Ziram

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Apr 1994 - 30 Jan 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Version / remarks:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 83-6 (Developmental Neurotoxicity Study)

Version / remarks:

not specified

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

adopted in 2001

Deviations:

yes

Remarks:

- Not all required organ weights were determined. Sperm parameters were not assessed for all animals; methodological limitations (limited information on sexua maturation and estrous cyclicity available)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ziram

EC Number:

205-288-3

EC Name:

Ziram

Cas Number:

137-30-4

Molecular formula:

C6H12N2S4Zn

IUPAC Name:

ziram

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Michigan, USA
- Age at study initiation: (P) approximately 6 weeks; (F1) at weaning
- Weight at study initiation: (P) Males: 144 - 218 g; Females: 117 - 174 g
- Housing: in clean, wire-mesh cages or plastic maternity cages with nesting material as follows:
Acclimatisation (F0): individually
Mating (F0): one female and one male animal
Littering (F0): one dam + litter
Lactation (F0): one dam + litter
Following weaning (F1 and F2 maternal females): individually
Pups (F1 and F2): individually
From weaning (F1): by litter
- Diet: Purina® Certified Rodent Chow® #5002 in meal form, ad libitum
- Water: Municipal water from the public supply, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26
- Humidity (%): 20 - 92
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 May 1994 To: 31 Mar 1995

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
An appropriate amount of test article was weighed for each test group into tared glass weighing containers and was added to 5 kg of rodent meal and was mixed for 5 min. The premix was then mixed for 10 min with enough rodent meal (weight/weight) to obtain a sufficient batch (17 kg) of homogeneous diet for the appropriate concentration per test group. Due to the limited stability of the formulated diets at the low and mid dietary concentrations when stored at room temperature, the test article diets were prepared weekly and were stored refrigerated.

Details on mating procedure:

- M/F ratio: 1:1
- Length of cohabitation: Up to 15 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- Any other deviations from standard protocol: Litters were culled to 8 pups/litter on lactation Day 4.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate samples were collected from the top, middle and bottom of the preparations for each group, including the control group, prior to the initiation of dosing. One set of samples was analysed for homogeneity. The second set was combined and stored refrigerated for 10 days and then analysed for stability. A sample from the middle of each dietary concentration (including control), was collected weekly throughout the study and a duplicate sample for study weeks 0, 1, 2 and 3 and once per month during the remainder of the treatment period and were analysed for concentration. Data from the analyses confirmed that the diet preparations were stable for 10 days refrigerated, were homogeneous and contained the designated amounts of test article.

Duration of treatment / exposure:

F0 parents: from study initiation until scheduled sacrifice
F1 parents: from weaning (Day 22) until scheduled sacrifice
F2 pups: from weaning (Day 22) until scheduled sacrifice

Duration of exposure before mating (F0 / F1 parents): 10 weeks

Frequency of treatment:

Continiously via the diet.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

60 ppm

Remarks:

The concentration of test material in the low dose level was increased by 20% above nominal (72 ppm) to compensate losses during storage.
In total this corresponded to:
5 mg/kg bw/day prior breeding and 3 mg/kg bw/day after breeding for (P) males
6 mg/kg bw/day prior breeding and 5 mg/kg bw/day during gestation for (P) females
12 mg/kg bw/day during lactation and 5 mg/kg bw/day after weaning for (P) females
For further details, please refer to "Details on study design".

Doses / concentrations 2

Dose / conc.:

180 ppm

Remarks:

The concentration of test material in the low dose level was increased by 15% above nominal (207 ppm) to compensate losses during storage.
In total this corresponded to:
15 mg/kg bw/day prior breeding and 10 mg/kg bw/day after breeding for (P) males
17 mg/kg bw/day prior breeding and 13 mg/kg bw/day during gestation for (P) females
33 mg/kg bw/day during lactation and 15 mg/kg bw/day after weaning for (P) females
For further details, please refer to "Details on study design".

Doses / concentrations 3

Dose / conc.:

540 ppm

Remarks:

corresponding to:
37 mg/kg bw/day prior breeding and 25 mg/kg bw/day after breeding for (P) males
43 mg/kg bw/day prior breeding and 33 mg/kg bw/day during gestation for (P) females
85 mg/kg bw/day during lactation and 37 mg/kg bw/day after weaning for (P) females
For further details, please refer to "Details on study design".

No. of animals per sex per dose:

30

Control animals:

yes, plain diet

Details on study design:

Due to the purity of the test substance, a correction factor of 1.022 was used for purposes of concentration calculation.

- Actual doses received:
F0 ♂:
5, 15, 37 mg/kg bw/day (prior to breeding)
3, 10, 25 mg/kg bw/day (after breeding)

F0 ♀: 6, 17, 43 mg/kg bw/day (prior to breeding)
5, 13, 33 mg/kg bw/day (gestation)
12, 33, 85 mg/kg bw/day (lactation)
5, 15, 37 mg/kg bw/day (after weaning)

F1 ♂:
6, 18, 46 mg/kg bw/day (prior to breeding)
3, 10, 26 mg/kg bw/day (after breeding)

F1 ♀:
7, 20, 51 mg/kg bw/day (prior to breeding)
5, 13, 32 mg/kg bw/day (gestation)
11, 30, 79 mg/kg bw/day (lactation)
5, 14, 34 mg/kg bw/day (after weaning)

Positive control:

none

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: for appearance, behavior, pharmacotoxic signs, moribundity, mortality and signs of dystocia, prolonged labor, delayed labor or other difficulties during the period of expected parturition.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during treatment and prior to terminal sacrifice for males. Confirmed mated females were weighed on presumed gestation Days 0, 7, 10, 14, and 20. Nursing dams were weighed on Days 1, 4, 7, 14, and 21 post partum. After weaning (day 22) once weekly until scheduled sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: The mean amounts of test substance consumed was calculated from the mean food consumption (g/kg bw/day) and the nominal concentration of the test substance in the diet (ppm).
Food consumption was recorded daily until pairing for all animals. Male food consumption was measured after mating again daily until scheduled sacrifice. Female food consumption was measured daily throughout gestation and lactation period. No food consumption data was obtained during the mating period.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

Oestrous cyclicity (parental animals):

- Daily during mating.

