1,3,5-triazine-2,4,6(1H,3H,5H)-trione, compound with 1,3,5-triazine-2,4,6-triamine (1:1)

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity – with F2 generation and both developmental neuro- and immunotoxicity (Cohorts 1A, 1B with extension, 2A, 2B, and 3)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Nov 2018 - 26 Feb 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

Principles of method if other than guideline:

OECD guidance document supporting OECD test guideline 443 on the extended one-generation reproductive toxicity test, No. 151, July 2013.

GLP compliance:

yes

Limit test:

no

Justification for study design:

The design of this study was based on the final decision on a compliance check of MELAMINE by ECHA (Decision no. TPE-D-2114373433-50-01/F, 22-Nov-2017).

Test material

Test material information

Constituent 1

Reference substance name:

Melamine

EC Number:

203-615-4

EC Name:

Melamine

Cas Number:

108-78-1

Molecular formula:

C3H6N6

IUPAC Name:

1,3,5-triazine-2,4,6-triamine

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for reproduction and developmental toxicity testing and for neurotoxicity and immunotoxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental/neurological/immunological historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive, neurological and immunological toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 11 wks
- Weight at study initiation: (P) Males: 263-342 g; Females: 179-229 g
- Fasting period before study: No
- Housing:
On arrival, prior to mating and during the post-weaning period, animals were group housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages.
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages.
During the post-mating phase, males were housed in their home cage with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages.
During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam until termination (unscheduled deaths, spares, and pups of Cohorts 2B and Surplus) or until weaning on PND 21 (Cohorts 1A, 1B, 2A and 3; positive controls).
During locomotor activity monitoring, F1-Cohort 2A animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water for a maximum of 2 hours.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles.
Animals were separated during designated procedures/activities.
- Diet: ad libitum, except during designated procedures (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: ad libitum, except during designated procedures (tap water).
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 45 - 63
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water was performed. Results of the analysis are on file at the Test Facility. It is considered that there were no known contaminants in the feed and the water that would interfere with the objectives of the study.

IN-LIFE DATES: From: 12 Nov 2018 To: 21 May 2019

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used to prepare pelleted diets. The test item was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Water (approximately 15% in total) was added to aid pelleting. The pellets were dried for approximately 24 hours at 35°C before storage. The control animals received similarly prepared pellets but without the test item.
Diets were prepared at least 3 weeks in advance of first use, and were stored in the freezer (≤ - 15°C) for maximally 3 weeks, if not used on the day of preparation. If stored in the freezer, diets were taken out of the freezer and kept at room temperature for at least overnight to allow acclimatizing to room temperature before first use. Diets were used for maximally 11 days at room temperature. Any remaining diet left after filling the food hoppers was stored at room temperature for supplementing food during the respective food consumption measurement interval. However, the diet was not used after storage at room temperature for longer than 11 days.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate sets of diet preparation samples were collected in week 1, 11, 22, and 26 for concentration (all dose groups) and homogeneity (groups 2 and 4) analysis.

Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration.
Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.

Duration of treatment / exposure:

F0-males were treated for 11 weeks, including 2 weeks prior to mating and during the mating and post-mating period, up to and including the day before scheduled necropsy.
F0-females were treated for a minimum of 8 weeks, including 2 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 21 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 6 weeks.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, via spilled diet from the food hopper or when they commence eating for themselves.
From weaning onwards (PND 21), F1-animals of Cohorts 1A, 1B, 2A and 3 were treated up to and including the day before scheduled necropsy. Cohort 1A was treated for 10-11 weeks. Cohort 1B males were treated for 13-14 weeks and females for 17-19 weeks. Cohort 2A animals were treated for 8 weeks and Cohorts 3 animals for 5 weeks. The F1-animals of Cohort 2B, Cohort Surplus and Spares (not assigned to one of the cohorts) remained with their mothers until scheduled necropsy and had access to the dietary concentrations used during the lactation.

Frequency of treatment:

continuously through diet

Details on study schedule:

- Mating of F1 parental animals started after at least 10 weeks of treatment (ie at least 13 weeks of age).
- Selection of parents from F1 generation when pups were 21 days of age.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 ppm

Remarks:

Group 2. During lactation: 500 ppm.

Doses / concentrations 2

Dose / conc.:

4 000 ppm

Remarks:

Group 3. During lactation: 2000 ppm.

Doses / concentrations 3

Dose / conc.:

12 500 ppm

Remarks:

Group 4. During lactation: 6250 ppm.

No. of animals per sex per dose:

F0: 28 animals/sex/dose
F1 cohort 1A: 20 animals/sex/dose
F1 cohort 1B: 25 animals/sex/dose
F1 cohort 2A: 10 animals/sex/dose
F1 cohort 2B: 10 animals/sex/dose
F1 cohort 3: 10 animals/sex/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
The dose levels were selected based on the results of a preliminary reproductive toxicity study (reproduction/developmental toxicity screening test) with dietary exposure of MELAMINE in rats, and in an attempt to produce graded responses to the test item. In this study, MELAMINE was administered to groups of 10 male and 10 female rats at dietary concentrations of 0, 4000 or 12500 ppm two weeks before mating, through mating and lactation until necropsy on PND 28. A trend towards lower body weight gain was noted at the high dose of 12500 ppm (both sexes). Food consumption was lower in females treated with 12500 ppm during the first two weeks. Clinical chemistry examinations revealed higher urea and creatinine concentrations in males at 12500 ppm.
Microscopic examinations showed findings in the kidneys of males and females treated at 12500 ppm, which were considered adverse in males. In females, some of the kidney findings were also present, but based on the lower severity, the incidence and the absence of additional degenerative changes, the kidney changes in the high dose females were considered as non-adverse.
The kidney findings were unilateral (left side), multifocal (mainly in the cortex and outer medulla) and considerably more severe in males. Findings consisted of:
- Basophilia, tubular: increased incidence and severity in males up to marked degree and females up to slight degree.
- Infiltrate inflammatory cell, mononuclear: increased incidence and severity in males up to moderate degree and in females up to slight degree.
- Tubular dilation: in males up to marked degree. The minimal dilation in some males treated at 4000 ppm was considered to be within normal limits in rats of this age and strain.
- Fibrosis, interstitial, mainly peritubular: in males up to moderate degree and in two females at minimal degree.
- Necrosis, tubular (consisting of single cell necrosis and sloughing of tubular cells into the lumen); in males up to slight degree.
No parental toxicity was observed at 4000 ppm.
No reproductive toxicity was observed at 4000 and 12500 ppm.
Developmental toxicity was seen at 12500 ppm only.
Mean body weights of male pups of the high dose group were slightly, but statistically significantly lower on PND 21 and 28 when compared to the concurrent control group. This is after pups started to consume test diet by themselves. As changes were relatively slight (6-7%), it was not considered adverse.

As food intake is considerably higher in lactating females, dietary concentrations in the current study were lowered by 50% during the lactation period (i.e. animals of Groups 2, 3 and 4 received a dietary concentration of 500, 2000 and 6250 ppm, respectively).

Positive control:

Positive control animals (Group 5, 10 animals/sex) were treated with cyclophosphamide via intraperitoneal injection once daily on five consecutive days prior to necropsy (i.e. starting between PND 48-54).

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- General health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were conducted in a standard arena once before the first administration of the test item and at weekly intervals during the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight with a maximum of 24 hours before blood sampling, but water was available.
- How many animals: 10 selected animals/sex/group F0-animals
- Parameters checked: according to OECD TG 443

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. on the day of scheduled necropsy
- Animals fasted: Yes, overnight with a maximum of 24 hours before blood sampling, but water was available.
- How many animals: 10 selected animals/sex/group F0-animals
- Parameters checked: according to OECD TG 443

URINALYSIS: Yes
- Time schedule for collection of urine: Overnight (15-20 hrs)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: according to OECD TG 443

THYROID HORMONE
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. on the day of scheduled necropsy
- Animals fasted: Yes, overnight with a maximum of 24 hours before blood sampling, but water was available.
- How many animals: 10 selected animals/sex/group F0-animals
Blood samples at a target volume of 1.0 mL were collected into tubes without anticoagulant. Blood samples were processed for serum and used for measurement of both T4 and TSH. Any remaining sample was discarded.

Oestrous cyclicity (parental animals):

Estrous stages were determined by examining the cytology of vaginal lavage samples.
Daily vaginal lavage was performed for all F0-females beginning 14 days prior to mating and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of scheduled necropsy, a vaginal lavage was also taken.

Sperm parameters (parental animals):

For all surviving F0-males and Cohort 1A and 1B F1-males, the following assessments were performed:
Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples. Based on the results from this study, it was decided in consultation with the Sponsor not to examine these smears for cohort 1B animals, but to retain them in the archive after report finalization.
One epididymis (left) was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.
Due to agenesis of the left epididymis in one Group 1 and one Group 2 male (F0 generation), the right side organ was fixed in modified Davidson's solution for histopathological examination. Consequently, no organ was available for sperm number evaluation.

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); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring until weaning:
- Mortality/morbundity: twice daily until weaning. The number of live and dead pups was determined on PND 1 and daily thereafter.
- Clinical observations: at least once daily.
- Body weights: Live pups were weighed individually on PND 1, 4, 7, 13 and 21.
- Sex: externally determined for all pups on PND 1 and 4.
- Anogenital distance (AGD): measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
- Areola/Nipple Retention: all male pups on PND 13.

The following parameters were examined in F1 offspring from weaning onwards (Cohorts 1A, 1B, 2A and 3):
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- General health/mortality, simultaneously with the mortality/moribundity check of the dam

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were conducted in a standard arena at weekly intervals during the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed weekly from weaning onwards. In addition, the body weight was recorded of each female on the day of acquisition of vaginal patency and of each male on the day of acquisition of balanopreputial separation. For animals of Cohorts 1A, 1B, 2A, 2B and Surplus and F2-animals of Cohort 1B (10 selected litters/group; one male and one female), a terminal weight was recorded on the day of scheduled necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was quantitatively measured weekly, from weaning onwards up to the day prior to scheduled necropsy.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

VAGINAL PATENCY: Yes
- Time schedule for examinations: monitored daily for alle females from PND 25 onwards until vaginal patency was present, by visual inspection of the vaginal area.

