1,3,5-Triazine-2,4-diamine, N2-[(1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-(1-fluoroethyl)-

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EU Guidelines on Reproductive Toxicity Studies, 91/414/EEC

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: J.M.A.F.F. 12 Nousan No 8147

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Health Canada, Guidelines on Reproduction Toxicity Studies

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]-6-[(1R)-1-fluoroethyl]-1,3,5-triazine-2,4-diamine

EC Number:

619-749-5

Cas Number:

730979-19-8

Molecular formula:

C16H20FN5

IUPAC Name:

N-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]-6-[(1R)-1-fluoroethyl]-1,3,5-triazine-2,4-diamine

Test animals

Species:

rat

Strain:

other: Wistar Hanover (Crl:WI(Glx/BRL/Han)IGS BR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation: (P) 8 wks; (F1) approx. 9 wks (initiation of second generation)
- Weight at study initiation: (P) Males: 203.6-261.1 g; Females: 136.2-176.0 g
- Fasting period before study: not applicable, feeding study
- Housing: Individually (except during the mating phase and as noted below for the F1 and F2 pups) in suspended stainless steel cages with deotized cage board in the bedding trays. During gestation and lactation, individual dams (and litters) were housed in polycarbonate cages with corn-cob bedding.
A single Nylabone® was placed in the cage of each animal (to provide environmental enrichment). Adult males were given the Nylabone® for the duration of the study except for the cohousing phase. Adult females were given the Nylabone® during the premating phase only. Pups passing vaginal patency and preputial separation were transferred to individual cages and provided a Nylabone®.
- Diet: ad libitum – Purina Mills Rodent Lab Chow 5002 meal
- Water: ad libitum – pressure-activated water nipples or water bottles
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): minimum of 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
The test compound was mixed directly with the feed. Treated diet was mixed at room temperature; aliquots of the chemical were taken from the original test batch and transferred to the mixing area. The control test diet was taken directly from the bag without mixing. A sample of each batch of feed mixed was taken and retained in the freezer until the study was complete and the analytical data were deemed satisfactory.
The concentration of the test substance in the feed, for the females only, was reduced during the lactation period (Days 0-21) by 50%. Samples from the first batch of adjusted feed for each dietary level were analyzed to measure the concentration. During the lactation phase, a substantial increase in food consumption is observed in all dams which results in greatly increased intake of test substance (normal occurrence). A decrease in the dietary concentration of the test substance offsets this increased food consumption, thereby maintaining an approximately constant test substance intake (mg/kg body weight/day) throughout the study.

Calculation of test substance intake:
Mean analytical concentration (ppm) specific for each phase / 1000 X mean weekly food consumption (g/kg bw/day) for each phase.

- Rate of preparation of diet (frequency): Replacement admixtures for each treatment group were prepared weekly (or at greater intervals, if within freezer stability limits) during the entire study. Additionally, the entire batch of replacement admixture for each treatment group was used for subsequent weekly feeding if within freezer stability limits.
- Mixing appropriate amounts with (Type of food): Purina Mills Rodent Lab Chow 5002 meal.
- Storage temperature of food: Stored at freezer conditions until presented to the animals.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 consecutive days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Females found to be inseminated were placed in a polycarbonate nesting cage.
- Any other deviations from standard protocol: In order to evaluate those females which might have been inseminated without exhibiting sperm in the vaginal smear or an internal vaginal plug, all remaining females were placed in polycarbonate nesting cages, following the 14-day mating period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of test substance in the various test diets were analytically verified for batches intended for weeks 1, 2, 3 and at monthly intervals thereafter (Bayer Crop science LP, Residue and Environmental Chemistry Group, 17745 S. Metcalf, Stilwell, KS). The homogeneity and stability of the test substance when mixed in the rodent feed were characterized, as well.
Homogeneity:
The mean concentrations of test substance in the feed, sampled from three distinct layers in the mixing bowl and containing a nominal concentration of either 20- or 10000-ppm, were determined to be 18.0 ppm (range 16.9-19.1 ppm; %RSD = 3.2) and 9079 ppm (range 8663-9443 ppm; %RSD = 2.9), respectively. Based on a %RSD <10%, test substance was judged to be homogeneously distributed in the feed over a concentration range of 20-10000 ppm (Jensen, 2007).
Stability:
Following 7 days of room temperature storage, the analytically determined concentration of the AI of the test substance in the 20- and 10000-ppm admixture was determined to be 19.0 ppm (18.0 ppm on Day 0) and 9376 ppm (8991 ppm on Day 0), respectively. Following 63 days of freezer storage, the analytically determined concentration of the AI of the test substance in the 20- and 10000-ppm admixtures was determined to be 18.4 ppm (18.0 on Day 0) and 9250 ppm (9079 on Day 0), respectively. The test substance mixed in rodent ration was judged to be stable at room temperature for at least seven days and following freezer storage for a minimum of 63 days, over a concentration range of 20-10000 ppm (Jensen, 2007).
Concentration:
Mean analytical concentrations for each dose group were 154, 1045, 4143 (high dose reduced from 8000 to 4000 ppm due to marked toxicity) and 8143 ppm, ranging from 102–105% of the corresponding nominal concentrations of 150, 1000, 4000 and 8000 ppm, respectively. During lactation the concentration of the test substance in the feed for the females was adjusted by 50%. During the lactation phase, a substantial increase in food consumption is observed in all dams which results in greatly increased intake of test substance (normal occurrence). A decrease in the dietary concentration of the test substance offsets this increased food consumption, thereby maintaining an approximately constant test substance intake (mg/kg bw/day) throughout the study. Mean analytical concentrations for each dose group during lactation were 79.3, 520, 2059 and 4143 ppm, ranging from 103-106% of the corresponding nominal concentrations of 75, 500, 2000 and 4000 ppm, respectively. The AI of the test substance was not detected in the control diet. Mean recovery was 102% and ranged from 95-112% for rodent ration spiked with 75, 150, 500, 1000, 2000, 4000 or 8000 ppm of test substance (Moore and Neal, 2008).