Sperm parameters (parental animals):

A qualitative evaluation of spermatogenesis was conducted on F0 and F1 male rats which were paired, but failed to sire a litter. Immediately following euthanization, spermatozoa were removed from the caudal segment of an incised epididymis and placed in a drop of warm saline on a microscope slide (maintained on a warming tray at approximately 35 °C). Gross morphology, an estimate of sperm numbers and the presence or absence of sperm motility were evaluated by visual estimation. Sperm smears were retained for possible future morphological examination.

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 / F2 offspring:
number and sex of pups, stillbirths, live births, presence of gross anomalies, weight gain, physical or behavioural abnormalities and pup weight. In addition, males were observed for balanopreputial separation beginning on post partum Day 40 and females were observed for vaginal perforation on post partum Day 30.

GROSS EXAMINATION OF DEAD PUPS:
A detailed gross necropsy was performed on any pup dying after lactation day 4 and prior to weaning; tissues were saved for histopathological examination only as deemed necessary by the gross findings.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Yes
The following investigations were used to assess the maturation and behavioural development of the selected F2 pups. These procedures were concluded at 62 days of age. Cumulative litter values included pups which tested positive for the event but were found dead prior to the end of the testing period. The selected F2 pups were randomized into three study replicates to allow for reasonable conduct of the behavioural evaluations. Each dose group and sex were approximately equally represented within a study replicate. Motor activity test, auditory startle test and biel maze swimming trials were performed.

Motor activity:
Motor activity observations were made on 10 rats per sex per group on postnatal Days 13, 17, 21 and 60 (+ 2). The testing of treatment groups was done according to replicate sequence. Data were collected in one minute epochs (test session was 41 minutes in duration) for each animal. Animal placement into the activity cage initiated the data collection process. The first epoch was often incomplete due to the placement of the animal in the activity cage. For this reason, the first minute of data was deleted. Motor activity was divided into two categories: total and ambulatory activity. Total motor activity was defined as a combination of fine motor skills (i.e. grooming; interruption of one or two adjacent photobeams) and ambulatory motor activity (interruption of three or more consecutive photobeams).

Auditory startle test:
An auditory startle response test was performed on 10 rats/sex/group on postnatal Days 22 + 2 and 60 + 2. The box was lined with approximately 2.6 inches of foam padding to establish a sound proof barrier to the external environment. Background noise was emitted by an approximately 6.6 inch round speaker
centered in the bottom of the sound chamber. The startle sound was emitted by an approximately 5.0 inch round speaker centered in the lid of the sound chamber. On the bottom surface of the sound chamber were four weight sensitive platforms that measured the response or movement of the animal to the noise burst. The test session on each day of testing consisted of a five min acclimation period with a background noise level of 70 + 10 db (decibels), followed by 50 presentations of a 50-msec 120 + 10 db short noise burst. Mean latency to peak (msec), response duration (msec), average response (grams) and peak
amplitude (grams) on each block (1-5) of 10 trials were recorded.

Biel maze swimming trials:
Swimming ability and learning and memory recall were assessed in one male and one female pup per litter, when available. The first testing interval initiated for each animal between postnatal Days 19 and 23; the second testing interval initiated for each animal between postnatal Days 58 and 62. Each testing interval was composed of the following three phases. Each animal was tested for swimming ability on the first day of testing and then tested for maze-learning ability on the second through fifth day of testing. On Days 2 and 3 each animal was tested in the Path A direction (forward route through the maze to the escape ramp) and on Days 4 and 5 in the Path B direction (reverse of Path A). These five test days were consecutive. After a three day rest period each animal was tested for memory recall (sixth day of testing) of the Path A and B directions. On Day 1, the straight channel was partitioned from the rest of the maze to prevent access to any other channel. The test animal was placed in the straight channel opposite the escape ramp. The time required to reach the escape ramp was recorded. Each animal was allowed four consecutive trials and an average time was reported as the measure of swimming ability. The maze partition was removed and animals were tested for maze-learning ability on test days 2 through 5. Each test day consisted of four trials per day with a minimum rest period of 1 h between each trial, with the following exceptions. For each trial, the test animal was placed in the start position of the maze and the time required to reach the exit platform was recorded, allowing a maximum of three minutes. If the animal did not escape the maze within the allotted three minutes, the animal was guided through the maze to the goal position and allowed to climb the escape ramp. If the animal deviated from the correct channel during a trial and entered an alternate channel with all four feet, an error was recorded. The mean number of errors and the mean escape time for each test day were considered measures of maze-learning ability. After a three day rest period, each rat was tested for memory recall on a sixth test day by swimming two consecutive Path A trials followed by two consecutive Path trials.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
All F0 adults were euthanized following the selection of the F1 generation and completion of a detailed clinical observation. All F1 adults were euthanized following weaning of the F2 pups. A complete necropsy and selective histopathological examination were conducted.

All surviving non-selected F1 weanlings were euthanized and necropsied with emphasis on developmental morphology on postnatal Day 28. Prior to weaning, 30 F2 pups/sex/group were randomly selected for developmental landmarks and behavioural testing, neuropathology and brain weight measurements. All remaining F2 weanlings were euthanized and necropsied with emphasis on developmental morphology on postnatal Day 22. Selected F2 pups not allocated for neuropathology and brain weight measurements received a complete necropsy on postnatal Day 70.