BALANOPREPUTIAL SEPARATION
- Time schedule for examinations: monitored daily for all males from PND 35 onwards until balanopreputial separation was present, by visual inspection of the genital area.

STAGE OF ESTROUS CYCLE
Estrous stages were determined by examining the cytology of a vaginal lavage sample, taken on the day of scheduled necropsy.

The following parameters were examined in cohort 1A animals:
ESTOUS CYCLE DETERMINATION
Estrous stages were determined by examining the cytology of vaginal lavage samples, taken during two periods.
During the first period, daily vaginal lavage was performed for all Cohort 1A females starting on the day of onset of vaginal patency and was minimally continued until the first estrus was determined, in order to determine the time interval between these two events. During the second period, daily vaginal lavage was performed from PND 75 to 88.

HAEMATOLOGY
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight with a maximum of 24 hours before blood sampling, but water was available.
- How many animals: 10 selected animals/sex/group
- Parameters checked: according to OECD TG 443

CLINICAL CHEMISTRY
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. on the day of scheduled necropsy
- Animals fasted: Yes, overnight with a maximum of 24 hours before blood sampling, but water was available.
- How many animals: 10 selected animals/sex/group
- Parameters checked: according to OECD TG 443

URINALYSIS
- Time schedule for collection of urine: Overnight (15-20 hrs)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: according to OECD TG 443

THYROID HORMONE
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. on the day of scheduled necropsy (cohort 1A), at culling (F1 culled pups), PND22 (F1 cohort surplus animals), PND4 (F2 culled pups)
- Animals fasted: Yes, overnight with a maximum of 24 hours before blood sampling, but water was available (cohort 1A only).
- How many animals: 10 selected animals/sex/group (cohort 1A), on PND 4 from two surplus pups per litter, on PND22 all F1 surplus pups (10/sex/group) and on OND4 2 pups per litter for the F2 culled pups.
Blood samples at a target volume of 1.0 mL (F1-animals of Cohort 1A), 0.5 mL (PND 4 pups; pooled samples) and 1.0 mL (Cohort Surplus PND 22 pups) were collected into tubes without anticoagulant. Blood samples were processed for serum.
Serum of Cohort 1A animals and Cohort Surplus animals (i.e. PND 22 pups) was used for measurement of both T4 and TSH. Pooled serum of culled PND 4 pups of both generations was used for measurement of T4 only.


The following parameters were examined in cohort 1B animals:
BODY WEIGHT: Yes
- Time schedule for examinations: Mated females were weighed individually on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was not determined for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was quantitatively measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Until 17 Feb 2019, inclusive, water consumption was monitored on regular basis by visual inspection of the water bottles. As a treatment-related effect was suspected based on this subjective appraisal, quantitative measurement of water consumption was introduced on 18 Feb 2019 (Week 6 of premating) and continued until the day of scheduled necropsy. Water consumption was measured overnight (24 ± 3 hours) on two separate days each week, except for males and females which were housed together for mating and for females without evidence of mating.

STAGE OF ESTROUS CYCLE
Estrous stages were determined by examining the cytology of vaginal lavage samples. Daily vaginal lavage was performed from start of the mating period until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.

MATING PROCEDURE (cohort 1B):
- M/F ratio per cage: 1:1
- Length of cohabitation: 14
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually

GENERAL ROPRODUCTION DATA (cohort 1B)
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.
Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.


ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY (cohort 2A):
- Acoustic startle response:
Acoustic startle response (habituation) was assessed using the StartleMonitor System (Kinder Scientific, Poway, USA). This was performed once between PND 23-25 in a sound-attenuated room.
To the extent possible, treatment groups were balanced across devices and the time of testing was counterbalanced across dose group and sex. The animals were tested in sets of up to 3. The test sessions consisted of a five-minute acclimation period with a 65 ± 5-dB broadband background white noise. The startle stimulus for each trial was a 115 ± 5-dB mixed frequency noise burst stimulus (approximately 20 milliseconds in duration). Responses were recorded during the first 250 milliseconds following onset of the startle stimulus for each trial. The test session consisted of 50 trials with an eight-second intertrial interval. Average response amplitude (AveN), average maximum response amplitude (MaxN) and average latency to achieving the maximum response amplitude (Tmax) was analyzed in five blocks of 10 trials each.

- Learning and memory test (Biel Maze)
The learning and memory test was performed for all animals in the period from PND 62-68, except for the last born Group 3 Female, which was tested from PND 61-67 due to practical considerations.
A water-filled Biel Maze was used. The time to escape and number of errors (animal deviates from the correct channel with all four feet) were recorded. Animals were allowed three minutes to escape (except for the straight channel swimming test for which the animals were allowed two minutes) after which they were removed. On Days 2-7, the minimum inter-trial interval was one hour.
Each animal was tested for seven consecutive days as follows:
Day 1: Four consecutive trials in the straight channel.
Day 2 and 3: Two trials per day in Path A.
Day 4, 5 and 6: Two trials per day in Path B (reverse Path A).
Day 7: Two trials per day in Path A.


- Functional observation battery: tests were conducted once between PND 69-75 in the order of sequence indicated below and were divided between several days.
1. Detailed clinical observations, consisting of a number of tests conducted in- and out-side the home cage
2. Rectal temperature, measured immediately after the detailed clinical observations.
3. Locomotor activity, tested using the Kinder Scientific Motor Monitor System. Recording period was one hour under normal laboratory light conditions.
To the extent possible, treatment groups were balanced across devices and the time of testing was counterbalanced across dose groups. Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.
4. Hearing ability.
5. Fore- and hindlimb grip strength, recorded per animal as the mean of three measurements, using a grip strength meter (Series M4-10, Mark-10 Corporation).
6. Pupillary reflex (both eyes).
7. Landing (hind) foot splay, recorded per animal as the mean of three measurements.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY (cohort 3 and positive control animals):
All F1-animals of Cohort 3 and all positive control animals were immunized once, 5 days before scheduled necropsy (i.e. once between PND 48-54) via intravenous injection into the tail vein (approximately 1 mL/min) with 1.0 mL of the 300 µg/ml of Keyhole Limpet Hemocyanin KLH solution.
For the positive control animals, this immunization was performed 2 to 4 hours after treatment with cyclophosphamide.
For Cohort 3 animals, this immunization was performed at least 2 hours after treatment with the test item formulations/vehicle.
The animals were restrained during the injection procedure and injected using a disposable needle and butterfly needle. After injection, the injection site was marked with indelible ink.
On the day of KLH injection, clinical sign observations was performed after this injection for animals of Cohort 3 and the positive control animals.

Inject site evaluation: at least daily, starting one day after KLH injection up to and including the day before scheduled necropsy
The irritation scores and a description of all other (local) effects were recorded as part of the “1-hour post-dose” observation. Adjacent areas of skin of each animal served as controls.
The irritation was assessed according to the following numerical scoring system. At each observation, the highest scores given were recorded:

Erythema and eschar formation:
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) 4
Where signs of necrosis or corrosion (injuries in depth) prevent erythema scoring, the maximum grade for erythema (= 4) is given.

Oedema formation:
Minimal oedema 1
Slight oedema 2
Moderate oedema 3
Severe oedema 4

In-life procedures positive control animals:
- Mortality/moribundity checks: twice daily
- Clinical observations: at least once daily, up to the day prior to necropsy.
- Body weight: Animals were individually weighed once, on the first day of cyclophosphamide treatment.
- Food and water consumption was not determined for positive control animals.

T-Cell Dependent Antibody Response (TDAR) Assay (cohort 3 and positive control animals)
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. pre-immunisation and 5 day after immunization (PND 53-59)
- Animals fasted: No
- How many animals: 10 selected animals/sex/group (cohort 3) and 10 selected animals/sex (positive control animals)
Blood samples at a target volume of 0.3 mL were collected into tubes without anticoagulant. Blood samples were allowed to clot for at least 30 minutes and centrifuged within 2 hours after collection. Within 1 hour after centrifugation, serum of these samples were divided into 2 aliquots and subsequently stored in labeled polypropylene tubes at ≤-75°C until analysis.
For the first aliquot of serum samples, the antibody response to the immunization with KLH was determined by measuring the anti-KLH IgM levels in serum using Rat Anti-KLH IgM ELISA Kits (Life Diagnostics, Inc., West Chester, PA, USA) and an EL808™ Absorbance Microplate reader including Gen5 Secure software version 1.11.5 (BioTek Instruments, Inc., Winooski Vermont, USA) according to a validated method. Since the results obtained with these analyses were considered not valid (data retained in the raw data), the second aliquot of serum samples was analysed using a different validated ELISA method using a goat anti-rat IgM polyclonal antibody with peroxidase (HRP) conjugate (Jackson lmmunoResearch Laboratories Inc.).

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after successful mating and a minimum of 10 weeks of treatment.
- Maternal animals: All surviving animals on LD 22-25, or after total litter loss, or failure to deliver

GROSS NECROPSY
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

Organ Weights and Tissue Collection/Preservation – F0-Generation
Organs were weighed at necropsy for all scheduled euthanasia animals according to the guideline. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.
Representative samples of the tissues were collected from all animals, preserved, processed and evaluated according to the guideline.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at:
Cohort 1A: PND 89-95
Cohort 1B: Males after successful mating; females which delivered LD21-23; females which failed to deliver without evidence of mating: approximately 24-26 days after the last day of the mating period.
Cohort 2A: PND 76-90
Cohort 2B: PND 21-22
Cohort 3: PND 53-59
Positive control animals: PND 53-59

Unscheduled Deaths– F1 -Generation
Pups sacrificed in extremis on or after PND 7 were euthanized by an intraperitoneal injection of sodium pentobarbital.
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally) and externally examined with emphasis on developmental morphology. For pups found dead or sacrificed in extremis from PND 14 onwards a limited necropsy was performed including sex determination (both externally and internally).
Descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Culled Pups (PND 4) – F1-Generation
On PND 4, the pups scheduled for culling (> 8 pups per litter) were euthanized by decapitation. Sex was determined both externally and internally. Pups were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities were recorded.