Duration of treatment / exposure:

approx. 39 weeks (10 weeks prior to mating, 14 days mating, approximately 22 days gestation, 21 days lactation until weaning; F1-pups 6 weeks after weaning prior to initiation of second generation. F2-pups were maintained after weaning for evaluation of developmental landmarks until PND 70.)

Frequency of treatment:

continuously

Details on study schedule:

- F1 parental animals not mated until 6 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 18 weeks (P); 9 weeks (F1)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
150, 1000, 8000 ppm (corresponding to 10.2, 68.9, 560.1 mg/kg bw/day for males and 12.6, 83.2, 656.2 mg/kg bw/day for females during premating and 11.9, 80.3, 637.4 mg/kg bw/day for females during gestation)
Basis:
nominal in diet
P-generation premating/gestation

Doses / concentrations 2

Remarks:

Doses / Concentrations:
75, 500, 4000 ppm (corresponding to 13.7, 87.7, 730.5 mg/kg bw/day)
Basis:
nominal in diet
P-generation lactation (reduction of dietary concentration due to increased food comsumption during lactation)

Doses / concentrations 3

Remarks:

Doses / Concentrations:
150, 1000, 4000 ppm (corresponding to 10.6, 69.6, 317.6 mg/kg bw/day for males and 13.1, 87.2, 355.5 mg/kg bw/day for females during premating and 11.1, 80.8, 355.8 mg/kg bw/day for females during gestation)
Basis:
nominal in diet
F1-generation premating/gestation

Doses / concentrations 4

Remarks:

Doses / Concentrations:
75, 500, 2000 ppm (corresponding to 13.3, 84.9, 363.1 mg/kg bw/day)
Basis:
nominal in diet
F1-generation lactation (reduction of dietary concentration due to increased food consumption during lactation)

No. of animals per sex per dose:

30

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
Based upon the preliminary results which emerged in the rat over the course of a pilot reproductive toxicity testing study conducted with the test chemical at doses of 0, 200, 1,000, 3,000 and 8,000 ppm (Milius, 2008). In that study, clinical observations considered to be attributed to the test article were not observed at any dietary level tested. Declines in body weight for the females of the 8000 ppm dose group were observed throughout the premating, gestation and lactation phases. Females of the 8000 ppm dose group also exhibited declines in food consumption during the premating period with initial declines observed during the first weeks and continuing through week eight. No test article-related effect on body weight or food consumption was observed in the males at any dietary level tested. In the 8000 ppm dose group, declines in female terminal body weights (-7%) and uterine weights (absolute, -41%; relative, -37%) were observed, as well as a trend toward increased thyroid weights (absolute, +29%; relative, +44%). Adult male organ weight effects included increased absolute and relative liver weights in both the 3000 and 8000 ppm dose groups and increased absolute and relative adrenal and kidney weights in the 8000 ppm dose group. Males showed no effect on terminal body weight at any dietary level tested. In the 8000 ppm dose group, female pup weight declines were observed on Day 21 (7%) with a decline in gain observed Days 7-14 (8%) and Days 14-21 (14%). Organ weight declines were observed in the spleen (absolute, -21%; relative, -15%) and uterus (absolute, -19%; relative, -13%) of the female pups of this same dose group. Body and/or organ weight effects were not observed in the male pups at any dietary level tested. Based on these interim results, the doses selected for the two-generation reproduction toxicity study are 0, 150, 1000 and 8000 ppm. This dose range is intended to produce evidence of toxicity at the highest dietary concentration and no parental or reproductive effects at the lowest dietary concentration.