GROSS NECROPSY
A complete necropsy examination was conducted on all parental animals (F0 and F1) euthanized at termination. The same procedure was performed on randomly selected F2 pups not allocated for neuropathology or brain weight measurements. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities including viscera. Organ and tissue samples from the selected F2 pups were collected and preserved at the postnatal Day 70 necropsy only as deemed necessary by the gross findings.
At the time of necropsy the following F0 and F1 parental tissues and organs were collected: adrenals, aorta, bone with marrow (sternebrae), brain (forebrain, midbrain, hindbrain), coagulating gland, eyes with optic nerve, gastrointestinal tract, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, heart, kidneys, liver (sections of two lobes) lungs (including bronchi, lymph node (mesenteric), ovaries and oviducts, pancreas, peripheral nerve (sciatic), pituitary, prostate, salivary gland, seminal vesicles, skeletal muscle (vastus medialis), skin with mammary gland, spinal cord (cervical), spleen, testes with epididymides and vas deferens, thymus, thyroids with parathyroids, trachea, urinary bladder, uterus with vagina, all gross lesions.

HISTOPATHOLOGY / ORGAN WEIGHTS
The testes, epididymides or ovaries and the brain, pituitary gland, kidneys and liver were weighed fresh for all F0 and F1 parental animals. Absolute weights and organ to final body weight ratios were recorded.
Microscopic tissue evaluations were performed on the following tissues for all F0 and F1 adult animals from the control and high dose groups: cervix, coagulating gland, epididymides, kidneys, liver, ovaries, pituitary gland, prostate, seminal vesicles, testes, uterus, vagina, vas deferens, all internal gross lesions.

Postmortem examinations (offspring):

NEUROPATHOLOGICAL EXAMINATIONS
Brain weights and brain region dissections:
At the scheduled evaluations (postnatal Days 11 and 70), 10 and 16 F2 pups/sex/group, respectively, were selected for neuropathology and/or brain weight measurements. The brain was excised from the skull and weighed for each animal. The brain was then dissected into the following regions: cerebellum, forebrain and hindbrain. Each brain region was weighed, with the following exception. At the postnatal Day 11 evaluation, cerebellum weights were included in the hindbrain weight, due to the size of the cerebellum at this stage of development. Absolute and relative (to final body weight) whole brain weights were reported. Regional brain weights (absolute and relative to whole brain weight) were also presented.

Neuropathology:
At the postnatal Day 11 evaluation, 6 selected F2 pups/sex/group were allocated for neuropathological analysis. A qualitative histopathological examination was performed on the brains from the control and high dose group males and females. At study termination (postnatal Day 70), 6 selected F2 pups/sex/group were euthanized and then perfused in situ for neuropathological analysis. The central and peripheral tissues were dissected and preserved. Brain weights and brain dimensions (length and width) were recorded. Any observable gross changes, abnormal coloration or lesions of the brain and spinal cord were recorded. The following nerve tissues were sampled for a qualitative histopathological examination from males and females in the control and high dose groups:
Central nervous system tissues: Brain - forebrain, center of cerebrum, midbrain, cerebellum and pons and the medulla oblongata, spinal cord - at cervical swellings C3 — C8 and at lumbar swellings T13 - L4, gasserian ganglion/trigeminal nerves, lumbar dorsal root ganglion at T13 - L4, lumbar dorsal root fibers at T13 - L4, lumbar ventral root fibers at T13 - L4, cervical dorsal root ganglion at C3 - C8, cervical dorsal root fibers at C3 — C8, cervical ventral root fibers at C3 — C8, optic nerves, eyes.
Peripheral nervous system tissues: Sciatic nerves (mid-thigh region and at sciatic notch), sural nerves, tibial nerves, peroneal nerves.

Statistics:

STATISTICAL TEST
- Chi-square test with Yates' correction factor
Pup Sex Ratios, Parental Mating and Fertility Indices, Numbers of Stillborn and Dead Pups, Pup Viability Indices

- One-way ANOVA with Dunnett's test
Parental Weekly Body Weights and Weight Changes, Gestation and Lactation Body Weights and Body Weight Changes, Parental Food Consumption, Mean Gestation Length, Pup Body Weights, Absolute and Relative Organ Weights, Live Litter Size

- Kolmogorov-Smimov test ( one-tailed test)
Histopathological findings

Reproductive indices:

Mating and fertility indices were calculated as follows:
Male (Female) Mating Index (%) = (No. of Males (Females) with Evidence of Mating X 100)/Total No. of Males (Females) Used for Mating
Female Fertility Index (%) = (No. of Females with Confirmed Pregnancy X 100)/Total No. of Females Used for Mating
Male Fertility Index (%) = (No. of Males Siring at Least | Litter X 100)/Total No. of Males Used for Mating

Offspring viability indices:

Litter parameters were defined as follows:
Live Litter Size = Total Viable Pups Day 0/No. Litters With Viable Pups Day 0
Viability Index (%) = (Pups Viable on Days | or 4 Before Culling/Pups Viable on Day 0) x 100 (Before Culling)
Viability Index (%) = (Pups Viable on Day (N)/ Pups Viable on Day 4 After Culling where N = (After Culling) (7, 14 or 21)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical findings with relationship to test article administration were apparent.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All F0 parental animals survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Weekly:
72 and 207 ppm dose groups:
Mean weekly body weights and body weight gains in the 72 and 207 ppm group males and females were comparable to the control group values throughout the F0 generation (Weeks 0 - 20). Any changes in mean body weight gains were transient and not interpreted to be adverse effects of test article administration.
540 ppm dose group:
The mean body weight in the 540 ppm group males at Week 0 was comparable to the control group value. The mean body weights at Weeks 1 (following the initial week of test article administration), 2 and 3 were reduced (statistically significant) in comparison to the control group values. Throughout the remainder of the study (Weeks 4 - 20), mean weekly body weights were comparable to the control group values. Mean body weight gain during Week 0 - 1 was reduced in comparison to the control group value (statistically significant). Mean weekly body weight gains were either comparable to or slightly higher than the control group values throughout the remainder of the study (Weeks 1 - 2 through 19 - 20). Increased mean body weight gains in this group during Weeks 4 - 5 and 6 - 7 were statistically significant when compared to the control group values. Mean weekly body weights in the 540 ppm group females prior to breeding (Weeks 1 - 10) and during Weeks 17 - 20 were significantly reduced when compared to the control group values. Mean body weight gains were significantly reduced during Weeks 0 - 1 and 1 - 2. Mean body weight changes females were similar to the control group values during Weeks 2 - 3 through 9 - 10 and 18 - 19 through 19 - 20. A reduced mean body weight gain during Week 6 - 7 was statistically significant when compared to the control group value.