Cohort surplus (10/sex/group)
Scheduled necropsy of Cohort Surplus was conducted on PND 22. Cohort Surplus animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all external abnormalities were recorded. Representative samples of the tissues were weight and collected.

Spare F1-animals which were not assigned to one of the Cohorts were sacrificed between PND 22-24 by intraperitoneal injection of sodium pentobarbital. Animals were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development, and sex was determined (both externally and internally). Descriptions of all external abnormalities were recorded.

Cohort 1A
Cohort 1A animals surviving to scheduled necropsy were deprived of food overnight (with a maximum of 24 hours) before necropsy, weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all external abnormalities were recorded.
HE stained step sections of ovaries and corpora lutea at a thickness of 5 micrometers (5 step sections in total, including the routine section) were prepared for the Cohort 1A females of Group 1 and 4 for quantitative evaluation of follicles (one ovary; primordial and small growing follicles counted together), as well as corpora lutea.

Sperm Analysis – Cohort 1A
For all males of Cohort 1A, the following assessments were performed: Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples.
One epididymis (left) was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.

Splenic Lymphocyte Subpopulation Analysis – Cohort 1A
From 10 selected animals/sex/group of Cohort 1A, splenic lymphocyte subpopulation analysis was performed at termination. One pup (male or female) was selected per litter (20 litters in total).
One half of the spleen was kept on ice until splenic lymphocytes were isolated using 70 µm cell strainers. The other half of the spleen was preserved for histopathological evaluation. Splenocytes were counted with the Coulter Counter Z1. The following subpopulations were determined in isolated splenic lymphocytes using the BD FACSCanto™ flow cytometer system on the day of necropsy:

Parameter (Abbreviation) Unit Markers for identification
T-cells % Lymphoid cells CD3+/CD45RA-
T-helper cells % Lymphoid cells CD3+/CD4+/CD8-
T-cytotoxic cells % Lymphoid cells CD3+/CD4-/CD8+
B-cells % Lymphoid cells CD3-/CD45RA+
NK-cells % Lymphoid cells CD3-/CD161a+
Ratio T-helper cells/ T-cytotoxic cells (Th/Tc) - -

Cohort 1B
Cohort 1B animals were not deprived of food overnight before necropsy. These animals were weighed weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

Cohort 2A and 2B
The animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. The animals were first anaesthetized using isoflurane and subsequently sacrificed by whole body (in situ) perfusion using heparinized saline (0.9% NaCl) followed by a 4% paraformaldehyde solution (adjusted to pH 7.4; HCl, KCl, NaH2PO4 x H2O, Na2HPO4 x 2H2O, paraformaldehyde and NaOH, aqua dest.).
All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.
After perfusion, the cranium was removed, exposing the brain. The skull including the brain was placed in 10% buffered formalin and allowed to fix for at least 7 days prior to removal from the skull.
The fixed brains were removed and weighed, and the length and maximum width of the brain was measured for all animals selected for neuropathology. Subsequently, the brain was fixed in 10% buffered formalin together with selected PNS tissues.
Morphometric (quantitative) analyses of CNS tissues was performed for Cohort 2A and 2B animals of Groups 1 and 4.

Cohort 3 and Positive Control Animals
Scheduled necropsy of Cohort 3 was conducted on PND 53-59 (except for one animal which was necropsied on PND 49). Positive control animals were euthanized on the same date(s). The animals were deeply anaesthetized using isoflurane and subsequently exsanguinatedand were subjected to a limited examination, with special attention being paid to the reproductive organs.

F2 generation
Scheduled necropsy of the F2-animals of Cohort 1B was conducted on PND 21-23. The animals were not deprived of food overnight.
From 10 selected litters/group, terminal body weight was determined for one male and one female pup. Subsequently, these pups were deeply anaesthetized using isoflurane and subsequently exsanguinated. The animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The organs identified for weighing and representative samples of the tissues were weighed and collected.
All remaining pups were sacrificed using Euthasol®20% by intraperitoneal (ip) injection. Also these pups were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means was applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).

Incidence
An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

Mating index males (%): Number of males mated/Number of males paired x 100
Mating index females (%): Number of females mated/Number of females paired x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index males(%): Number of pregnant females/Number of males mated x 100
Fertility index females(%): Number of pregnant females/Number of females mated x 100
Gestation index (%): Number of females with living pups on Day 1/Number of pregnant females x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Post-implantation survival index (%): Total number of offspring born/Total number of uterine implantation sites x 100
Live birth index (%): Number of live offspring on Day 1 after littering/Total number of offspring born x 100
Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
Viability index (%): Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering x 100
Weaning index (%): Number of live offspring on Day 21 after littering/Number live offspring on Day 4 (after culling) x 100
Percentage live males at weaning (%): Number of live male pups on Day 21 after littering/Number of live pups on Day 21 after littering x 100
Percentage live females at weaning (%): Number of live female pups on Day 21 after littering/Number of live pups on Day 21 after littering x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No test item-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
One female at 1000 ppm was observed with yellow discoloration of her urine from treatment Day 17 onwards. There was no correlating necropsy finding. As the yellow discoloration of the urine was an isolated finding in the low dose group only, no toxicological relevance was attached to it.
Piloerection was noted on a few occasions in Weeks 4, 5 and/or 6 of treatment in one male at 12500 ppm and two females at 4000 ppm. At the low incidence observed and in the absence of a dose-related effect (females), this finding was considered to be of no toxicological relevance.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred that was considered to be attributable to treatment with the test item.
One female in the 4000 ppm group was sacrificed on Day 4 of lactation, because she had a total litter loss.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For males at 12500 ppm, body weight gain was statistically significantly decreased from start of treatment onwards (values ranged between 0.62x and 0.82x of controls), without affecting the mean absolute body weight (terminal mean body weight was 0.96x of controls; not statistically significant).
Body weights and body weight gain of males up to and including 4000 ppm and females up to and including 12500 ppm remained in the same range as controls over the treatment period.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In males and females at all dose levels, both absolute and relative food consumption was statistically significantly higher on most occasions from Week 3 (males) or Week 1 (females) of treatment onwards. Changes compared to the control group were slight (mean values for relative food consumption in treated groups ranged between 1.04x and 1.15x of controls) and occurred in absence of a dose-response.

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

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological parameters of treated rats were not affected by treatment with the test item.
A statistically significant lower white blood cell count (WBC) was noted in females at 12500 ppm, which was likely the result of the lower lymphocytes mean level observed in these animals. As mean values remained within normal range, it was considered unrelated to treatment with the test item.
The statistically significantly lower mean value for neutrophils in females at 1000 and 12500 ppm was unrelated to treatment as no dose-response could be established. It was regarded to be the result of a slightly high control value.
Coagulation parameters of treated rats were not affected by treatment with the test item.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes distinguished animals treated at 12500 ppm from control animals. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.
- Increased urea concentration in males (1.33x) and females (1.18x)
- Increased potassium levels in females (1.14x)
- Increased chloride levels in females (1.02x)
Changes were slight only with all group mean values within internal control data or just above the 95th percentile (mean value for urea in males: 6.5 mmol/L; control data Urea (mmol/L): mean = 4.8; P5–P95 = 3.3-6.4 (males, n=80)). However, in light of the effects seen at the kidney organ level they were considered treatment-related.
Any other statistically significant changes in clinical biochemistry parameters were unrelated to treatment as these occurred in the absence of a dose-related trend.

Thyroid hormone analyses:
Serum level of total T4 was significantly reduced in males at 12500 ppm. At the observed magnitude (0.78x of control), a possible relation to treatment with the test item cannot be excluded. However, since the group mean level remained within normal limits, and no treatment-related histopathological changes were observed in the thyroid gland, it was regarded as non-adverse. Serum levels of T4 in females, and TSH (thyroid stimulating hormone) in both sexes, were unaffected by treatment up to 12500 ppm.
The slightly lower mean value for T4 observed in females at 12500 ppm was not regarded as treatment-related. The change was relatively small (0.90x of control), reaching no statistical significance, and group mean value (4.70 µg/dL) was very close to the mean of the internal control data (4.41 µg/dL).
The slightly (not statistically significantly) higher group mean level for TSH noted in females at 12500 ppm was mainly attributed to two females, whose values were 3.7x and 4.4x the mean control value, respectively. When excluding these two females, the group mean value (0.3975 µg/dL) for the remaining 8/10 females was well within the internal control range (TSH (uIU/mL): mean = 0.191; P5 – P95 = 0.030-0.614 (females, n=77)). Therefore, it was considered not to be toxicologically relevant.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

An increased presence of red blood cells was noted in the urine of males at 12500 ppm (score 3 vs. score 0 in controls).
Urinary parameters in males up to 4000 ppm and females up to 12500 ppm were unaffected by treatment with the test item. Any statistically significant changes in urinalysis parameters occurred in the absence of a dose-related trend.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in both sexes in the kidney starting at 4000 ppm, and in the urinary bladder at 12500 ppm in males, and at 4000 and 12500 ppm in females. An increased incidence of testicular and epididymal findings compared to background was noted in the males treated at 12500 ppm.
Kidney: A dose-related increased incidence and severity of microscopic alterations in the kidney of males (up to massive) and females (up to moderate) were observed at 4000 ppm and 12500 ppm. Dependent on the severity, the findings were extending from papilla to cortex and were diagnosed as retrograde nephropathy. Retrograde nephropathy is a diagnosis representing a combination of degenerative changes including tubular dilation, tubular basophilia, degeneration of tubular cells, infiltration of mononuclear inflammatory cells, fibrosis which in many cases surrounded atrophic tubules, and in some animals amorphous hyaline material was present in some tubules.

Urinary bladder: Diffuse hyperplasia was present in males up to massive degree treated at 12500 ppm, and in females at 4000 and 12500 ppm up to moderate degree.

Testes and epididymis: An increased incidence in tubular degeneration/atrophy (up to moderate) was noted in males treated at 12500 ppm with related minimal cellular debris in the epidydimis. An incidence of 1/28 males with slight testicular degeneration/atrophy in the 1000 ppm treatment group was regarded within background incidences.