Positive control:

none

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (AM and PM) and once daily on weekends and holidays.
- Cage side observations included: Mortality, morbidity, behavioral changes, signs of difficult or prolonged delivery, and overt toxicity by viewing the animal in the cage. In the event a possible clinical sign was observed during the cageside evaluation, the animal was removed from the cage and a detailed assessment conducted.

DETAILED CLINICAL OBSERVATIONS: Yes, including observation of the animal in the cage and removal of the animal to perform a physical examination.
- Time schedule: at least one per week throughout the entire in-life phase of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Once per week during the 10-week premating period, during mating and until sacrifice for males and unmated females. During gestation, dam body weight was measured on days 0, 6, 13 and 20; during lactation, dam body weight was measured on days 0, 4, 7, 14 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, once per week during the 10-week premating period, during gestation and la. During mating, food consumption was not measured. During lactation exept week one of lactation
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as mean daily intake for each phase from the consumption and body weight data: Yes, calculation for test substance intake is: Mean analytical concentration (ppm) specific for each phase / 1000 X mean weekly food consumption (g/kg/body weight/day) for each phase.

Oestrous cyclicity (parental animals):

The estrous cycle (determined by examining daily vaginal smears) was characterized for all P- and F1-generation females, over a three-week period prior to mating. Additionally, the estrous cycle stage was determined for all females just prior to termination.

Sperm parameters (parental animals):

Parameters examined in P and F1 male parental generations (one testis and one epididymis per male):
High dose + control groups: Sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm morphology
All groups: Sperm motility

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); grossly abnormal pups underwent a gross internal and external examination, and all culled pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
Number of live pups (PND 0-21), pup weight, sex of pups, stillbirths, postnatal mortality (PND 0-21), presence of gross anomalies, gross abnormalities, preputial separation, vaginal patency

GROSS EXAMINATION OF DEAD PUPS:
Yes, grossly abnormal pups for external and internal abnormalities; possible cause of death was determined for pups born or found dead or terminated in moribund condition.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals as soon as possible after the last litters in each generation were produced.
- Maternal animals: All surviving animals after the last litter of each generation was weaned (lactation day 22).

GROSS NECROPSY
- Gross necropsy of males consisted of: Determination of terminal body weights and recording of all gross pathologic alterations, weighing designated organs, and saving all gross lesions.
-Gross necropsy of females consisted of: Gross external examination of each dam (both P- and F1-generations). Terminal body weights were measured and the recording of all gross pathologic alterations, weighing designated organs, and saving all gross lesions was conducted on all females. The uterus was excised and the implantation sites, if present, were counted. Females that were sperm positive and/or had an internal vaginal plug but did not deliver were sacrificed after gestation day 24. Females that were never observed as being inseminated and/or with an internal vaginal plug and did not deliver at least 24 days after the completion of the mating phase, were sacrificed and necropsied. A gross necropsy was performed on these animals as described above. In addition, patency of the cervical/uterine os (opening) in these females was examined via flushing of the uterine horns with 10% buffered formalin.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated were prepared for microscopic examination and weighed, respectively:
Organ weights: Brain, pituitary, liver, kidney, spleen, thyroid, thymus, adrenal, cervix, epididymis, coagulating gland, ovary, prostate, seminal vesicle, testis, uterus, vagina, epididymis cauda.
Micropathology: Pituitary, liver, kidney, spleen, adrenal, cervix, epididymis, coagulating gland, oviduct, prostate, seminal vesicle, testis, uterus, vagina, epididymis cauda, gross lesions.
Additionally, the lung and the physical identifier were collected, but not subjected to any further determination.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Macroscopical examination and determination of organ weight. One pup/sex/litter for each generation had tissues collected and evaluated for any structural abnormalities or pathological changes. Organs collected: Brain, thymus, gross lesions, uterus, ovary, vagina, cervix, oviduct, testis, epididymis, prostate, coagulating gland, seminal vesicle.