Gestation:
Mean body weights in the 540 ppm group females were statistically significantly reduced in comparison to the control group values throughout gestation (Days 0, 7, 10,14 and 20). Mean body weight gains were similar to the control group values during gestation days 0 - 7, 7 - 10, 14 - 20 and 0 - 20. The mean body weight gain in the high dose group for gestation days 10 - 14 was significantly reduced when compared to the control group value. Mean gestation body weights and body weight gains in the 72 and 207 ppm groups were unaffected by treatment. No other differences between the treated and control group values were statistically significant.

Lactation:
Mean body weights in the 540 ppm group females were significantly reduced when compared to the control group values during lactation days 1, 4, 7 and 14. Mean body weight in this group was comparable to the control group value on lactation day 21. Mean body weight gains were similar to the control group values during lactation days 1 - 4 and 4 - 7. Mean body weight gain in the 540 ppm group during lactation days 7 - 14 was reduced (not statistically significant) when compared to the control group value. During the remainder of lactation and for the overall lactation period (Days 14 - 21 and 1 - 21, respectively), mean body weight gains were significantly increased in comparison to the control group values. Mean lactation body weights and body weight changes in the 72 and 207 ppm groups were similar to the control group values. None of the differences were statistically significant.

For details, please refer to attachment 1 under "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Weekly:
In the 540 ppm group males food consumption was statistically significantly reduced during Week 0 - 1 when compared to the control group value. Food consumption (g/animal/day) in the high dose group males continued to be statistically significantly reduced for Weeks 1 - 2 through 3 - 4; the g/kg/day food consumption values for these weeks were comparable to the control group values. Food consumption values were comparable to the control group values during Weeks 4 - 5 through 7 - 8. Significantly reduced food consumption values were noted during Weeks 8 - 9 through 10 - 11 when compared to the control group values, with the exception of the Weeks 8 - 9 and 9 - 10 g/kg/day values which were comparable to the control group values. After the breeding period (Weeks 13 - 14 through 19 - 20) food consumption were comparable to the control group values, with the following exceptions. The g/animal/day values for Weeks 14 - 15, 18 - 19 and 19 - 20 were significantly reduced in comparison to the control group values. Food consumption in the 540 ppm group females was significantly reduced during Week 0 - 1 in comparison to the control group value. Throughout the remainder of the F0 generation (Weeks 1 - 2 to 10 - 11, 18 - 19 and 19 - 20, only g/animal/day) food consumption values in the high dose group females were significantly reduced in comparison to the control group values. Food consumption values in the 207 ppm group males were comparable to the control group values throughout the F0 generation. Food consumption in the 207 ppm group females was statistically significantly reduced during Week 0 - 1 when compared to the control group values. However, the differences were slight and no adverse effect on body weight gain was observed during this interval. In addition, a similar effect was not reproduced in the 207 ppm group males. Therefore, no relationship to the test article was evident. Throughout the remainder of the F0 generation (Weeks 1 - 2 to 10 - 11, 18 - 19 and 19 - 20), food consumption values in the mid dose group females were comparable to the control group values. Food consumption values in the 72 ppm group males and females were comparable to the control group values throughout the F0 generation. None of the differences were statistically significant.

Gestation:
Food consumption in the 540 ppm group females was reduced (usually significant) throughout gestation (Days 0 - 7, 7 - 10, 10 - 14, 14 - 20 and 0 - 20) when compared to the control group values. In the 207 ppm group females food consumption was slightly reduced during gestation days 0 - 7; (only g/animal/day) statistically significant. Food consumption values in the mid dose group females during gestation Days 7 - 10 and 10 - 14 were comparable to the control group values. During the remainder of gestation and for the overall gestation period (Days 14 - 20 and 0 - 20, respectively), food consumption was significantly reduced in comparison to the control group values. Food consumption in the 72 ppm group females was comparable to the control group values throughout gestation. None of the differences were statistically significant.

Lactation:
Food consumption in the 540 ppm group females was comparable to the control group values during lactation Days 1 - 4. Reduced food consumption values were noted during lactation Days 4 - 7 (only g/animal/day) and 7 - 14; these differences from the control group values were significant. Throughout the remainder of lactation (Days 14 - 21), food consumption values were comparable to the control group values. When the overall lactation period (Days 1 - 21) was evaluated, g/animal/day food consumption in the high dose group females was slightly, but statistically significantly reduced in comparison to the control group value. Food consumption values in the 207 ppm group females were similar to the control group values throughout gestation. Observed reductions were slight and not sustained throughout gestation; no adverse effect of treatment was apparent. Food consumption in the 72 ppm group females was similar to the control group values throughout lactation. No statistically significant differences were observed.

For details, please refer to attachment 2 under "Overall remarks, attachments".

