross findings in rats of this strain and age.

Microscopic observations
F0-generation:
Males
In male rats, no treatment-related histopathological changes could be detected.
Females
Histopathological analysis revealed that most female rats of the control group were in the pro-estrus or estrus phase (22/28). One rat was in a persistent anestrus phase.
In the low-dose group, the number of rats in a persistent anestrus phase (10/28) was increased in comparison to the control group (1/28). In the mid-dose group, the number of rats in a persistent anestrus phase further increased (17/28).
Of one animal, of the mid-dose group, the reproductive phase could not be determined. The vaginal epithelium of this rat showed slight atrophy, characterised by the presence of 1 layer of low epithelial cells. In addition the uterine lumen was lined by optically clear tall columnar cells, with oval instead of round nuclei.
In the high-dose group, the reproductive cycle could not be determined in the majority of rats (25/28). These rats showed slight to moderate atrophy of the vaginal epithelium, characterised by the presence of 1 layer of low epithelial cells. In addition, the majority of these rats showed slight atrophy of the uterus (19/28) and a uterine lumen that was lined by optically clear tall columnar cells, with oval instead of round nuclei (26/28).
Semi-quantitative analysis indicated that the average number of corpora lutea in the ovaries, was statistically significantly lower in the high-dose group in comparison to average number in the control group.

F1-generation:
Males
In male rats, no treatment-related histopathological changes could be detected.
Females
Similar to the control females of the F0-generation, most control females of the F1-generation were in the pro-estrus or estrus phase (22/28). Two rats were in a persistent anestrus phase.
In the low-dose group, the number of rats (6/28) in a persistent anestrus phase was slightly increased as compared to the controls. In the mid-dose group, this number remained approximately the same (8/28).
One animal of the mid-dose group showed slight atrophy of the vaginal epithelium.
Similar to the F0-generation, the reproductive cycle could not be determined in the majority of rats of the high-dose group (18/28). Most rats (19/28) showed slight to moderate atrophy of the vaginal epithelium, characterised by the presence of just 1 layer of low epithelial cells. In addition, the majority of these rats showed slight atrophy of the uterus (18/28) and a uterine lumen that was lined by optically clear, tall columnar cells, with oval instead of round nuclei (18/28).
Semi quantitative analysis indicated that the average number of corpora lutea in the high-dose group was statistically significantly lower in comparison to the average number of the control group.

Effect levels (P0)

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Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
800 other: mg/kg diet (57.4 mg/kg bw/day)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: decreased body weight and reduced food consumption
Remarks on result:
other: Generation: F0 and F1 (migrated information)
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
800 other: mg/kg diet (70.7 mg/kg bw/day)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: decreased body weight and reduced food consumption
Remarks on result:
other: Generation: F0 and F1 (migrated information)
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
3 200 other: mg/kg diet (236.7 mg/kg bw/day)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no treatment related effects
Remarks on result:
other: Generation: F0 and F1 (migrated information)
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
1 600 other: mg/kg diet (139.2 mg/kg bw/day)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: decreased absolute and relative weight of the uterus and ovaries and histopathological changes in the ovaries, uterus and vagina
Remarks on result:
other: Generation: F0 and F1 (migrated information)
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
800 other: mg/kg diet (57.4 mg/kg bw/day)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: decreased pup weight and decreased spleen weight in the pups
Remarks on result:
other: Generation: F0 and F1 (migrated information)
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
800 other: mg/kg diet (70.7 mg/kg bw/day)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: decreased pup weight and decreased spleen weight in the pups
Remarks on result:
other: Generation: F0 and F1 (migrated information)

Results: F1 generation

Details on results (F1)

LITTER SIZE AND SEX
F0-generation:
The mean number of pups delivered was statistically significantly increased in the dosed groups when compared to the control group; mean number of pups delivered was 9.2, 10.5, 10.3 and 10.8 for the control, low-, mid- and high-dose group respectively. The mean number of pups in the control groups was relatively low but within the historical control. The lower mean number of live pups in the control group resulted also in a statistically significant increased number of pups in the dosed groups on PN 1 and PN 4 (preculling). Pup mortality (PN 1-4) was statistically significantly decreased in the low- and mid-dose groups. Between PN 5 and PN 7, 4 pups of the control and 1 pup of the low-dose group died. The total number of pups alive on PN 21 was statistically significantly increased in the mid-dose group when compared to the control group; the mean number of pups was comparable between all groups. Sex ratio was comparable between the groups.
F1-generation:
No statistically significant difference was observed in the mean number of pups delivered between the dosed groups and the control group. The number of live pups was statistically significantly increased in the low- and mid-dose group; the number of stillborn pups was statistically significantly decreased in the low- andmid-dose group. In the low-dose group no pups died between PN 1 and 4; in the control and the mid- and high-dose group 6, 5 and 5 pups died during this period. Two dams of the control group lost their litters between PN 1 and 4: the only pup of a dam was found dead on PN 4 and 5 pups of a dam were missing on PN2. In all groups a low mortality was observed and the differences were of no toxicological significance. The mean number of pups on PN 4 of the high-dose group was statistically significantly decreased when compared to the control group. This was considered of no toxicological significance as pup mortality was low, the control value was increased due to litter loss of 2 abovementioned dams and the value was within the historical control. Pup mortality after PN 4 was low. Two pups of a dam of the low-dose group were missing on PN 21, one pup of a dam of the mid-dose group was missing on PN 14 and 1 pup of a dam of the high-dose group was missing on PN 21.
In summary, no effect on pup mortality, number or sex ratio was observed.