There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were unaffected by treatment with the test item.
Most females had regular cycles of 4 to 5 days. An irregular cycle of 8 days was noted for one female at 4000 ppm and one female at 12500 ppm (both with normal litter). Given their incidental nature and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Spermatogenesis staging
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. Few males treated at 12500 ppm showed degeneration/atrophy of germ cells in the testes. In all other males examination of the testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Sperm analysis
The following statistically significant changes distinguished males treated at 4000 and/or 12500 ppm from control animals. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.
- Lower percentage of motile sperm at 4000 ppm (0.85x) and 12500 ppm (0.84x)
- Lower percentage of progressive sperm at 4000 ppm (0.73x) and 12500 ppm (0.76x)
- Higher number of cells with a detached head at 12500 ppm (3.6x; mean of 18 cells in the high dose group vs. 5 cells in the control group).
Note: at the individual level, 18/27 males at 12500 ppm had a score higher than 10, compared to 1/28 males in the concurrent control group.
The higher number of sperm cells with a detached head noted in males at 12500 ppm was considered treatment-related for the following reasons: The mean value of 18 cells with detached head was far above the available internal control range with 18/27 males having a score higher than 10, compared to 1/28 males in the concurrent control group. Moreover, similar effects were seen in males of the F1-generation (see results Cohort 1A).
The lower percentages of motile sperm and progressive sperm in males at 4000 and 12500 ppm as compared to the concurrent control were considered unrelated to treatment. Mean values were close to the historical mean, no dose-related response could be established and no correlating findings were noted in males of Cohort 1B. Statistical significance was reached due to the relatively high mean of the control group.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Of the 28 couples of each dose group, one control couple, two couples at 1000 ppm and two couples at 4000 ppm did not succeed in producing healthy offspring. One additional couple treated at 4000 ppm had total litter loss on Day 4 of lactation. For one male in the 1000 ppm group, agenesis of the left testis and epididymis was noted, the right testis showed moderate tubular atrophy with related cellular debris in the epididymal lumen. This was regarded the cause of infertility of one couple at 1000 ppm. Based on the low severity of the testes and epididymis findings of the other 1000 ppm male (slight degeneration/atrophy in the testes and minimal luminal cellular debris in the epididymis), these findings were not considered to be related to the lack of healthy offspring. Despite a mild increased incidence (above background) in degenerative/atrophic changes of germ cells in few testicular tubules of males treated at 12500 ppm, fertility was not affected. The morphology of female reproductive organs was unaffected by the treatment with test item.

Mating index, precoital time, number of implantation sites, fertility index, gestation index, duration of pregnancy, the total number of offspring born compared to the total number of uterine implantations, litter size, sex ratio, live birth index, viability index and weaning index were not affected by treatment with the test item.
All females showed evidence of mating, resulting in mating indices of 100% for all groups.
Most females showed evidence of mating within 4 days, except for one control female and one female at 4000 ppm, for which mating took 12 and 6 days, respectively. At the incidence observed and in the absence of a dose-relationship, this was considered to be unrelated to treatment.
Mean number of implantation sites was 12.8, 13.2, 12.3 and 12.7 in the control, 1000, 4000 and 12500 ppm groups, respectively.
The fertility indices were 96, 93, 93 and 100% for the control, 1000, 4000 and 12500 ppm groups, respectively.
The number of non-pregnant females versus mated females was 1/28, 2/28, 2/28 and 0/28 in the control, 1000, 4000 and 12500 ppm groups, respectively. As these cases of non-pregnancy showed no dose-related incidence across the dose groups, this was considered unrelated to treatment.
All pregnant females had live offspring, resulting in a gestation index of 100% for all groups.
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 92, 96, 94 and 92% for the control, 1000, 4000 and 12500 ppm groups, respectively.
Mean litter sizes were 11.8, 12.4, 12.2 and 11.7 living fetuses/litter for the control, 1000, 4000 and 12500 ppm groups, respectively.
The live birth indices were 100, 98, 99 and 99% for the control, 1000, 4000 and 12500 ppm groups, respectively. One pup of the control group, six pups at 1000 ppm, two pups at 4000 ppm and two pups at 12500 ppm were found dead at first litter check. No toxicological relevance was attributed to these dead pups as the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Viability indices were 100, 98, 99 and 98% for the control, 1000, 4000 and 12500 ppm groups, respectively. One pup of the control group, six pups at 1000 ppm , three pups at 4000 ppm and eight pups at 12500 ppm were found dead or missing between PND 2 and 4. Pups found missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
The weaning indices were 100, 100, 98* and 100% for the control, 1000, 4000 and 12500 ppm groups, respectively.
* Due to an accident during animal handling, four pups at 4000 ppm (all from one litter) were lost on PDN 9; three pups died immediately (necropsy findings: blue spots on different parts of the body and/or scabs on the back) and one pup was sacrificed in extremis, based on its poor condition, including blue spots on head and chest and a swollen left jaw. None of these deaths was related to treatment with the test item. After correction for this accidental loss, the weaning index for the 4000 ppm group reached 100%.
One pup at 12500 ppm went missing on PND 20. Most likely it was cannibalised. No toxicological relevance was attributed to this dead pup since the mortality incidence remained within the range considered normal for pups of this age.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Adverse kidney effects in both males and females at 4000 ppm

Remarks on result:

other: corresponding to 65 mg/kg bw/day in males and 87 mg/kg bw/day in females of the F0-generation

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

>= 12 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of adverse effects up to and including the highest dose level tested.

Remarks on result:

other: corresponding to 833 mg/kg bw/day in males and 1124 mg/kg bw/day in females of the F0-generation

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

System:

urinary

Organ:

kidney

Treatment related:

yes

Results: P1 (second parental generation)

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A: Length and regularity of the estrous cycle were not affected by treatment up to 12500 ppm. For all females for which estrous cycle regularity could be determined, regular cycles of 4 to 5 days were recorded between PND 75 and 88.
No cycle classification was possible for 2/20 females at 12500 ppm, as only one complete cycle was observed during the 2-weeks observation period. Given its low incidence and the fact that all remaining 18/20 high dose females had regular cycles of 4-5 days, this findings did not indicate a relation with treatment with the test item.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A
The following statistically significant changes distinguished males of Cohort 1A treated at 12500 ppm. Relative differences in mean values as compared to the concurrent control group are indicated between parentheses.
- Lower percentages of motile sperm and progressive sperm (means of 59% and 21%; 0.79x and 0.62x control, respectively)
- Lower epididymal sperm count (mean of 305.6x10E6/gram; 0.58x of control)
- Lower number of cells with normal morphology (mean of 145; 0.86x of control)
- Higher number of cells with detached head (mean of 24 cells vs. 2 cells in the control group; 12.0x of control). Note: at the individual level, 16/20 males at 12500 ppm had a score higher than 10, compared 0/20 control males.
- Higher number of cells with abnormal neck (mean of 5 cells vs.2 cells in the control group; 2.5x of control)
The higher number of sperm cells with detached head noted at 12500 ppm was considered treatment-related for the following reasons: The mean value of 24 cells with detached head was far above the available internal control range with 16/20 males having a score higher than 10, compared to 0/20 males in the concurrent control group. Moreover, similar effects were seen previously in males of the F0-generation.
The decreased number of cells with normal morphology was a direct consequence of this marked increase in cells with detached head at 12500 ppm.

The slightly higher number of cells with abnormal neck at 12500 ppm was unrelated to treatment. Mean value was within the internal control range, and no correlating findings were noted in males of the previous F0-generation, and when compared to the concurrent control group, the number of cells with abnormal neck was only higher in two high dose Cohort 1A-males which had respectively 13 and 12 cells with abnormal neck, compared to a maximum of 9 cells observed in control Males.
The lower percentages of motile sperm and progressive sperm as compared to the concurrent control were unrelated to treatment. Mean values were well within the internal control range, and no correlating findings were noted in males of Cohort 1B. Statistical significance was reached due to the relatively high mean of the control group.
Also the lower epididymal sperm count at 12500 ppm was unrelated to treatment. Mean values were well within the internal control range, and no correlating findings were noted in males of either the F0-generation or Cohort 1B. At the individual level, a very low sperm count was observed in a single high dose male (10.0x10E6/gram). No toxicological relevance was attached to this isolated observation.

Cohort 1B
No treatment-related changes were noted on sperm motility and epididymal sperm count in Cohort 1B males treated up to 12500 ppm. The significantly higher epididymal sperm count (1.20x of control) at 4000 ppm occurred in the absence of a dose-related trend and was therefore unrelated to treatment.
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. Except for the males that showed degeneration/atrophy of germ cells the testis revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Note: Sperm analysis in Cohort 1B males did not include evaluation of sperm morphology.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1B:
The mating indices were 100% for the control group, and 1000 and 4000 ppm groups. One female at 12500 ppm did not show evidence of mating, resulting in a mating index of 96% in this group.
Mean precoital time (i.e. days before mating) was 3.0, 2.3, 2.9 and 3.8 days for the control group, and 1000, 4000 and 12500 ppm groups, respectively.
Most females showed evidence of mating within 5 days, except for one control female, and two females at 12500 ppm for which mating took 13 days. As this variation in precoital time remained within the normal range of biological variation and occurred at low incidence, it was considered to be unrelated to treatment.
Number of implantation sites was considered not to be affected by treatment up to 12500 ppm.
Mean number of implantation sites was 12.7, 12.6, 11.8 and 11.4 in the control, 1000, 4000 and 12500 ppm groups, respectively. The slightly lower mean values observed in the 4000 and 12500 ppm groups were considered to be unrelated to treatment as changes compared to the concurrent control group were relatively small (not statistically significant), and most individual values remained within the range of available internal control data . One female at 12500 ppm had 3 implantation sites only. As such low numbers of implantation sites are occasionally seen in rats of this strain and age, no toxicological relevance was attached to this isolated finding.
For one control female, the number of pups was slightly higher than the number of implantations. This phenomenon is observed occasionally in this type of rat studies.
The fertility indices were 100% for the control group, and 1000 and 4000 ppm groups. One female at 12500 ppm was not pregnant, resulting in a fertility index of 96% in this group. At the isolated incidence, it was regarded as unrelated to treatment with the test item.