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study: reproductive performance (mating and fertility indices), oestrous cyclicity during mating, spermatogenesis (only on F0 and F1 male rats which were paired, but failed to sire), litter number and sex of pups, stillbirths, live births, presence of gross anomalies, weight gain, physical or behavioural abnormalities and pup weight. For details, please refer to the respective result fields

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No microscopic lesions attributed to test article administration were observed in any 540 ppm F0 parental tissues upon histopathological examination. The lesions observed in the treated group males and females were noted infrequently and/or similarly in the control group.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Microscopic examination of gross lesions noted at the scheduled necropsy in the 540 ppm group did not reveal any effects of treatment.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Qualitative evaluations performed on 4, 4, 3 and 2 males failing to sire a litter in the control, 72, 207 and 540 ppm groups, respectively, did not reveal any test article-related changes in gross sperm morphology, apparent relative numbers or motility. One male in the control group had non-motile, abnormal sperm in the left epididymis. One male (each in control group and 207 ppm group) had motile, abnormal sperm in the left epididymis. All other examined males had normal, motile sperm in the epididymides.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Reproductive performance:
Reproductive performance was unaffected by test article administration at concentrations of 72, 207 and 540 ppm. Fertility and mating indices for the F0 males and females were comparable in all dose groups. Males which did not sire a litter numbered 4, 4, 3 and 2 in the control, 72, 207 and 540 ppm groups, respectively. Three females in the control and two females each in the 72 and 207 ppm groups respectively, had evidence of mating but did not deliver; all females were nongravid. One female each in the control, 72, 207 ppm group and two females in the 540 ppm group, had no evidence of mating. In the control and 207 ppm group females each delivered a litter and the 72 and 504 ppm group females were nongravid. The mean numbers of days between pairing and coitus in the treated groups were comparable to the control group value.

For details, please refer to attachment 6 under "Overall remarks, attachments".

Gestation length and parturition:
The mean lengths of gestation were comparable between the F0 treated groups and the control group. None of the observed differences were statistically significant within the reproductive historical control data. No signs of dystocia were noted.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

207 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 10 mg/kg bw/day (post-breeding intake; lowest value throughout study period)

2

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

540 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: corresponding to ca 25 mg/kg bw/day (lowest value throughout study period)

3

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 540 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to the highest dose level tested

Remarks on result:

other: corresponding to ca. 25 mg/kg bw/day (lowest value throughout study period)

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Description (incidence and severity):

No test article-related clinical signs were observed in animals at any concentration.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All F1 parental animals survived to the scheduled necropsies.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Weekly
Mean weekly body weights in the 540 ppm group males were statistically significant reduced when compared to the control group beginning with administration of the test diets at Week 18 and continuing through Week 39 (scheduled necropsy). Mean body weight gains in males were reduced from Weeks 18 - 19 through 21 - 22; the differences from the control group for Weeks 18 - 19, 20 - 21 and 21 - 22 were statistically significant. Mean body weight gains were statistically significantly reduced during Weeks 33 - 34 and 35 - 36 and comparable to the control group values during Weeks 34-35. Mean body weights were comparable to the control group values throughout the remainder of the F1 generation (Weeks 36 - 37 to 38 - 39). Mean weekly body weights for the 540 ppm group females were statistically significantly reduced prior to breeding (Weeks 18 - 30) and during Weeks 38 - 39. Mean body weight gain was statistically significantly reduced during Week 18 - 19 in comparison to the control group value. During Weeks 19 - 20 through 22 - 23, mean body weight gains were comparable to the control group values. Mean body weight gains were statistically significantly reduced during Weeks 23 - 24 and 24 - 25 when compared to the control group values. Throughout the remainder of the period prior to breeding (Weeks 25 - 26 to 29 - 30) and during Week 38 - 39, mean body weight gains in the 540 ppm group females were comparable to the control group values. Mean weekly body weights and body weight gain in the 207 ppm group males were comparable to the control group values throughout the F1 generation (Weeks 18 through 39). One isolated reduction near the end of treatment of the F1 mid dose males was not observed in the F0 generation and was not attributed to test article administration. Mean weekly body weights and body weight gains in the 207 ppm group females were comparable to the control group values throughout the F1 generation. None of the differences were statistically significant. Mean weekly body weights and body weight gain in the 72 ppm group males were comparable to the control group values throughout the F1 generation. One isolated reduction near the end of treatment was not interpreted to be related to administration of the test article. Mean weekly body weights and body weight gains in the 72 ppm group females were comparable to the control group values throughout the F1 generation. None of the differences were statistically significant.

Gestation
Mean body weights in the 540 ppm group females were reduced (statistically significant) throughout gestation. Mean body weight gains in the high dose group females were comparable to the control group values during gestation Days 0 - 7, 7 - 10 and 10 - 14. During the remainder of gestation and for the overall gestation period (Days 14 - 20 and 0 - 20, respectively), mean body weight gains in the 540 ppm group females were statistically significantly reduced in comparison to the control group values. Mean body weights and body weight gains in the 207 ppm group females were similar to the control group values throughout gestation. One single reduction during gestation was not interpreted to be an adverse effect of treatment. Mean gestation body weights and body weight gains in the 72 ppm group females were unaffected by treatment. None of the differences from the control group values were statistically significant.

Lactation
Mean body weights in the 540 ppm group females were statistically significantly reduced in comparison to the control group values throughout lactation. Mean body weight gains in the 540 ppm group throughout lactation were similar to the control group values. Mean lactation body weights and body weight gains in the 72 and 207 ppm groups were unaffected by treatment.