GROSS NECROPSY
Pups found dead or terminated in moribund condition underwent a gross necropsy for possible defects and/or cause of death.
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated were prepared for microscopic examination and weighed, respectively:
Micropathology: Gross lesions, uterus, ovary, vagina, cervix, oviduct, testis, epididymis, prostate, coagulating gland, seminal vesicle.
Organ weight: Brain, spleen, thymus, uterus.

Statistics:

The data were analyzed using applications provided by DATATOX (Instem Computer Systems), SAS (SAS Institute, Inc.), or TASC (Toxicology Analysis Systems Customized, 1993). Parametric data (including body weight and food consumption, as well as body weight gain for pups and females during gestation were analysed using a univariate Analysis of Variance (ANOVA), and if significant differences were observed, a Dunnett’s Test was performed. Nonparametric data (e.g., number of estrous cycles, litter size, and number of implantation sites) were first analysed by the Kruskal-Wallis test and then subjected to Dunn’s Test if significant differences were identified. Nonparametric dichotomous data (e.g. fertility and gestation indices) were initially analysed by the Chi-Square Test, and if significance was observed between groups, then by the Fisher’s Exact Test with the Bonferroni adjustment. To the extent possible, the frequency of gross lesions was first examined visually, then, in the event of questionable distribution, by statistical analysis using the Chi-Square and Fisher’s Exact tests. Differences between the control and test substance-treated groups were considered statistically significant when p≤0.05 or p≤0.01.

Reproductive indices:

Mating index, fertility index, gestation index,

Offspring viability indices:

Birth index, livebirth index, viability index, lactation index, gestation length

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

adverse

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

adverse

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

adverse

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

adverse

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

secondary effect in F1 parents due to severe toxicity

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
P-generation: There were no mortalities at any dietary level tested. A test substance-related increase in the incidence of coarse tremors was observed in the P-generation females of the 8000 ppm dose group. There were no other clinical observations considered to be test substance-related in the females at any other dietary level or in the males at any dietary level tested.

F1-pups (PND 0-21): There was no test substance-related effect on the viability of the pups prior to weaning at any dietary level tested. Based on clinical observations observed in the F1-pups after day 21, the clinical observations of urine stains and nasal stains in the 8000 ppm dose group can not be ruled out as test substance-related. There were no test substance-related clinical observations observed at any other dietary level tested.

F1-pups (PND 22- 61): Prior to reducing the dose from 8000 ppm to 4000 ppm, F1-pups were severely affected by the test substance at this high dietary level. Two pups from one litter were sacrificed in a moribund condition on their PND 23, and two pups from another litter were found dead on their PND 27. Many pups from the majority of the litters exhibited severe clinical signs. These observations included increased activity, increased reactivity, myo-clonus (jerking movements), tremors, distended abdomen, labored breathing, diarrhea, soft stool, and various stains (perianal, urine, and nasal). There were no test substance-related effects observed on clinical observations at any other dietary level tested.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males:
P-generation males of the 8000 ppm dose group exhibited a slight decline in body weight gain (declined 6.6%) after 14 weeks of exposure when compared to controls. There were no test substance-related effects on absolute body weight at any dietary level tested. Incidental increases in absolute body weight and body weight gain occurred in the 150 and 1000 ppm dose group. This finding did not show a dose response relationship and is more probably due to animals being a little heavier (although within ±20% of the mean for all males at randomization) at start of treatment.

A statistically significant increase in food consumption was observed in the males of the 8000 ppm dose group on a g/kg bw/day basis beginning week 6 of premating (mean increase of 6.6%). Food consumption in this dose group was comparable to the controls on a g/animal/day basis. Incidental declines in food consumption were observed for the males of the 1000 and 8000 ppm dose groups during the first week of premating and are considered to be due to initial palatability of the test substance. By the second week of premating the food consumption values for males were comparable to controls.

F1-generation males of the 4000 ppm dose group showed a decline in absolute body weight beginning on day 0 (declined 21.2% compared to controls) and continuing throughout the 14-week period, ending in a decline at that time of 10.1%, relative to controls. This decline in absolute body weight is considered to be due to the significant declines in weight of these animals when they were pups. In this same dose group, the absolute body weight gain was increased from controls 12.3%. Also noted was a significant increase in food consumption (mean increase of 17.0%), relative to controls. These findings exhibit some compensation in growth after lowering the dose to 4000 ppm. Body weight effects were not observed at any other dietary level tested. There were no effects observed on food consumption at any dietary level tested that were considered to be attributed to the test substance.