Histopathological examination of reproductive tissues revealed no evidence of a treatment-related effect on reproduction.
Of the 25 couples of each dose group (Group 3 contained 23 males), two couples at 12500 ppm did not succeed in producing healthy offspring. One additional couple treated at 12500 ppm had total litter loss on Day 2 of lactation. Based on the low severity of the testes and epididymis findings of two males at 12500 ppm (slight degeneration/atrophy in the testes), this finding was not considered to be related to the lack of healthy offspring/total litter loss. Despite a mild increased incidence and/or severity (above background) in degenerative/atrophic changes of germ cells in few testicular tubules of males treated at 4000 and 12500 ppm, fertility was not affected. The morphology of female reproductive organs was unaffected by the treatment with test item.

Gestation index, duration of gestation, total number of offspring born compared to the total number of uterine implantations, litter size, live birth index, viability index and the number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) were not affected by treatment with the test item.
All pregnant females had live offspring, resulting in a gestation index of 100% for all groups. Mean duration of gestation ranged from 21.2 to 21.3 days across the dose groups. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Post-implantation survival index was 92, 94, 93 and 89% for the control, 1000, 4000 and 12500 ppm groups, respectively. The slightly lower value in the 12500 ppm group was mainly caused by one high dose female which had 10 implantation sites, but delivered only three pups. When excluding this female, a post-implantation index of 91% is reached which is very close to the 92% in the control group. Therefore, it was considered not to be treatment-related.
A trend towards a lower mean litter size was noted in the mid and high dose group at first litter check: mean values of 11.6, 11.8, 10.8 and 10.0 live pups per litter were recorded in the control, 1000 ppm, 4000 ppm and 12500 ppm groups, respectively. In both the mid and high dose group a few small litters were observed. However, this finding was considered unrelated to treatment, as both groups contained also many relatively large litters, small sized litters are occasional seen in this type of study, and group mean values remained within the available control range.
The live birth indices were 99% in the control, 4000 and 12500 ppm groups, and 100% in the 1000 group. Two pups of the control group, one pup at 1000 ppm, two pups at 4000 ppm and two pups at 12500 ppm were found dead at first litter check. No toxicological relevance was attributed to these dead pups as the mortality incidence did not show a dose related trend and remained within the range considered normal for pups of this age.
Viability indices were 98, 100, 99 and 99% for the control, 1000, 4000 and 12500 ppm groups, respectively. Five pups of the control group, one pup at 1000 ppm, two pups at 4000 ppm and two pups at 12500 ppm were found dead or missing between PND 2 and 4. Pups missing were most likely cannibalised.
The weaning indices were 99, 98, 100 and 100% for the control, 1000, 4000 and 12500 ppm groups, respectively. One pup of the control group was found dead on PND 19, and in the 1000 ppm group one pup was found dead on PND 20, and three went missing on PND 9 or 10. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups as the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Effect levels (P1)

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction F1

Effect level:

>= 12 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of adverse effects up to and including the highest dose level tested

Remarks on result:

other: corresponding to 1200 mg/kg bw/day in males and 1227 mg/kg bw/day in females of the F1-generation

Target system / organ toxicity (P1)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

pre-weaning:
No clinical signs occurred among pups that were related to treatment with the test item.
One control pup was observed with a swollen abdomen on several days between PND 15 and PND 20. A relation to treatment could be excluded as pups of the control group had not been exposed to the test item.
The nature and incidence of other clinical signs remained within the range considered normal for pups of this age.

post weaning:
No test item-related clinical signs were noted during daily detailed clinical observations or weekly arena observations.
In the 4000 ppm group, three males in Cohort 3 presented with piloerection from Days 7-12 of treatment, together with significant body weight loss on Day 8. The poor condition of these males was caused by a defect water bottle.
All clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Pre-weaning: see reproductive performance P0.
No mortality occurred that was considered to be attributable to treatment with the test item.
Two males at 4000 ppm in Cohort 3 died on Day 8 of treatment. Due to a manufacturing error in the nozzle of their water bottle, these pups had no access to water for one day. One pup was found dead and one pup died shortly after the incident had been discovered. Gross findings for these two dead pups included purple discoloration of the brain, beginning autolysis (of the gastro-intestinal tract), liver with several gray-white foci, dark-red discoloration of the testes and/or lungs that were not collapsed.
One control female in Cohort 1B had to be sacrificed for humane reasons on PND 36. On the day of sacrifice, hunched posture, slightly decreased locomotor activity, slightly laboured respiration, swelling of the abdomen, piloerection, ptosis, and a lean and slightly dehydrated appearance were noted for this animal. Gross findings at necropsy included irregular surface of the forestomach with many reddish foci, intussusception of the jejunum, right medial lobe of the liver with isolated black-brown foci and grown together with the diaphragm, and several black-brown foci on the kidneys. As this female was a control animal, a relation to treatment with the test item could be excluded.
In addition, one female at 12500 ppm in Cohort 1B was sacrificed on Day 2 of lactation, because she had a total litter loss.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Pre-weaning:
Body weights of pups were not affected by treatment with the test item.

Post weaning:
No test item-related effects on body weight and body weight gain were observed in males and females at 1000 ppm (Cohorts 1A, 1B, 2A and 3). All values remained remained within the normal range of biological variation.
At 12500 ppm, lower body weights and/or body weight gains were noted in both males and females (not always statistically significant). When compared to the concurrent control group changes in mean body weight gain were highest after the first week of treatment (ranging from 0.72-0.75x and 0.70-0.78x of control in males and females, respectively) of Cohorts 1A, 1B, 2A and 3), followed by recovery. After 10 weeks of treatment (Cohorts 1A and 1B), mean body weight was 0.94x and 0.91-0.95x of control in males and females, respectively. On Day 1 of the mating period (Cohort 1B), mean body weight at 12500 ppm was 0.95x of control (males) and and 0.94x of control (females). In females at 12500 ppm (Cohort 1B), mean body weights were 0.91x of control and 0.93x of control at the end of the post-coitum and lactation phase, respectively. Body weight gain in these females was 0.90x of control at the end of the post-coitum phase (not statistically significant), and remained comparable to controls throughout the lactation phase.
A similar trend was observed at the mid dose level of 4000 ppm (both sexes; Cohorts 1A, 1B, 2A), reaching statistical significance on some occasions. However, changes were relatively slight with overall no significant effects on mean values for absolute body weight.
The relatively low mean value for body weight observed in Cohort 3 males of the 4000 ppm group could be related to a defect water bottle in one cage. Two males did not survive this incident and for the remaining three males a body weight loss of 32-41% was recorded from treatment Days 1-8. After replacing the water bottle, the three males recovered again, but their body weights remained approximately one week behind the body weight of other males in the same age.
Terminal body weight was statistically significantly lower for Cohort Surplus male pups at 12500 ppm (0.90x of controls; PND 22). As no treatment-related effects on pup body weight was observed in the period from PND 1-21, no toxicological significance was attached to this finding.
Any other statistically significant changes in body weights were considered unrelated to treatment with the test item as no trend was apparent regarding dose and/or duration of treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the entire observation period, there was a general trend towards higher food intake (both absolute and relative to body weight) in males and females of all treated groups (Cohorts 1A, 1B, 2A and 3). Changes compared to the concurrent control group reached often, but not always statistical significance during the treatment (Cohorts Cohorts 1A and 1B) and post-coitum (Cohort 1B) period in all treated groups. During lactation, only the high dose group had a significantly higher food intake (Cohort 1B).

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

effects observed, treatment-related

Description (incidence and severity):

As a test item-related effect on water intake was suspected in males, quantitative measurement was introduced for Cohort 1B (both sexes) from Week 6 of premating onwards. In males at 12500 ppm, water intake was higher compared to controls during premating and mating, reaching statistical significance on most occasions. A similar trend was observed in females at 12500 ppm, but changes compared to the control group were less pronounced than in males and reached statistical significance on a few occasions only (combined mean of means: 1.44x and 1.10x of control, for males and females, respectively).

Haematological findings:

no effects observed

Description (incidence and severity):

Haematological parameters of treated rats were not affected by treatment with the test item.
Coagulation parameters of treated rats were not affected by treatment with the test item.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant changes distinguished animals treated at 12500 ppm from control animals. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.
- Increased aspartate aminotransferase (ASAT) activity in males (1.17x)
- Increased alkaline phosphatase (ALP) activity in females (1.38x)
- Increased inorganic phosphate (Inorg.Phos) levels in males (1.08x)
These alterations in clinical biochemistry parameters were considered unrelated to administration of the test item due to the relatively low magnitude of the change and/or absence of effects on correlating parameters. Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Thyroid hormone analyses:
Serum T4 levels in male and female pups, culled at PND 4, and T4 and TSH levels in male and female pups of Cohort Surplus at PND 22 were considered to be unaffected by treatment.
Serum levels of total T4 in male and female pups, culled at PND 4, were statistically significantly lower at 12500 ppm when compared to controls (0.86x). As the mean value remained within the internal control range, this effect was considered unrelated to treatment with the test item.
The slightly higher levels of total T4 and TSH in PND 22 males (Cohort Surplus) at 12500 ppm and PND 22 females (Cohort Surplus) at 4000 ppm and 12500 ppm were considered to be unrelated to treatment, as differences were minimal (i.e. no statistical significance was reached), mean values remained within the available internal control range and/or occurred in the absence of a dose-related trend.
Serum levels of T4 and thyroid stimulating hormone (TSH) in F1-animals at End of Treatment were not affected by treatment with the test item.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis parameters of treated rats were not affected by treatment with the test item.
The significantly lower value for crystals in males at 12500 ppm was not toxicologically relevant, as the opposite effect would be expected in case of toxicity.