For details, please refer to attachment 3 under "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Weekly
Food consumption, evaluated as g/animal/day, was significantly reduced in the 540 ppm group males throughout the F1 generation (Weeks 18 - 19 to 30 - 31 and 33 - 34 to 38 - 39). G/kg bw/day food consumption values in the high dose group males were slightly reduced during Weeks 18 - 19 and 19 - 20; the difference from the control group for Week 19 - 20 was significant. Throughout the remainder of the F1 generation (Weeks 20 - 21 to 30 - 31 and 33 - 34 to 38 - 39), g/kg bw/day food consumption values in the 540 ppm group males were comparable to the control group values, with the following exception. The Week 35 - 36 g/kg bw/day food consumption value in the high dose group males was statistically significantly reduced in comparison to the control group values. Food consumption, evaluated as g/animal/day, in the 540 ppm group females was reduced (generally significant) throughout the F1 generation (Weeks 18 - 19 to 30 - 31 and 33 - 34 to 38 - 39). G/kg bw/day food consumption values in the high dose group females were comparable to the control group values throughout the F1 generation. Food consumption (g/animal/day and g/kg bw/day) in the 207 ppm group males was slightly reduced during Weeks 18 - 19 and 19 - 20; the g/animal/day differences from the control group values were statistically significant. Throughout the remainder of the F1 generation (Weeks 20 - 21 to 30 - 31 and 33 - 34 to 38 - 39), food consumption values were comparable to the control group values, with the exception of a slightly increased g/kg/day value during Week 25 - 26 (statistically significant). Food consumption values (g/animal/day and g/kg bw/day) in the 207 ppm group females were comparable to the control group values throughout the F1 generation (Weeks 18 - 19 to 30 - 31 and 33 - 34 to 38 - 39), with the following exceptions. Reduced g/animal/day (Week 38 - 39) and g/kg/day (Week 30 - 31) values were statistically significant when compared to the control group values. Food consumption values (g/animal/day and g/kg bw/day) in the 72 ppm group males and females were comparable to the control group values throughout the F1 generation. Isolated reductions were not attributed to test article administration. Food consumption values (g/animal/day and g/kg bw/day) in the 72 ppm group females were comparable to the control group values throughout the F1 generation. Isolated reductions were not attributed to test article administration.

Gestation
Food consumption in the 540 ppm group females was reduced throughout gestation (Days 0 - 7, 7 - 10, 10 - 14, 14 - 20 and 0 - 20); all of the differences from the control group values were statistically significant, with the exception of the gestation Day 0 - 7 g/kg bw/day value (not significant). Mean food consumption values in the 207 ppm group females were similar to the control group values during gestation. Single reductions in the mid dose group females were slight and were not sustained throughout gestation; no adverse effect of treatment was apparent. Food consumption in the 72 ppm group females was unaffected by test article administration throughout gestation. None of the differences from the control group values were statistically significant.

Lactation
Food consumption, evaluated as g/animal/day, in the 540 ppm group females was reduced (statistically significant) throughout lactation when compared to the control group values. When evaluated on a g/kg bw/day basis, food consumption in the high dose group females was comparable to the control group values during lactation Days 1 - 4 and 4 - 7 and reduced during lactation Days 7 - 14, 14 - 21 and 1 - 21. Food consumption in the 207 ppm group females was comparable to the control group values during lactation. Single reductions in the mid dose group were slight and were not sustained throughout lactation; no adverse effect of treatment was evident. Food consumption (g/animal/day and g/kg bw/day) in the 72 ppm group females was unaffected by treatment throughout lactation. One isolated, slight decrease in the low dose group females was not interpreted to be an adverse effect of test article administration.

For details, please refer to attachment 4 under "Overall remarks, attachments".

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study: reproductive performance (mating and fertility indices), oestrous cyclicity during mating, spermatogenesis (only on F0 and F1 male rats which were paired, but failed to sire), litter number and sex of pups, stillbirths, live births, presence of gross anomalies, weight gain, physical or behavioural abnormalities and pup weight. For details, please refer to the respective result fields.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test article-related effects on mean absolute and relative organ weights were observed in the F1 treated males and females. Mean absolute brain weight in the 540 ppm group males was statistically significantly lower than the control group value. However, the difference was slight (4%) and mean brain weight relative to final body weight in the high dose group males was increased in comparison to the control group value; no relationship to treatment was apparent. Mean absolute kidney weights in the mid dose group females and high dose group males and females were statistically significantly lower than the control group values. However, no corresponding decreases in mean kidney weights relative to final body weight were observed in these groups and no adverse effect of treatment was evident. Other statistically significant differences occurred when relative organ weights were compared to the control group values. These changes were not accompanied by corresponding absolute organ weight changes and were not attributed to test article administration.

Gross pathological findings:

no effects observed

Description (incidence and severity):

All F1 parental animals survived to the scheduled necropsy; no test article-related macroscopic lesions were noted. Macroscopic findings were limited to singular or infrequent occurrences and/or were noted similarly in the control group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No microscopic lesions attributable to test article administration were observed in any 540 ppm F1 parental tissues upon histopathological examination. The lesions observed in the treated group males and females were noted infrequently or similarly in the control group. Microscopic examination of gross lesions noted at the scheduled necropsy in the 540 ppm group did not reveal any effects of test article administration.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Qualitative evaluations performed on 7, 6, 4 and 1 males failing to sire a litter in the control, 72, 207 and 540 ppm groups, respectively, did not reveal any test article-related changes in gross sperm morphology, apparent relative numbers or motility. One male each in the 72 and 207 ppm groups, had normal sperm with decreased motility. One male in the 207 ppm group had no sperm present in either epididymis. This male had soft and small testes upon macroscopic examination. All other examined males had normal, motile sperm in the epididymides.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Reproductive performance:
Reproductive performance was unaffected by test article administration at concentrations of 72, 207 and 540 ppm. Fertility indices for the F1 males and females were as well unaffected by the test article administration. Male and female mating indices were comparable within all dose groups. Males which did not sire a litter numbered 7, 6, 4 and 2 in the control, 72, 207 and 540 ppm groups, respectively. Females which had evidence of mating but did not deliver numbered 6, 5, 2 and 2 in these respective groups; all were nongravid. One and two females in the control and 207 ppm groups, respectively, had no evidence of mating and were nongravid. One, two and one females in the control, 72 and 540 ppm groups, respectively, had no evidence of mating but delivered a litter. The mean numbers of days between pairing and coitus in the treated groups were similar to the control group value.