Females (Premating):
P-generation females of the 8 000 ppm dose group exhibited statistically significant declines in absolute body weight beginning the second week of treatment and continuing throughout the 10-week premating period (mean decline weeks 2-10 was 7.8%). Body weight gain was declined 27.4% in this same dose group when compared to controls. Slight declines in body weight were observed in the 1000 ppm dose group beginning week 5 of treatment with statistical significance noted only during week 8. Body weight effects were not observed at the 150 ppm dietary level.

Beginning week 4 of treatment, sporadic declines in food consumption were observed in both the 1000 and 8000 ppm dose groups on a g/kg bw/day basis. Declines in food consumption on a g/animal/day basis were also noted in these same dose groups with more consistent declines in the 8000 ppm dose group (mean decline weeks 4-10 of 10.1%). No test substance-related findings were observed on food consumption at the 150 ppm dietary level. Incidental declines in food consumption were observed for the females of the 8000 ppm dose group during the first week of premating and are considered to be due to initial palatability of the compound. Food consumption was comparable to controls by the second week prior to the test substance-related declines beginning week 4.

F1-generation females of the 4000 ppm dose group showed a decline in absolute body weight beginning on day 0 (declined 13.2% compared to controls) and continuing throughout the premating period ending in a decline at that time of 10.7%, relative to controls. This decline in absolute body weight is considered to be partially due to the significant declines in weight of these animals when they were pups. In this same dose group, the absolute body weight gain was comparable to controls.

Food consumption evaluation for the females of this level showed sporadic increases on a g/kg bw/day basis but overall the food consumption on a g/animal/day basis was decreased when compared to controls. Based on these findings, the females seem to be having a more difficult time compensating after lowering the dose to 4000 ppm. Body weight effects were not observed at any other dietary level tested. There were no effects observed on food consumption at any other dietary level tested.

Females (Gestation):
P-generation females of the 8000 ppm dose group showed statistically significant body weight declines during gestation beginning day 0 (decline of 8.8%) and continuing throughout gestation. By Day 20 of gestation decline in body weight, relative to controls, was 14.8%. Body weight gain throughout gestation was declined 29.7%. Body weight effects were not observed at any other dietary level tested.

There were no effects on food consumption observed at any dietary level tested.

F1-generation females of the 4000 ppm dose group showed statistically significant body weight declines during gestation beginning day 0 (decline of 10.2%) and continuing throughout gestation. By day 20 of gestation decline in body weight, relative to controls, was 12.7%. Body weight gain throughout gestation was declined 19.1%. In the 1000 ppm dose group, nonstatistical declines in body weight were observed beginning on day 0 with statistical declines by day 20. There was no effect on body weight gain in the 1000 ppm dose group. Body weight effects were not observed at the 150 ppm dietary level.

Food consumption on a g/kg bw/day basis was increased in the 4000 ppm dose group with significance observed on days 13-20 (increased 12%, relative to controls). There were no effects on food consumption considered to be test substance-related at any other dietary level tested.

Females (lactation):
P-generation females of the 8000 ppm dose group demonstrated statistically significant body weight declines, relative to control, throughout lactation (mean decline days 0-21 of 14.3%). Body weight effects were not observed at any other dietary level tested.

In the 8000 ppm dose level food consumption on a g/animal/day basis was statistically declined days 4-21 (mean decline of 14.7%), relative to the controls. There were no test substance-related effects on food consumption observed at any other dietary level tested.

F1-generation females of the 4000 ppm dose group showed statistically significant body weight declines, relative to control, throughout the lactation period (days 0-21; mean decline of 10.5%). There were no test substance-related effects on body weight at any other dietary level tested, although trends of lower body weight when compared to the controls were noted in the 1000 ppm dose group.

In the 4000 and 1000 ppm dose levels, food consumption on a g/animal/day basis was statistically declined, relative to controls, on days 4-21 (mean decline of 8.9 and 13%, respectively). There were no food consumption effects considered to be test substance-related at the 150 ppm dietary level.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Based on food consumption, body weight and dietary analyses results, the doses expressed as mean daily mg test substance/kg body weight during the pre-mating period (10 weeks for males and females) were calculated according to the following formula: Mean analytical concentration (ppm) specific for premating / 1000 X mean weekly food consumption (g/kg/body weight/day) during premating. Results can be found above under "Doses/concentrations".

