Ziram

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Key, report number 480-1-04-26106, EOGRTS (rat): 

NOAEL P0 (general toxicity): 70 ppm (corresponding to 5.41 – 11.65 mg/kg bw/day in males and females)

LOAEL P0 (general toxicity): 200 ppm (corresponding to 14.89 – 31.72 mg/kg bw/day in males and females)

NOAEL P0 (fertility, reproductive toxicity): ≥ 550 ppm (corresponding to 38.24 – 106.02 mg/kg bw/day in males and females)

NOAEL P1 (general toxicity, cohort 1A): 70 ppm (corresponding to 7.19 – 8.87 mg/kg bw/day)

LOAEL P1 (general toxicity, cohort 1A): 200 ppm (corresponding to 19.69 – 23.78 mg/kg)

NOAEL P1 (fertility, reproductive toxicity, cohort 1A): ≥ 650 ppm (corresponding to 68.81 – 83.19 mg/kg bw/day)

NOAEL P1 (general toxicity, cohort 1B): 70 ppm (corresponding to 5.95 – 13.04 mg/kg bw/day)

LOAEL P1 (general toxicity, cohort 1B): 200 ppm (corresponding to 15.73 – 36.03 mg/kg bw/day)

NOAEL P1 (fertility, reproductive toxicity, cohort 1B): ≥ 650 ppm (corresponding to 52.07 – 120.91 mg/kg bw/day)

NOAEL F1/F2 (developmental toxicity): 200 ppm

LOAEL F1/F2 (developmental toxicity): 650 ppm

Key, report number WIL-223003, 2-generation (rat): 

NOAEL P0 (general toxicity): 207 ppm (corresponding to 10 mg/kg bw/day post-breeding intake; lowest value throughout study period)

LOAEL P0 (general toxicity): 540 ppm (corresponding to ca 25 mg/kg bw/day; lowest value throughout study period)

NOAEL P0 (fertility): ≥ 540 ppm (corresponding to ca 25 mg/kg bw/day; lowest value throughout study period)

NOAEL P1 (general toxicity): 207 ppm (corresponding to 10 mg/kg bw/day post-breeding intake; lowest value throughout study period)

LOAEL P1 (general toxicity): 540 ppm (corresponding to ca 25 mg/kg bw/day; lowest value throughout study period)

NOAEL P1 (fertility): ≥ 540 ppm (corresponding to ca 25 mg/kg bw/day; lowest value throughout study period)

NOAEL F1 (developmental toxicity): 207 ppm (corresponding to 10 mg/kg bw/day post-breeding intake; lowest value throughout study period)

LOAEL F1 (developmental toxicity): 540 ppm (corresponding to ca 25 mg/kg bw/day; lowest value throughout study period)

NOAEL F2 (developmental toxicity): 207 ppm (corresponding to 10 mg/kg bw/day post-breeding intake; lowest value throughout study period)

LOAEL F2 (developmental toxicity): 540 ppm (corresponding to ca 25 mg/kg bw/day; lowest value throughout study period)

NOAEL F2 (neurotoxicity): ≥ 540 ppm, corresponding to ca 25 mg/kg bw/day; lowest value throughout study period)

Conclusion: No specific effects on fertility were observed.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Version / remarks:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPP 83-6 (Developmental Neurotoxicity Study)

Version / remarks:

not specified

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

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

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.

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".

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".

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

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

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: 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.

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)

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)

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)

Key result

Critical effects observed:

no

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: 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.

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)

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)

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)

Key result

Critical effects observed:

no

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.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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)

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)

Key result

Critical effects observed:

no

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.

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:

not examined

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.

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)

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)

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)

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Toxicity to reproduction was assessed in rats according to OECD TG 443 (EOGRTS, RL1) and OECD TG 416 (2-generation study, RL2) under GLP conditions. The studies are considered of reliable quality and validity, and to fulfill the criteria of key studies and thus, are sufficiently conclusive for the assessment of the present endpoint.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Toxicity to reproduction was assessed in rats according to OECD TG 443 (EOGRTS, RL1) and OECD TG 416 (2-generation study, RL2) under GLP conditions. The studies are considered of reliable quality and validity, and to fulfill the criteria of key studies and thus, are sufficiently conclusive for the assessment of the present endpoint.

 

Reproductive toxicity studies via the oral route

Toxicity to reproduction was assessed in rats according to OECD TG 443 (EOGRTS, RL1) and OECD TG 416 (2-generation study, RL2) under GLP conditions.

Extended One-Generation study

An extended one-generation study was performed under regulation (EC) No 1107/2009 to meet the EFSA request for additional information on endocrine properties.