For details, please refer to attachment 6 under "Overall remarks, attachments".

Gestation length and parturition:
The mean lengths of gestation were comparable between the F1 treated groups and the control group. No differences were statistically significant. The mean gestation lengths in the 72, 207 and 540 ppm groups were within reproductive historical control data. No signs of dystocia were noted.

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

207 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 10 mg/kg bw/day (lowest value throughout study period)

2

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

540 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: corresponding to 26 mg/kg bw/day (lowest value throughout study period)

3

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 540 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: corresponding to 26 mg/kg bw/day (lowest value throughout study period)

Target system / organ toxicity (P1)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The numbers of dead pups (F1) on lactation Day 0 were not affected by treatment at any dose level. Pup viability indices (F1) were comparable to the values in the control group, with one exception on lactation Day 4 (before selection) the pup viability index in the 72 ppm group was statistically significantly lower than the control group value. As no dose-relationship was observed at the higher dose levels, this decrease was not attributed to treatment. No other statistically significant differences were observed.
Pups which were found dead during postnatal Days 1 - 28 numbered 5, 17, 7 and 3 in the control, 72, 207 and 540 ppm groups, respectively. In these same respective dose groups, 5, 7, 5 and 2 pups, respectively, were missing and presumed cannibalized.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean F1 pup body weights in the 540 ppm group were comparable to the control group values for lactation Days 1, 4 (before and after selection) and 7. Mean pup body weight on lactation Day 14 in the 540 ppm group (28.8 g) was statistically significantly reduced in comparison to the control group value (31.4 g). Although the high dose group value was within the range of values noted in the historical control data (23.7 - 36.8 g), this reduction was interpreted to be treatment-related in view of the corresponding sustained reductions in the F2 pups. Mean pup body weight in the high dose group on lactation Day 21 continued to be slightly reduced (not statistically significant) when compared to the control group value. No adverse effects on mean pup body weights were apparent at concentrations of 72 and 207 ppm; no statistically significant differences were observed.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not specified

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At necropsy:
At the postnatal day 28 scheduled necropsy, no treatment-related internal findings were observed in any dose group. One F1 pup in each of the control and 72 ppm groups had dilated renal pelves. One pup in the control group had a distended and gas-filled stomach And another pup dark red contents in the jejunum. One pup in the 207 ppm group had enlarged iliac lymph nodes. Malformations were noted for single pups in the control and 207 ppm groups. One pup in the control group had unilateral anophthalmia. Situs inversus was noted for one pup in the 207 ppm group.

Pups found dead during postnatal period:
With the exception of the presence or absence of milk in the stomach, remarkable necropsy findings for pups found dead during the postnatal period were as follows. One pup in the 207 ppm group had a distended bladder with red fluid contents and evidence of mechanical trauma (hemorrhaging of the brain).
Two other pups in this litter had dark red contents in the stomach and one other pup had multiple white areas on the liver.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Litter size:
Mean F1 live litter size in the 540 ppm group (12.0 pups) was slightly reduced (statistically significant) in comparison to the concurrent control group value (13.6 pups). However, the high dose value was within the range of values noted in the reproductive historical control data (11.7 - 15.9 pups) and this effect was not reproduced in the F2 litters; thus, no relationship to treatment was evident. Mean live litter sizes in the 72 and 207 ppm groups were comparable to the control group value.

Sex ratio:
F1 pup sex ratios were not adversely affected at any dose level. The pup sex ratios in the 207 and 540 ppm groups were slightly skewed toward females when compared to the control group sex ratio; the difference for the 540 ppm group was statistically significant. However, these changes were not interpreted to be adverse effects of treatment as the control group pup sex ratio was skewed toward males. In addition, sex ratios which were skewed toward females at a similar level have been previously observed in the historical control data. The pup sex ratio in the 72 ppm group was comparable to the control group ratio.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

207 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 10 mg/kg bw/day (lowest value throughout study period)

2

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

540 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: corresponding to 25 mg/kg bw/day (lowest value throughout study period)

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The numbers of dead pups on lactation Day 0 were unaffected by treatment at concentrations of 72, 207 and 540 ppm. Pup viability indices were comparable to the values noted in the control group; no statistically significant differences were observed.
Pups which were found dead and available for necropsy between postnatal days 1-21 numbered 6, 6, 3 and 4 in the control, 72, 207 and 540 ppm groups, respectively. In these same dose groups, 1, 2, 3 and 5 pups, respectively, were missing and presumed cannibalized.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean pup body weights in the 540 ppm group were slightly reduced (generally statistically significant) on lactation Days 1, 4 (before and after selection), 7, 14 and 21 in comparison to the control group values. Mean body weights in the 540 ppm group F2 male and female pups selected for neuropathological evaluation on postnatal day 70 continued to be reduced (statistically significant) throughout the remainder of the study (Weeks 38 - 44). Mean pup body weights in the 207 ppm group were comparable to the control group values throughout lactation and the remainder of the study. One single observed increase was not interpreted to be an adverse effect of test article administration. No adverse effects were apparent on mean pup weights at a concentration of 72 ppm.

For details, please refer to attachment 5 under "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Balanopreputial separation:
Balanopreputial separation was not affected in any of the F2 pups. All male pups (100%) were observed with balanopreputial separation by postnatal day 50.

Vaginal perforation:
Vaginal patency was not affected in any of the F2 pups. All female pups (100%) had vaginal opening by postnatal day 40.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

For details on neuropathology, please refer to "Details on results (F2)).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At necropsy:
At the postnatal day 22 scheduled necropsy, no macroscopic findings in correlation with parental treatment were observed in any dose level.
No test article-related macroscopic lesions were noted at the postnatal day 70 necropsy examination of selected F2 pups which were not allocated for neuropathology or brain weight measurements. Internal findings noted in the treated groups were limited to occurrences in one or two animals per test group and/or were noted similarly in the control group.