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Results from the evaluation of vaginal smears in both the P- and F1-generation females did not indicate any test substance-related findings at any dietary level tested.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no test substance-related effects observed on any sperm parameter evaluated at any dietary level tested for either generation. An isolated decline in testicular counts was observed in the P-generation males of the 8000 ppm dose group and is considered to be incidental to treatment based on the following. There was no concomitant reduction in epididymal counts, in fact the epididymal count for this generation was higher in the 8000 ppm dose group, relative to control. Also, there was no effect on testicular weight, no associated micropathology in the testes, nor was there any reproductive consequence. A decline in testicular count was not observed at the 4000 ppm dose level of the F1-generation.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
P-generation:
Overall reproductive performance was not affected for any parameter (e.g., mating, fertility or gestation indices, days to insemination, gestation length, or the median number of implants) at any dietary level tested.

F1-generation:
A decrease in the mean number of implantation sites was noted in the 4000 ppm dose group. This finding corresponds to a slight decrease in litter size (mean of 9.3, relative to controls) observed in this same dose group. The mean litter size is just outside of historical control values for this laboratory in this strain of rat (range 9.4-12.8). These reproductive findings are considered to be due to the significant toxicity observed in these animals during the sensitive period of growth and development when they were pups as discussed under F1-Offspring. It is also taken into consideration that there were no reproductive effects in the P-generation adults when exposed to the higher concentration of 8000 ppm. Overall reproductive performance was not affected for any other parameter (e.g., mating, fertility or gestation indices or days to insemination) at any other dietary level tested.

ORGAN WEIGHTS (PARENTAL ANIMALS)
P-Generation Adults:
In the 8000 ppm dose group, females exhibited significant declines in terminal body weight (mean decline of 11.46%, relative to controls). Terminal body weight effects were not observed at any other dietary level tested in the females. There were no test substance-related effects on terminal body weights in the P-generation males at any dietary level tested. Organ weight changes considered to be test substance-related in the 8000 ppm dose group included increased absolute and relative liver and kidney weights in the males and decreased absolute and relative spleen and uterus weights in the females. In the 1000 ppm dose group, increased absolute and relative liver weights were observed in the males. No other organ weight changes considered to be test substance-related were observed.

F1-Generation Adults:
Statistically significant terminal body weight declines, relative to controls, were observed in the 4 000 ppm dose group of both genders (males decreased 10.6% and females decreased 8.5%). Terminal body weight effects were not observed at any other dietary level tested. There were no organ weight changes considered to be directly test substance-related in either the males or females of the F1-generation. The decrease noted in the 4000 ppm female pituitary weights (both absolute and relative) was not associated with morphological changes and is of equivocal biological significance. This finding may also be associated with the severe toxicity these animals suffered from during the sensitive growth and development of the brain when they were pups.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no test substance-related gross necropsy findings observed at any dietary level tested in either generation.

HISTOPATHOLOGY (PARENTAL ANIMALS)
P-Generation:
Minimal to moderate centrilobular hepatocellular hypertrophy extending into the midzonal lobular region in some animals was noted in the livers of 1000 and 8000 ppm males and 8000 ppm females. In the 8000 ppm male kidneys, minimal to slight “hyaline degeneration” was noted in the renal cortices, principally within the proximal convoluted tubules of the kidney. In addition, there was an increase in the background incidence of minimal to slight “tubular regeneration” (focal or multifocal) within the renal cortex. In the females of the 8000 ppm dose group there was a subtle, generally minimal to mild increase in “cytoplasmic vacuolation” of the zona glomerulosa (outer zone) of the adrenal cortices. All other microscopic lesions were considered to be incidental and/or background and were not considered to be test substance-related.

F1-generation:
Minimal to moderate centrilobular hepatocellular hypertrophy extending into the midzonal lobular region in some animals was noted in the livers of 4000 ppm males and females. In the 4000 ppm male kidneys, minimal to slight “hyaline degeneration” was noted as described for the males of the P-generation. All other microscopic lesions were considered to be incidental and/or background and were not considered to be test substance-related.