Sexual maturation:

no effects observed

Description (incidence and severity):

Balanopreputial separation (prepuce opening) in males and vaginal patency (vaginal opening), occurrence of first estrus and time between vaginal opening and first estrus in females were not affected by treatment with the test item.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment.
The statistically significantly higher mean normalised anogenital distance of female pups at 12500 ppm was only minimal and all individual values remained below the upper limit of the available internal control data. This difference was therefore considered not to be related to treatment with the test item.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 12500 ppm had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Fresh weights of brain, thymus, kidneys or spleen in Cohort Surplus pups (PND 22; both sexes) were unaffected by treatment up to 12500 ppm.
The observed slightly lower mean organ/body weight ratios for thymus (males) and spleen (females) in the high dose group was considered not to be toxicologically relevant as all group means and individual values remained within the internal control range.
Any other statistically significant changes in absolute and/or relative organ weights between treated and control animals were considered not to be related to treatment with the test item as these occurred in the absence of a dose-related trend.

Organ weights of Cohort 1A and 1B animals were not affected by treatment.
The following statistically significant changes distinguished treated from control animals. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.
Cohort 1A
- Lower absolute heart weight in males at 4000 ppm (0.95x) and 12500 ppm (0.94x).
- Lower absolute liver weight in males at 4000 ppm (0.93x) and 12500 ppm (0.91x).
- Lower absolute adrenals weight in males at 12500 ppm (0.86x).
- Lower absolute and relative thymus weight in females at 4000 ppm (0.88x for both absolute and relative weight) and 12500 ppm (0.87x for absolute weight, and 0.92x for relative weight (not statistically significant)).
- Higher relative brain weight in males at 4000 ppm (1.05x) and 12500 ppm (1.11x) and in females at 12500 ppm (1.06x).
- Higher relative thyroids weight in males at 12500 ppm (1.20x).
- Higher relative kidneys weight in males at 12500 ppm (1.13x).
Cohort 1B
- Higher relative thyroids weight in males (1.25x) and females (1.20x) at 12500 ppm
- Higher relative kidneys weight in males at 12500 ppm (1.12x) and lower absolute kidneys weight in females at 12500 ppm (0.95x)
- Lower absolute testes weight in males at 12500 ppm (0.94x)
- Higher absolute and relative uterus weight in females at 12500 ppm (1.19x and 1.32x, respectively).
These changes observed in Cohort 1A and 1B animals were relatively slight only, were secondary to the lower terminal body weight of high dose animals and/or occurred without any macroscopic or microscopic correlate. Therefore, they were considered to be of no toxicological relevance.
Any other statistically significant changes in absolute and/or relative organ weights between treated and control animals were not related to treatment with the test item as these occurred in the absence of a dose-related trend.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be related to treatment with the test item. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered unrelated to treatment.

Test item-related macroscopic alterations at the end of the treatment period were recorded for the kidney in animals of Cohort 1A (PND 89-95), Cohort 1B (≥PND 90), and Cohort 2A (PND 76-90), treated at 4000 and/or 12500 ppm (both sexes).
These alterations consisted of:
- Cohort 1A: Irregular surface in the kidneys of 3/20 males and 1/20 females at 4000 ppm, and 15/20 males and 7/20 females at 12500 ppm. The microscopic correlate was retrograde nephropathy.
- Cohort 1B: Irregular surface in the kidneys of 4/23 males at 4000 ppm, and 21/25 males and 18/25 females at 12500 ppm (and a single high dose female sacrificed on Day 2 of lactation).
- Cohort 2A: Irregular surface in the kidneys of 3/9 males at 12500 ppm.
In the kidneys of males treated at 4000 ppm (Cohort 1A) and 12500 ppm (Cohorts 1A, 1B and 2A), also an increased incidence of pelvic dilation was observed. Although this increase may indirectly be related to the retrograde nephropathy, it is not present in females despite the presence of retrograde nephropathy. Moreover, this finding is commonly seen as background finding and in most cases unilateral (e.g. an incidence of 3/20 in control females of Cohort 1B). Therefore, the pelvic dilation is not considered to be test item-related.
There were no test item-related gross observations in the brain of animals in Cohort 2A (PND 76-90) and Cohort 2B (PND 21-22), and peripheral nervous system organs of animals in Cohort 2A.
There were no test item-related macroscopic findings in animals of Cohort 3 (incl. positive controls; PND 53-59).
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A: Test item-related microscopic findings after treatment with the test item were noted in both sexes in the kidney starting at 4000 ppm and an increased incidence and/or severity of testicular and epididymal findings compared to background were noted in the males treated at 4000 and 12500 ppm.
Kidney: A dose-related increased incidence and severity of microscopic alterations in the kidney of males (up to marked) and females (up to moderate) were observed at 4000 ppm and 12500 ppm. Dependent on the severity, the findings were extending from papilla to cortex and were diagnosed as retrograde nephropathy. Retrograde nephropathy is a diagnosis representing a combination of degenerative changes including tubular dilation, tubular basophilia, degeneration of tubular cells, infiltration of mononuclear inflammatory cells, fibrosis which in many cases surrounded atrophic tubules, and in some animals amorphous hyaline material was present in some tubules.
Testes and epididymis: An increased incidence and/or severity in tubular degeneration/atrophy in the testes was noted in males treated at 4000 and 12500 ppm (up to moderate) with related cellular debris in the epididymis (up to marked).
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Ovarian Follicle Counts (Cohort 1A)
There were no test item-related effects on the ovarian follicle counts in the F1-females (Cohort 1A) at 12500 ppm when compared to control group females. Any variations between group mean counts represented biological variability and were not statistically significant.

Other effects:

no effects observed

Description (incidence and severity):

Splenic Lymphocyte Subpopulation – F1-Generation (Cohort 1A): There were no test item-related effects on splenic lymphocyte subpopulations observed.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Acoustic startle response was considered not affected by treatment with the test item. The slightly higher average response amplitude for males at 12500 ppm (not statistically significant) was caused by two males, that had mean values of 0.17 and 0.28, respectively. This slight difference in average acoustic startle response was considered not related to treatment as the difference to control was minimal only.
There were no toxicologically relevant effects on learning and memory up to 12500 ppm. In general, all groups had similar baseline swimming ability when tested in the straight channel on Day 1. Additionally, rats of all groups were able to learn each path, indicated by decreasing time and fewer errors made from the first trial over the subsequent trials. Time and errors increased when animals switched to Path B, which is a normal response when switching from one path to another. These decreased over time as animals learned the rules of the reverse path. The only exception to this were Trials 8 and 10 in Path B when males in Group 4 suddenly needed longer times again to reach the platform (with statistical significance in Trial 10 only) and made slightly more errors (Path 10). As these were two isolated observations, and time and errors in the remaining trials were comparable for Group 4 males and control males, this finding was considered unrelated to treatment. Animals also remembered Path A when switched back, indicating they were able to remember the original path learned. Any statistically significant changes in Groups 2 and 3 were considered not to be related to treatment as a dose response relationship was lacking.
Detailed clinical observations revealed no symptoms that were considered to be related to treatment with the test item. The clinical symptoms that were observed were considered to be within the normal range of behavioural findings for this type of study, and were generally also observed in control animals. These findings were therefore considered not to be related to treatment.
Rectal temperature was not affected by treatment with the test item. All values remained within the concurrent control and internal control data range (except for one female each in the control and high dose group whose rectal temperature of 39.4°C was just above the higher limit).
Motor activity was not affected by treatment with the test item. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Functional observation parameters (hearing ability, pupillary reflex, foot splay and grip strength) were not affected by treatment with the test item. The statistically significant changes in footsplay and grip strength of the foreleg for males at 4000 ppm were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Neuropathology and Morphometry (Cohorts 2A and 2B)
Fixed brain weights (absolute and relative to body weight) of Cohort 2B animals (PND 21-22) and Cohort 2A animals (PND 76-90) were not affected by treatment up to 12500 ppm.
The statistically significant lower mean value for absolute fixed brain weight noted in the 4000 ppm group when compared to the concurrent control group was not related to treatment with the test item as it occurred in the absence of a dose-related trend.
Examination of the H&E stained sections of brain or peripheral nervous system (control and high-dose group) did not reveal any test item-related effects in males and females of Cohort 2B (PND 21-22) or Cohort 2A (PND 76-90).

Administration of the test item in the diet to male and female Wistar Han rats throughout gestation and lactation (Cohorts 2A and 2B) and then directly in the feed at 0, 1000, 4000 and 12500 ppm (Cohort 2A), with appropriate feed adjustments during lactation, was not associated with any evidence of developmental neurotoxicity in the brain. There were no changes in morphometric measurements.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

An initial analysis of pre- and post-immunization samples (Aliquot 1) unexpectedly showed no or only very limited induction of anti-KLH IgM post-immunization in the concurrent Control group for Cohort 3. Mean post/pre-immunization ratio for the concurrent Control group was 1.4 for both males and females. Since a ratio of at least 2 was considered required to confirm adequate performance of the assay within this study, the results obtained from Aliquot 1 were considered not valid. Therefore, these results were not reported, but retained in the raw data.
After review of the study data it was concluded that the unexpected low ratios were most likely attributable to the ELISA kit used. Therefore, the second aliquot of serum samples was analyzed using a different (validated) ELISA method.
All animals were negative for the presence of anti-KLH IgM antibodies before the administration of KLH. There were no treatment related effects on the level of anti-KLH IgM antibodies detected at any dose level. All levels detected in test item-treated animals remained within the range of the vehicle control animals. In contrast, the positive control animals of both genders treated with Cyclophosphamide had a decreased group mean value of anti-KLH IgM antibodies compared to all other groups, and a higher incidence of non-responding animals (4/10 for males and 4/10 for females), thus revealing a relevant degree of immunosuppression caused by the Cyclophosphamide treatment. No local clinical signs were noted at the KLH injection site in any group.

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Generation:

F1

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Adverse kidney effects in both males and females at 4000 ppm

Remarks on result:

other: on average corresponding to 89 mg/kg bw/day in males and 93 mg/kg bw/day in females of the F1-generation

2

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental General Toxicity

Generation:

F1

Effect level:

>= 12 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of adverse effects at this dose on developmental parameters.

Remarks on result:

other: on average corresponding to 1200 mg/kg bw/day in males and 1227 mg/kg bw/day in females of the F1-generation

3

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental Neurotoxicity

Generation:

F1

Effect level:

>= 12 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of adverse effects at this dose on parameters measuring development of the neurological system.