Pups found dead during postnatal period:
The only remarkable necropsy findings noted for pups found dead during the postnatal period were the presence or absence of milk in the stomach. One selected F2 pup in the 540 ppm group died postnatal day 35 due to mechanical trauma. Internal findings noted for this animal were skull fracture (frontal and parietal, bilateral), associated hematoma on dorsal head and brain damage due to the skull fracture. This female also had red matting on the external nasal area.
For details on neuropathology, please refer to "Details on results (F2)).

Histopathological findings:

not examined

Description (incidence and severity):

For details on neuropathology, please refer to "Details on results (F2)).

Other effects:

not examined

Description (incidence and severity):

Litter size:
Mean F2 live litter sizes in the 72, 207 and 540 ppm groups were comparable to the control group value. None of the differences were statistically significant.

Sex ratio:
F2 pup sex ratios were not adversely affected. No statistically significant differences between the control and treated group values were observed.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Motor activity:
Motor activity (total and ambulatory) was comparable between all study groups at days 13, 17, 21 and 60. No effects were observed which could be related to maternal treatment.

Auditory startle test:
No treatment-related trends were apparent at any concentration on responses to the auditory startle test (peak amplitude, latency to peak, response duration and average response). Differences between the treated and control group values were not observed in a dose-related manner and were not interpreted to be adverse effects of test article administration.

Biel maze swimming trials:
No adverse effects on swimming ability, learning and recall were noted in the treated groups when compared to the control group values.
For details on neuropathology, please refer to "Details on results (F2)).

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

Brain weights and brain measurement:
No adverse effects of test article administration were apparent on mean absolute and relative (to final body and whole brain weights) F2 pup brain, forebrain and hindbrain (including cerebellum) weights when evaluated on postnatal day 11. None of the differences between the control and treated group values were statistically significant. The only statistically significant difference from the control group on postnatal day 70 was a slightly increased mean brain weight relative to final body weight in the 540 ppm group males. However, the difference was slight (8%) and was attributed to the significantly lower mean final body weight in the high dose group males; no relationship to treatment was evident. Mean brain length and width measurements for the F2 males and females at postnatal day 70 were unaffected by treatment of the parental animals.

Histomorphological examinations:
No remarkable changes were apparent at the qualitative histomorphological examination of brains in the F2 control and 540 ppm group male and female pups on postnatal day 11. No microscopic lesions attributed to the administration of the test substance were observed in any central or peripheral nervous system tissues upon histopathological examination on postnatal day 70. Digestion chambers and swollen axons in the lumbarventral root fibers were observed for one control group male. Swollen axons in the lumbar dorsal root fibers were noted for another control group male. Digestion chambers were observed for two 540 ppm group males (sciatic nerve) and one control group female (peroneal nerve). Spontaneous nerve fiber degeneration characterized by digestion chambers has been previously noted in the central and peripheral nervous systems of control and treated rats and should not be considered uncommon.

Effect levels (F2)

1

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F2

Effect level:

207 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 10 mg/kg bw/day (lowest value throughout study period)

2

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Generation:

F2

Effect level:

540 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: corresponding to 25 mg/kg bw/day (lowest value throughout study period)

3

Key result

Dose descriptor:

NOAEL

Remarks:

neurotoxicity

Generation:

F2

Effect level:

>= 540 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse neurotoxic effect observed at the highest dose level tested

Remarks on result:

other: corresponding to 25 mg/kg bw/day (lowest value throughout study period)

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 7.8.1-A1        Body weight changes in reproductive toxicity study
Parameter	Genera­tion	Controls		60 ppm		180 ppm		540 ppm		Dose-response +/–
		♂	♀	♂	♀	♂	♀	♂	♀	♂	♀
Body weight [g]
average weight on gestation Day 14	F0 dams	/	348.4	/	338.6	/	356.0	/	343.8	/	−
	F1 dams	/	357.1	/	339.1	/	344.7	/	316.7*	/	−
average pup weight on lactation Day 21	F1 pups	44.8	42.8	44.6	41.8	46.3	43.5	40.2*	37.7*	−	−
	F2 pups	40.7	39.5	41.0	39.3	44.1	41.2	38.5	36.9	−	−
* statistically significant different from control p </= 0.05

Applicant's summary and conclusion

Conclusions:

The study was conducted under GLP conditions, according to EPA OPP 83-4 and similar to OECD 416. The study is considered reliable with restrtictions due to methodological limitations. This study was designed to investigate the potential adverse effects of the test substance on reproductive capabilities in two generations of rats (F0 and F1) and the potential of the test substance to cause functional and morphological changes to the nervous system of the developing rat (F2 litters) following continuous treatment of the F0 and F1 generations. In conclusion, parental toxicity in the F0 and F1 generations was exhibited at a dose level of 540 ppm by inhibition of body weight gain and reduced food consumption. No parental toxicity was observed at doses of 72 and 207 ppm. Reproductive performance was unaffected by test article administration at the 72, 207 and 540 ppm dose levels. Neonatal toxicity was expressed at 540 ppm by slightly reduced pup body weights (F1 and F2). No neonatal toxicity was observed at 72 and 207 ppm. Based on the results of this study, a dose of 207 ppm (corresponding to approximately 10 mg/kg bw/day) was considered to be the NOAEL for parental systemic toxicity, 207 ppm (corresponding to approximately 10 mg/kg bw/day) was considered to be the NOAEL for neonatal toxicity and 540 ppm (corresponding to approximately 25 mg/kg bw/day) was considered to be the NOAEL for reproductive and developmental neurotoxicity.