OTHER FINDINGS (PARENTAL ANIMALS)
Ovarian Follicle Counts from F1-Generation Females:
None of the mean primordial (preantral), antral, or corpora lutea follicular counts were statistically different from controls. The nonstatistical decrease in the mean numbers of corpora lutea of the 4000 ppm dose group were not associated with abnormal morphologic changes. All stages of corporal luteal development and regression were present. This finding could be associated with the slightly smaller litter size observed in the F1-females and may have been caused by the severe toxicity to these females as pups when exposed to the 8000 ppm dietary level.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

150 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse systemic effects observed

Remarks on result:

other: corresponding to 10.2 and 12.6 mg/kg bw/day for males and females, respectively

2

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 68.9 and 83.2 mg/kg bw/day for males and females, respectively

3

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

>= 8 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects on reproduction observed

Remarks on result:

other: corresponding to 560.1 and 637.4 mg/kg bw/day for males and females, respectively

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

adverse

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

adverse

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

adverse

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

secondary effects

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

secondary effects

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
F1-pups (PND 0-21): There was no test substance-related effect on the viability of the pups prior to weaning at any dietary level tested.

F1-pups (PND 22- 61): Prior to reducing the dose from 8000 ppm to 4000 ppm, F1-pups were severely affected by the test substance at this high dietary level. Two pups from one litter were sacrificed in a moribund condition on their PND 23, and two pups from another litter were found dead on their PND 27. Many of the remaining pups from the majority of the litters exhibited severe clinical signs, as described below. There were no test substance-related effects observed on clinical observations at any other dietary level tested.

F2-pups (PND 0-70): There were no test substance-related effects on the viability of the pups at any dietary level tested.

CLINICAL SIGNS (OFFSPRING)
F1-pups (PND 0-21): Based on clinical observations observed in the F1-pups after day 21, the clinical observations of urine stains and nasal stains in the 8000 ppm dose group can not be ruled out as test substance-related. There were no test substance-related clinical observations observed at any other dietary level tested.

F1-pups (PND 22- 61): Prior to reducing the dose from 8000 ppm to 4000 ppm, F1-pups were severely affected by the test substance at this high dietary level, as reflected by the observed mortality. Many pups from the majority of the litters exhibited severe clinical signs. These observations included increased activity, increased reactivity, myo-clonus (jerking movements), tremors, distended abdomen, labored breathing, diarrhea, soft stool, and various stains (perianal, urine, and nasal). There were no test substance-related clinical signs observed at any other dietary level tested.

F2-pups (PND 0-70): There were no test substance-related clinical observations observed at any dietary level tested. The outward rotation of the forepaws noted in 4 pups from 1 litter of the 4000 ppm dose group is considered to have been due to the extreme toxicity presented in the parents of this litter when they were pups and not a direct result of the test substance. In addition, this finding was not noted in the F1-pups when exposed to the higher dose of 8000 ppm. Misshapen forelimb and/or deformed forelimbs noted in 1 pup from 1 control litter and 3 pups from 3 litters of the 1000 ppm dose group was due to damage during the tattooing process.


BODY WEIGHT (OFFSPRING)
Lactation until weaning:
F1-Pups: Pup body weights at birth for all three treated groups were comparable to the control group. In the 8000 ppm dose group, statistically significant declines in body weight were observed by Day 4 and continued throughout lactation (mean decline days 4-21 of 17.1%). Overall body weight gain was declined 22.6%, relative to control. Declines in pup body weight were not observed at any other dietary level tested.

F2-Pups: Pup body weights at birth for all three treated groups were comparable to the control group. In the 4000 ppm dose group, declines in body weight were observed by day 21 (mean decline of 5.9% in males and 5.8% in females). Overall body weight gain was declined 6.7%, relative to control. In the 1000 ppm dose group, no effect on absolute body weight was observed. Changes in body weight gain, relative to the controls, were observed on lactation days 14-21 in both the 1000 (more significantly with the females) and 4000 ppm dose groups. No declines in pup body weight were observed at the 150 ppm dietary level.

Juvenile pups (after weaning PND 21):
F1-juveniles of the 8000 ppm dose group (dose was decreased to 4000 ppm between the pup age range of 26-38 days old) exhibited significant declines in body weight, relative to controls, upon passing criteria for balanopreputial separation and vaginal patency. Male body weights were declined 7.3% and female body weights were declined 13.6%. There was no effect on body weight at passing these developmental landmarks, compared to the control group, at any other dietary level tested.

F2-Juveniles: There was no effect on body weight, relative to controls, upon passing criteria for balanopreputial separation and vaginal patency for the F2-juveniles at any dietary level tested.

SEXUAL MATURATION (OFFSPRING)
F1 pups: Significant delays in balanopreputial separation and vaginal patency were observed in the 8000 ppm dose group. The average male passed criteria for balanopreputial separation 9.3 days later than control males with the average female passing criteria for vaginal patency 6.8 days later than control females. Delays in passing these developmental criteria are considered to be test substance-related but these delays are considered to be exacerbated by the severe toxicity observed in these pups, exhibited by the clinical observations and significant body weight declines during the lactation period prior to decreasing the dose to 4000 ppm. Delays in balanopreputial separation and vaginal patency were not observed at any other dietary level tested.