Remarks on result:

other: on average corresponding to 1200 mg/kg bw/day in males and 1227 mg/kg bw/day in females of the F1-generation

4

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental Immunotoxicity

Generation:

F1

Effect level:

>= 12 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of adverse effects at this dose on parameters measuring development of the immunological system.

Remarks on result:

other: average corresponding to 1200 mg/kg bw/day in males and 1227 mg/kg bw/day in females of the F1-generation

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment with the test item.
One pup in the 12500 ppm group that was found dead at first litter check had a cleft palate. At this isolated incidence, it was regarded as a background finding. The nature and incidence of remaining clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be not toxicologically relevant.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were not affected by treatment with the test item.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum levels of total T4 were considered not affected by treatment with the test item.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not affected by treatment.
Any statistically significant changes in anogenital distance normalized for body weight in female pups were minor in magnitude and lacked a dose relationship. These were therefore considered not to be toxicologically relevant.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant differences in organ weight of brain, thymus, kidneys and spleen were observed following treatment up to 12500 ppm.
The lower absolute and relative thymus weight observed in both males and females in the 12500 ppm group compared to the control group were relatively slight, reaching statistical significance for relative thymus weight in males only (0.82x of control), and remained within the available internal control range . Therefore, they were considered not toxicologically relevant.
Any other differences, including those that reached statistical significance were not test item-related due to the lack of a dose-related pattern.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio was not affected by treatment with the test item.

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2 (cohort 1B)

Effect level:

>= 12 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of adverse effects at this dose on developmental parameters.

Remarks on result:

other: average corresponding to 1200 mg/kg bw/day in males and 1227 mg/kg bw/day in females of the F1-generation

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Dose formulation analyses

Accuracy

The concentrations analyzed in the diets of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%).

A small response at the retention time of the test item was observed in the chromatograms of the control Group 1 diets prepared for use in Weeks 1, 11, 22 and 26. It was considered to derive from the diet matrix, as this was also observed in the blanc QC sample (which only contains diet matrix) analyzed during the validation project. The maximum contribution to the Group 2 samples was 3.8%, 4.5%, 4.2% and 7.2%, respectively.

Homogeneity

The diets of Groups 2 and 4 were homogeneous (i.e. coefficient of variation≤ 10%).

  Test Item Intake – F₀-Generation

			Mean over means intake [mg test item/kg body weight] (mean range indicated within brackets)
	Group No.	2	3	4
	Nominal dietary inclusion level (ppm)
Sex	Study period
Males	Pre-mating	72 (72-73)	290 (290-291)	890 (879-901)
	Post-mating	63 (57-69)	263 (240-283)	820 (769-870)
	Mean of means^a	65	268	833
Females	Pre-mating	84 (84)	325 (323-327)	1042 (1029-1054)
	Post-coitum	86 (81-98)	349 (330-385)	1083 (1000-1270)
	Lactation	91 (64-122)	379 (278-483)	1226 (863-1647)
	Mean of means^a	87	355	1124

^a    Mean of means of all periods, weighed for number of measurement intervals per period:

Males: ((2 x mean premating) + (9 x mean mating)) / 11
Females: ((2 x mean premating) + (6 x mean post-coitum) + (4 x mean lactation)) / 12

 Test Item Intake – F1-Generation

			Mean over means intake [mg test item/kg body weight] (mean range indicated within brackets)
	Group No.	2	3	4
	Nominal dietary inclusion level (ppm)
Sex	Study period
Males	Pre-mating	93 (67-121)	387 (284-499)	1250 (958-1563)
	Post-mating	64 (63-65)	275 (266-283)	927 (892-962)
	Mean of means^a	89	370	1200
Females	Pre-mating	100 (81-123)	401 (323-529)	1300 (1064-1592)
	Post-coitum	83 (78-90)	347 (317-373)	1127 (1020-1238)
	Lactation	89 (62-117)	356 (247-467)	1175 (833-1579)
	Mean of means^a	93	377	1227

^a    Mean of means of all periods, weighed for number of measurement intervals per period:

Males: ((11 x mean premating) + (2 x mean mating)) / 13

Females: ((11 x mean premating) + (6 x mean post-coitum) + (4 x mean lactation)) / 21

Applicant's summary and conclusion

Conclusions:

In an extended one generation reproductive toxicity study according to OECD TG 443 (including Cohorts 1, 2 and 3), the following no-observed-adverse-effect level (NOAEL) of MELAMINE were established:
General Toxicity NOAEL F0-generation and F1-generation: 1000 ppm (on average corresponding to 65 mg/kg bw/day in males and 87 mg/kg bw/day in females of the F0-generation, and 89 mg/kg bw/day in males and 93 mg/kg bw/day in females of the F1-generation). This NOAEL is based on adverse kidney effects in both males and females at 4000 ppm (both generations).
Reproduction Toxicity NOAEL: F0-generation 4000 ppm and F1-generation 1000 ppm (on average corresponding to 268 mg/kg/day in F0 males and 89 mg/kg/day F1 males, based on tubular degeneration/atrophy in the testis with related minimal cellular debris in the epididymis in F0 males at 12500 ppm (833 mg/kg bw/day) and in F1 males at and above 4000 ppm (370 mg/kg bw/day).
Developmental General Toxicity NOAEL F1-generations and F2-generation: at least 12500 ppm (on average corresponding to 1200 mg/kg bw/day in males and 1227 mg/kg bw/day in females of the F1-generation), in absence of adverse effects at this dose on developmental parameters.
Developmental Neurotoxicity NOAEL F1-generation: at least 12500 ppm (on average corresponding to 1200 mg/kg bw/day in males and 1227 mg/kg bw/day in females of the F1-generation), in absence of adverse effects at this dose on parameters measuring development of the neurological system.
Developmental Immunotoxicity NOAEL F1-generation: at least 12500 ppm (on average corresponding to 1200 mg/kg bw/day in males and 1227 mg/kg bw/day in females of the F1-generation), in absence of adverse effects at this dose on parameters measuring development of the immunological system.

Executive summary:

An Extended One Generation Reproductive Toxicity Study (including Cohorts 1, 2 and 3) was performed in Wistar Han rats in according to OECD TG 443 and in accordance with GLP principles.

The dose levels in this study were selected to be 0, 1000, 4000, 12500 ppm, based on the results of a preliminary reproductive toxicity study (reproduction/developmental toxicity screening test) with dietary exposure of MELAMINE in rats. The same dietary concentrations were used throughout the study, except for the lactation period. As food intake is considerably higher in lactating females, dietary concentrations were lowered by 50% for all treated groups during the lactation period (PND 1-21).
Chemical analyses of dietary preparations were conducted at four occasions during the study (i.e. in Weeks 1, 11, 22 and 26) to assess accuracy and homogeneity. Stability of dietary preparations with Melamine under conditions bracketing those used in the current study had been confirmed previously.
For the F0-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, estrous cycle determination, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations. For the F1-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, water consumption, vaginal patency and balanopreputial separation, day of first estrous, estrous cycle determination, functional observations including acoustic startle response, learning and memory Biel Maze test, immunotoxicity assessments using a TDAR assay, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations, neurohistopathological examinations and morphometric analysis of brain tissue.
In addition, the following reproduction/developmental parameters were determined for the F0- and F1-generation: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormones).

Dietary preparations were considered homogeneous at the concentrations tested, and analysis of accuracy and stability revealed acceptable levels.

Parental results – F0-generation

For males at 12500 ppm, body weight gain was statistically significantly decreased from start of treatment onwards (values ranged between 0.62x and 0.82x of controls), resulting in slightly but not statistically significantly lower mean absolute body weights compared to concurrent controls (terminal mean body weight was 0.96x of controls). It should be noted that there was a general trend towards higher food intake (absolute and relative to body weight) in all treated groups from Week 3 (males) or Week 1 (females) of treatment onwards. This higher food intake may be related to the sweet taste of the test item (information provided by the Sponsor). Changes compared to the control group were slight (mean values for relative food consumption in treated groups ranged between 1.04x and 1.15x of controls) and occurred in absence of a dose-response. The delayed growth of high dose males in the presence of higher food intake is indicative for a lower food efficiency, i.e. more food has to be ingested to reach the same growth in the animal’s mass. This effect was less pronounced in females, who had body weight gains in the same range as concurrent controls throughout the whole treatment period.

Serum level of total T4 was significantly reduced in F0-males at 12500 ppm. At the observed magnitude (0.78x of control), a possible relation to treatment with the test item cannot be excluded. However, since the group mean level remained within normal limits, and no treatment-related histopathological changes were observed in the thyroid gland, it was regarded as non-adverse. Treatment in F0-males started at adulthood (11 weeks of age) and continued for 11 weeks. It is important to note that serum T4 levels in males of the next generation (F1) were not affected by life-time treatment with MELAMINE up to 12500 ppm. At the organ level, treatment-related changes were observed in the kidney and urinary bladder in males and/or females starting at 4000 ppm.

A dose-related increased incidence and severity of retrograde nephropathy in the kidney was observed in both males and females at 4000 and 12500 ppm (with correlating irregular/granulated surface and increased weights in males at 12500 ppm; higher urea in plasma of males and females at 12500 ppm; higher potassium and chloride in plasma of females at 12500 ppm). Due to its degenerative character, retrograde nephropathy seen at 4000 and 12500 ppm was regarded as adverse in nature.
Retrograde nephropathy after dietary exposure to MELAMINE has been described earlier (Hard 2009). It appears to be related to reflux phenomenon associated with transient or partial obstruction or precipitation of material in the urinary tract resulting in irritation and perturbation along the nephron with degenerative effects such as tubular basophilia predominating along with dilation of tubules and ducts in the distal nephron. In the present study a similar pattern was noted and main histopathological hallmarks observed were tubular dilation, tubular basophilia, degeneration of tubular cells, infiltration of mononuclear inflammatory cells, fibrosis that in many cases surrounded atrophic tubules, and in some animals amorphous hyaline material was present in some tubules. The lesions extended from the papilla to the cortical areas and severities of the different features varied amongst the animals. The kidneys in males seemed to be more affected than those in females. In the F0-generation, the kidney lesions in males and females at 12500 ppm were seen in combination with diffuse hyperplasia of the urinary bladder urothelium (with correlating thickened/hardened wall and presence of red blood cells in the urine in males at 12500 ppm). Also latter finding in the urinary bladder was considered adverse in nature.