PUP CLINICAL OBSERVATIONS
F0-generation:
Clinical signs and abnormalities:
The number of sparsely haired pups was statistically significantly increased on PN 21 in the high-dose group; this finding was only observed in one litter of one dam and therefore not considered of toxicological significance.
F1-generation:
The number of sparsely haired pups was statistically significantly increased on PN 14 and 21 in the high-dose group; this finding was only observed in one litter of one dam and therefore not considered of toxicological significance. The other effects were normal for this strain and age and evenly distributed among the groups.

PUP WEIGHTS
F0-generation:
The following effects were observed on pup weight and pup weight change:
Low-dose group: statistically significant decrease in mean pup weight change (male and females separately and combined) between PN 14 and 21.
Mid-dose group: statistically significant decrease in mean pup weight (male and females separately and combined) on PN 21. Statistically significant decrease in pup weight change (both sexes combined) between PN 7 and 14 and a statistically significantly decrease in mean pup weight change (males and females separately and combined) between PN 14 and 21.
High-dose: statistically significant decrease in mean pup weight (males) on PN 4 and statistically significantly decreased in mean pup weight (males and females separately and combined) on PN 7, 14 and 21. Statistically significant decrease in pup weight change (male and females separately and combined) between PN 7 and 14 and between PN 14 and 21.
F1-generation:
The following effects were observed on pup weight and pup weight change:
Low-dose group: no statistically significant effects.
Mid-dose group: statistically significant decrease in mean pup weight (male and females separately and combined) on PN 21. Statistically significant decrease in pup weight change (males and females separately and combined) between PN 14 and 21.
High-dose: statistically significant decrease in mean pup weight (males and females separately and combined) on PN 7, 14 and 21. Statistically significant decrease in pup weight change (male and females separately and combined) between PN 1 and 4, between PN 7 and 14 and between PN 14 and 21.

MACROSCOPIC OBSERVATIONS OF STILLBORN AND DIED PUPS
F0-generation:
No abnormalities were observed.
F1-generation:
No abnormalities were observed.

PUP NECROPSY: MACROSCOPIC OBSERVATIONS
F0-generation:
At necropsy, macroscopic changes were found in 4 pups of 4 litters of the control group, 1 pup of the mid-dose group and 3 pups of 2 litters of the high-dose group. These macroscopic changes were only observed in a few pups and were considered normal for pups of this age and strain.
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F1-generation:
At necropsy, macroscopic changes were found in 2 pups of 2 litters of the low-dose group and 3 pups of 2 litters of the high-dose group. These macroscopic changes were only observed in a few pups and were considered normal for pups of this age and strain.

PUP ORGAN WEIGHTS
F0-generation:
Absolute brain weights of the pups (male or female pups or both sexes combined) of the high-dose group were statistically significantly decreased when compared to the brain weights of control group. Relative brain weights of the female pups of the mid-dose group and the male and female pups and the combined weights of both sexes of the high-dose group were statistically significantly increased. The effect on brain weight was not considered to be an adverse effect but a body weight related effect.
Absolute thymus weights of the female pups and the combined thymus weights of both sexes of the mid-dose group and of male and female pups separately and all pups of the high-dose group were statistically significantly decreased; as no effect was observed on relative thymus weight these effects were considered to be related to body weight.
Absolute and relative spleen weights of the pups of the mid- and high-dose groups were statistically significantly decreased; this effect was observed for the male and females pups separately and both sexes combined except for the relative spleen weight of the male pups. This effect was considered to be related to treatment.
F1-generation:
Absolute brain weights of the pups of the high-dose group were statistically significantly decreased when compared to the brain weights of control group. Relative brain weights of the female pups and the combined weights of both sexes of the mid-dose group and the brain weights of the male and female pups and the combined weights of both sexes of the high-dose group were statistically significantly increased. The effect on brain weight was not considered to be an adverse effect but a body weight related effect.
Absolute thymus weights of the male and female pups and the combined thymus weights of both sexes of the high-dose group were statistically significantly decreased.
As only a statistically significant effect (not dose-related increase) was observed on relative thymus weight in the low- (males and females combined) and mid-dose group (males and females combined and male pups separately) and no effect was observed on relative thymus weight in the high-dose group, these effects were considered not to be of toxicological concern.
Absolute and relative spleen weights of the pups of the mid- and high-dose groups were statistically significantly decreased. This effect was considered to be related to treatment (secondary effects due to reduced maternal body weight and food intake).

SEXUAL MATURATION
F1-generation:
Preputial separation was statistically significantly delayed in males of the high-dose group.
Vaginal opening of the females was statistically significantly delayed in the mid- and high-dose group.
These effects were most probably due to the decreased body weight of the animals of this group and the high substance intake during this period. The growth retardation (on the basis of body weight) of the animals of the mid- and high-dose groups was approximately 0.5 and 2 weeks, respectively, when compared to the animals of the control group at the time of sexual maturation.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The available data do not indicate that PERKALINK 900 causes effects on fertility.
Executive summary:

The NOAEL for parental toxicity was 800 mg/kg diet (57.4 and 70.7 mg/kg bw/day for males and females, respectively) based on decreased body weight and reduced food consumption.

There were no treatment related effects in males for reproductive toxicity, therefore the NOAEL 3200 mg/kg diet (236.7 mg/kg bw/day), the highest dose tested. In females decreased absolute and relative weight of the uterus and ovaries and histopathological changes in the ovaries, uterus and vagina were observed, resulting in a NOAEL of 1600 mg/kg diet (139.2 mg/kg bw/day).

The NOAEL for developmental toxicity was 800 mg/kg diet (57.4 and 70.7 mg/kg bw/day for males and females, respectively) based on decreased pup weight and decreased spleen weight in the pups. These effects on pups were considered to be secondary effects due to reduced maternal body weight and food intake.