F2-pups - Significant delays in balanopreputial separation and vaginal patency were observed in the 4000 ppm dose group. The average male passed criteria for balanopreputial separation 3.4 days later than control males with the average female passing criteria for vaginal patency 2.4 days later than control females. Delays in passing these developmental criteria are considered to be test substance-related but also correlate with pup body weight declines during lactation. Delays in balanopreputial separation and vaginal patency were not observed at any other dietary level tested.

ORGAN WEIGHTS (OFFSPRING)
F1-pups: Organ weight changes were observed on brain, spleen and thymus in both genders of the 8000 ppm dose group. These organ weight effects are considered to be secondary to the statistically significant pup weight declines observed during lactation at this dose level. Organ weight effects were not evident at any other dietary level tested.

F2-pups: Organ weight changes were observed on the brain and spleen in both genders of the 4000 ppm dose group. Spleen weights were also decreased in the female pups of the 1000 ppm dose group. These organ weight effects are considered to be secondary to the statistically-significant pup weight declines observed during lactation at this dose level. Organ weight effects were not evident at the 150 ppm dietary level.

GROSS PATHOLOGY (OFFSPRING)
There were no test substance-related gross necropsy findings observed at any dietary level tested in either the F1- or F2-pups.

HISTOPATHOLOGY (OFFSPRING)
There were no test substance-related microscopic findings observed at any dietary level tested in either the F1- or F2-pups.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F1

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse systemic effects observed

Remarks on result:

other: corresponding to 69.6 and 80.8 mg/kg bw/day for males and females, respectively

2

Dose descriptor:

LOAEL

Remarks:

systemic

Generation:

F1

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: corresponding to 317.6 and 355.8 mg/kg bw/day for males and females, respectively

3

Dose descriptor:

NOAEL

Remarks:

reproductive

Generation:

F1

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects on reproduction observed

Remarks on result:

other: corresponding to 80.8 mg/kg bw/day

4

Dose descriptor:

LOAEL

Remarks:

reproductive

Generation:

F1

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: number of implantation sites; litter size; other: number of corpora lutea; secondary effects due to severe toxicity

Remarks on result:

other: corresponding to 355.8 mg/kg bw/day

5

Dose descriptor:

NOAEL

Remarks:

reproductive

Generation:

F1

Effect level:

8 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects on reproduction observed

Results: F2 generation

Effect levels (F2)

1

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F2

Effect level:

150 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse systemic effects observed

Remarks on result:

other: corresponding to 13.3 mg/kg bw/day

2

Dose descriptor:

LOAEL

Remarks:

systemic

Generation:

F2

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Remarks on result:

other: corresponding to 84.9 mg/kg bw/day

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

4 000 ppm

Treatment related:

yes

Relation to other toxic effects:

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

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

CONCLUSION

For males, the parental systemic LOAEL is 1000 ppm (68.9 mg/kg bw/day), based on increased liver weights associated with centrilobular hepatocellular hypertrophy in the P-generation, and the systemic NOAEL is 150 ppm (10.2 mg/kg bw/day).

For females, the parental systemic LOAEL is 1000 ppm (83.2 mg/kg bw/day), based on decreased body weight and food consumption in the P-generation, and the systemic NOAEL is 150 ppm (12.6 mg/kg bw/day).

For males and females of the P-generation, the reproductive NOAEL is greater than 8000 ppm (≥560.1 mg/kg bw/day and ≥637.4 mg/kg bw/day, respectively), based on no adverse reproductive findings observed. For F1-males the reproductive NOAEL is greater than 8000 ppm, as well, as no adverse effects were observed, either.

For females of the F1-generation the reproductive LOAEL is 4000 ppm (355.8 mg/kg bw/day), based on decreased number of implants, corpora lutea and smaller litter size, considered to be secondary to severe toxicity in these females as pups. The reproductive NOAEL is 1000 ppm in F1-females (80.8 mg/kg bw/day).

The systemic LOAEL for the F2-generation is 1000 ppm (84.9 mg/kg bw/day), according to the study authors. The systemic LOAEL is based on decreased body weight gain with secondarily mediated spleen weight declines in F2-female pups. The systemic NOAEL for the F2-generation is 150 ppm (13.3 mg/kg bw/day).