No parental toxicity was observed at 1000 ppm.
No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. mortality/viability, clinical appearance, haematology and coagulation parameters, T4 (females) and TSH thyroid hormone levels (both sexes)).

Reproduction results – F0-generation
No reproductive toxicity was observed up to the highest dose level tested (12500 ppm). Sperm analysis revealed a marked increase in the number of sperm cells with detached head at 12500 ppm. Other sperm morphology parameters investigated were unaffected. Histopathological examination of the testis revealed increased incidence of tubular degeneration/atrophy (up to moderate) at 12500 ppm, with related minimal cellular debris in the epidydimis. This term ‘tubular degeneration/atrophy’ is used as the summative diagnosis when there are combinations of changes in the testicular tubules that include atrophy, degeneration, vacuolation, exfoliation and sperm retention (Ref. 6). The severity of the findings in the present study was generally low and did not affect the fertility of the males in both, the F0-generation and F1-generation (see below). Although degeneration/atrophy may occasionally be observed in control animals (as for example the single affected F0-male in the 1000 ppm treatment group which is regarded as incidental) the incidence in the present study is above background and therefore considered to be test item-related.

No treatment-related changes were noted in any of the other reproductive parameters investigated in this study (i.e. sperm motility parameters, epididymal sperm count, mating and fertility indices, precoital time, number of implantations, estrous cycle, and spermatogenic profiling).

Developmental results – F0-Generation / F1-Generation (pre-weaning)

No developmental toxicity was observed up to the highest dose level tested (12500 ppm). In pups of the F0-generation (i.e. F1-pups), no treatment-related or toxicologically relevant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration of gestation, parturition, live birth, viability and weaning indices, sex ratio, litter size, maternal care, and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels (total T4 in PND 4 and PND 22 pups, and TSH in PND 22 pups), macroscopic examination and brain and spleen weight).

Parental results – F1-Generation (post-weaning)
No treatment-related mortality occurred post-weaning up to 12500 ppm.
From weaning onwards, lower body weights and/or body weight gains were noted for both sexes at 12500 ppm (not always statistically significant). When compared to the concurrent control group changes in mean body weight gain were highest after the first week of treatment (ranging from 0.72-0.75x and 0.70-0.78x of control in males and females, respectively) of Cohorts 1A, 1B, 2A and 3), followed by recovery. After 10 weeks of treatment (Cohorts 1A and 1B), mean body weight was 0.94x and 0.91-0.95x of control in males and females, respectively. At the end of the post-coitum and lactation phase, mean body weights in high dose females (Cohort 1B) were 0.91x and 0.93x of control, respectively. Body weight gain in these females was 0.90x of control (not statistically significant), and remained comparable to controls throughout the lactation phase. Terminal mean body weights were 0.92-0.95x of control for animals of Cohort 1A and Cohort 1B (not statistically significant for Cohort 1A females). A similar trend was observed at the mid dose level of 4000 ppm (both sexes; Cohorts 1A, 1B, 2A and 3), reaching statistical significance on some occasions. However, changes were relatively slight with overall no significant effects on mean values for absolute body weight. Similar to the F0-generation, relative food consumption was higher in F1-males and F1-females of all treated groups (Cohorts 1A, 1B, 2A and 3) when compared to the concurrent control group on many occasions during treatment. During lactation, only the high dose group had a significantly higher food intake (Cohort 1B).

As also water consumption was suspected to be affected in high dose males (based on subjective appraisal), quantitative measurement was introduced for Cohort 1B (both sexes) from Week 6 of premating onwards. In males at 12500 ppm, water intake was higher compared to controls during the premating and mating period. A similar trend was observed in females at 12500 ppm, but changes compared to the control group were less pronounced than in males (combined mean of means: 1.44x and 1.10x of control, for males and females, respectively).

Exposure of the F1-generation to MELAMINE in utero, through nursing during lactation, and via dietary administration following weaning was associated in Cohort 1A with retrograde nephropathy in kidney of males and females starting at 4000 ppm (with correlating irregular/granulated surface in males and females starting at 4000 ppm). Similar to the F0-generation, these findings were considered adverse in nature. Besides an irregular surface, also an increased incidence of pelvic dilation was observed macroscopically in males at 4000 ppm (Cohort 1A) and 12500 ppm (Cohorts 1A and 1B). Although this increased incidence may indirectly be related to the retrograde nephropathy, it is not present in females despite the presence of retrograde nephropathy. Moreover, pelvic dilation is commonly seen as background finding and in most cases unilateral (e.g. an incidence of 3/20 in control females of Cohort 1B of the present study). Therefore, the increased incidence in pelvic dilation was not regarded as test item-related.

No treatment-related effects on the urinary bladder were observed.

No parental toxicity was observed at 1000 ppm.

No treatment-related effects were recorded for developmental parameters in F1-animals, including mortality/viability, clinical signs, balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first estrus, time between vaginal opening and first estrus, clinical pathology parameters (incl. haematology, coagulation, clinical biochemistry and T4/TSH thyroid hormone levels, urinanalysis). Histopathologically, no test-item related effects were noted at stage-dependent qualitative evaluation of spermatogenesis (Cohort 1A), and ovarian follicle and corpora lutea counts (Cohort 1A) and morphology of the female reproductive organs.

No treatment-related changes were observed in the developmental neurotoxicity endpoints tested, including fixed brain weights, brain dimensions, brain histopathology and morphometry (Cohorts 2A and 2B), and functional tests (Cohort 2A; tests included acoustic startle response, detailed clinical observations, rectal temperature, motor activity test, hearing ability, pupillary reflex, foot splay and grip strength), and the learning and memory Biel Maze test.

No treatment-related changes were observed in the developmental immunotoxicity endpoints tested. In the TDAR assay, it was demonstrated that the development of a humoral immune response to an injected antigen (KLH) was unaffected by treatment with MELAMINE. All levels of anti-KLH IgM antibodies in MELAMINE treated animals of Cohort 3 remained within the range of the vehicle control animals. In contrast, the positive control animals of both genders treated with Cyclophosphamide had a decreased group mean value compared to all other groups, and a higher incidence of non-responding animals with anti-KLH IgM levels below LLOQ (4/10 for males and 4/10 for females), thus revealing a relevant degree of immunosuppression caused by the Cyclophosphamide treatment. These results from the TDAR assay (Cohort 3) indicated that the test item did not induce any immunotoxic effect in young Wistar Han rats. In accordance with this, no pathology findings in the lymphoid organs (i.e. histopathology and organ weight) and no test item-related changes in splenic lymphocyte subpopulations (Cohort 1A) were noted.

Reproduction results – F1-Generation (Cohort 1B)

No reproductive toxicity was observed up to the highest dose level tested (12500 ppm). Similar to the F0-generation, a remarkable number of high dose males was observed with an unusual high number of sperm cells with detached head. This finding was considered to be treatment-related. Histopathological examination of the testis revealed an increased incidence and/or severity in tubular degeneration/atrophy (up to moderate; for definition, see above), starting at 4000 ppm, with related cellular debris in the epididymis (up to marked). The severity of the findings in the present study was generally low and did not affect the fertility of the males. Although degeneration/atrophy may occasionally be observed in control animals (as for example the single affected F0-male in the 1000 ppm treatment group which is regarded as incidental) the incidence in the present study is above background and therefore considered to be test item-related.

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. length and regularity of the estrous cycle, sperm motility, epididymal sperm count sperm, spermatogenic profiling, mating and fertility indices, precoital time, number of implantation sites, and histopathological examination of the female reproductive organs).

Developmental results – F1-Generation / F2-Generation (Cohort 1B)

No developmental toxicity was observed up to the highest dose level tested (12500 ppm). In pups of the F1-generation (i.e. F2-pups), no treatment-related or toxicologically relevant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration of gestation, parturition, live birth and viability indices, sex ratio, litter size, maternal care, and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels (total T4 on PND 4), macroscopic examination, and brain, thymus, kidney and spleen weight).

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1, 2 and 3), the following no-observed-adverse-effect level (NOAEL) of MELAMINE were established:

General Toxicity NOAEL: F0-generation and F1-generation 1000 ppm (on average corresponding to 65 mg/kg/day in males and 87 mg/kg/day in females of the F0-generation, and 89 mg/kg/day in males and 93 mg/kg/day in females of the F1-generation). This NOAEL is based on adverse kidney effects in both males and females at 4000 ppm (both generations).

Reproduction Toxicity NOAEL: F0-generation 4000 ppm and F1-generation 1000 ppm (on average corresponding to 268 mg/kg/day in F0 males and 89 mg/kg/day F1 males, based on tubular degeneration/atrophy in the testis with related minimal cellular debris in the epididymis in F0 males at 12500 ppm (833 mg/kg bw/day) and in F1 males at and above 4000 ppm (370 mg/kg bw/day).

Developmental General Toxicity NOAEL: F1-generations and F2-generation at least 12500 ppm (on average corresponding to 1200 mg/kg/day in males and 1227 mg/kg/day in females of the F1-generation), in absence of adverse effects at this dose on developmental parameters.

Developmental Neurotoxicity NOAEL: F1-generation at least 12500 ppm (on average corresponding to 1200 mg/kg/day in males and 1227 mg/kg/day in females of the F1-generation), in absence of adverse effects at this dose on parameters measuring development of the neurological system.

Developmental Immunotoxicity NOAEL: F1-generation at least 12500 ppm (on average corresponding to 1200 mg/kg/day in males and 1227 mg/kg/day in females of the F1-generation), in absence of adverse effects at this dose on parameters measuring development of the immunological system.

ref:

G. C. Hard, G. P. Flake, R. C. Sills (2009) Re-evaluation of Kidney Histopathology from 13-Week Toxicity and Two-Year Carcinogenicity Studies of Melamine in the F344 Rat: Morphologic Evidence of Retrograde Nephropathy. Vet Pathol 46:1248-1257.