The Extended One-Generation study (EOGRTS, Report number 480-1-04-26106, according to OECD TG 443 and CGLP) was conducted to evaluate specific life stages, to evaluate the potential adverse effects on the systemic, fertility, reproductive and developmental toxicity focused endpoints arising due to the repeated daily administration of the test substance for two successive generations. Male and female Wistar rats were exposed via diet to 0, 70, 200 and 550/650 ppm (parent generation (P)) or 650 ppm (first filial (F1) generation). Briefly, males (P and F1) were treated seventy-one (71) days before mating, during mating and post-mating period (until terminal sacrifice). Females (P and F1) were treated seventy-one (71) days before mating, the variable time to conception, pregnancy duration, and twenty-two (22) days after delivery. The mean percent test substance recovery obtained for the test diet was within the acceptance level of ±20% of the nominal concentration, demonstrating that the exposure concentrations were as intended by the study plan and the %CV was less than 20, suggesting that the test substance was homogeneously distributed in the diets.

In the parental P0 generation, no mortality or morbidity was observed in rats treated with the test substance at dietary concentrations up to 650 ppm. Statistically, a significant decrease in mean body weights, body weight gain, and food consumption of male and female rats was observed at 650 ppm dose level. The following parameters were found to be comparable between rats from the treated groups and the control group; oestrous cycle length and the number of normal oestrous cycles, sperm parameters, pre-coital interval, fertility, mating, gestation, and parturition indices, percentage of pregnant rats, duration of the gestation, the mean number and percent of pre- and postnatal losses, and the mean number of implants. Statistically, a significant decrease in serum T4 level of P0 female rats was observed in 200 and 650 ppm dose groups. Effects were not related to the test substance treatment in absence of alterations in TSH and T3 and also absence of lesions in histopathology of the thyroid glands. No effect on thyroid weights was observed. Further, a statistically significant increase in absolute and/or relative weight of the spleen was observed. This alteration was well correlated with the histopathological finding (extramedullary hematopoiesis (EMH), erythroid) observed in the spleen in the 200 and 650 ppm dose groups in a dose-dependent manner and considered as adverse effects related to the test substance treatment. Test substance treatment at the 650 ppm dose level was associated with a decrease in RBC in both sexes and haemoglobin and haematocrit in females. These effects led to an increase in reticulocyte count, MCV and MCH. All these alterations, except MCV and MCH, were also noted at the 200 ppm dose level with less severity and consistency. Treatment with the test substance at 200 ppm and 650 ppm dose levels led to a decrease in the total protein. A decrease in total protein was mainly owed to a decrease in the albumin level effects were also observed in the mid dose group. The macroscopic examination did not reveal any treatment-related lesion in parent rats. Histopathological examination revealed treatment-related lesions in the spleen (EMH, erythroid) and bone marrow femur (increased cellularity of erythroid cells) at 650 and 200 ppm dose levels.

In F1 pups, the following observations were made: 

No treatment-related clinical signs were observed in pups up to the dietary concentration of 650 ppm. Statistically, a significant decrease in mean body weights and body weight gain of male pups, female pups, and the composite of male and female pups were observed at the 650 ppm dose level. The following parameters in treated F1 pups were comparable to those observed in the control group; mortality index, live birth index, survival index, lactation index, litter size, male sex ratio, body temperature, anogenital distance, physical and sexual development landmarks, macroscopic and microscopic findings. Statistically, a significant decrease in serum T4 level of F1 male pups (PND-22) was observed in 200 and 650 ppm dose groups. As the values were within the range of historical control data and no effect on thyroid weight, these changes were considered as incidental. Statistically, a significant decrease was noted in the terminal body weight of pups in 650 ppm dose group. In addition, treatment-related decreases were also noted in absolute weights of the liver, spleen, brain and thymus of the high dose group. However, consistency was observed more in females than males.

Cohort 1A Rats:

No mortality or morbidity was observed in rats treated with the test substance at up to 650 ppm dose level. Statistically, a significant decrease in mean body weights, body weight gain, and food consumption of male and female rats was observed at the 650 ppm dose level. The following parameters were found to be comparable between treated rats and the control group, oestrous cycle length and the number of the normal oestrous cycle, sperm parameters, and splenic lymphocyte subpopulation. The splenic lymphocyte subpopulation of male and female rats was comparable with that of the control group. Serum T3, T4, and TSH levels of Cohort 1A male and female rats were comparable with those of the control group. Statistically, a significant increase in absolute and/or relative weight of the spleen was observed. Furthermore, this alteration was well correlated with the histopathological finding (EMH, erythroid) observed in the spleen at 650 ppm and 200 ppm in a dose-dependent manner and considered adverse effects related to the test substance treatment. Test substance treatment at the 650 ppm dose level was associated with a decrease in RBC in both sexes and haemoglobin and haematocrit in females. In addition, these effects led to an increase in reticulocyte count, MCV and MCH. Except MCV and MCH, all these alterations were also noted at 200 ppm dose level with less severity and consistency. Treatment with the test substance at a 650 ppm dose level led to a decrease in the total protein. A decrease in total protein was mainly owed to a decrease in the albumin level. Effects were also observed in the 200 ppm dose level. The macroscopic examination did not reveal any treatment-related lesion in the Cohort 1A rats. Histopathological examination revealed treatment-related lesions in the spleen (EMH, erythroid) and bone marrow femur (increased cellularity of erythroid cells) at 650 and 200 ppm dose levels.

Cohort 1B Rats:

No mortality or morbidity was observed in rats treated with the test substance at dietary concentrations up to 650 ppm dose level. Statistically, a significant decrease in mean body weights, body weight gain, and food consumption of male and female rats was observed at the 650 ppm dose level. The following parameters were found to be comparable between treated rats and the control group; pre-coital interval, fertility, mating, gestation, and parturition indices, percentage of pregnant rats, duration of the gestation; the mean number and percent of pre- and postnatal losses; and the mean number of implants. Statistically, a significant increase in absolute and/or relative weight of the spleen was observed at 650 ppm dose level. The macroscopic examination did not reveal any treatment-related lesions in Cohort 1B rats.

F2 Pups:

No treatment-related clinical signs were observed in pups up to 650 ppm. Statistically, a significant decrease in mean body weights and body weight gain of male pups, female pups, and the composite of male and female pups were observed at the 650 ppm dose level. Statistically, a significant increase in mortality index and decrease in survival index of the composite of male and female pups was observed on PND 4 in the 650 ppm dose group. Further, a statistically significant decrease in the mean number of composites of male and female pups was observed at all post-natal days in the 650 ppm dose group. In addition, reduced litter size was correlated with a decreased mean number of implants at all three dietary concentrations and increased mortality index on PND 4. The following parameters in F2 pups were comparable to those observed in the control group; litter size, male sex ratio, body temperature, anogenital distance, and physical development landmarks, macroscopic and microscopic findings. Statistically, a significant decrease was noted in the terminal body weight of pups in the high dose group. Treatment-related decreases were also noted in absolute weights of the liver, spleen, brain and thymus of the high dose group, though consistency was observed more in females than males. Effect of decrease in relative weights were also noted in high dose group in thymus, spleen and liver, while increase was noted in brain. The changes observed in the absolute and relative weights of organs were related to a decrease in terminal body weight.

Based on the results of this study, a dose of 70 ppm (corresponding to 5.41 - 13.04 mg/kg bw/day in males and females) was considered to be the NOAEL for parental systemic toxicity (P0, F1 and F2), 200 ppm was considered to be the NOAEL for developmental toxicity (F1 and F2 generation) and 550/650 ppm was considered to be the NOAEL for reproductive toxicity (P0 and P1, corresponding to 38.24 - 120.91 mg/kg bw/day in adult males and females).

 

Two-Generation study

The study (Report number WIL-223003) was conducted under GLP conditions, according to EPA OPP 83-4 and similar to OECD 416. The study is considered reliable with restrictions 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. Reproductive parameters (fertility, mating, days between pairing and coitus, gestation and parturition) were not adversely affected by test article administration at concentrations of 72, 207 and 540 ppm during the F0 and F1 generations. All F0 and F1 parental animals survived to the scheduled necropsies. No adverse clinical signs attributed to the test article administration were observed in the treated males and females. Mean body weights and body weight gains in the 540 ppm group F0 males were reduced early in the treatment period and generally reduced throughout the F1 generation. In F0 and F1 females mean body weights and body weight gains were generally reduced prior to breeding, during gestation and lactation and after weaning. No adverse effects were apparent in the 72 and 207 ppm group males and females in either generation on body weight development. Food consumption in the 540 ppm group F0 and F1 males was generally reduced throughout each respective generation and in females prior to breeding, during gestation and lactation and after weaning in the F0 and F1 generations. No adverse effects were observed in males and females in the 72 and 207 ppm dose group in either generation. No test article-related findings were noted in the F0 and F1 treated groups at the macroscopic examinations and no adverse effects on organ weights were observed at any concentration. No microscopic lesions attributed to test article administration were observed in the 540 ppm group tissues. Microscopic examination of gross lesions noted at the scheduled necropsies in the 540 ppm group did not reveal any adverse effects of test substance administration. Mean pup body weights in the 540 ppm group litters were slightly but statistically reduced during lactation (F1 and F2 pups) and throughout the remainder of the study until the postnatal day 70 neuropathology evaluation (selected F2 pups). F1 and F2 pup sex ratios, live litter sizes, numbers of dead pups on lactation Day 0, viability indices, the general physical condition of the pups and brain weights (selected F2 pups) were not adversely affected by parental treatment at any concentration. Necropsy findings for the F1 and F2 pups that died or were euthanized at the scheduled postnatal necropsies were not suggestive of any correlation with parental treatment. Various indicators of physical and functional development, as well as behavioral responses of the selected F2 treated pups in the developmental neurotoxicity phase were comparable to the control group values. At the postnatal Days 11 and 70 neuropathological examinations, no gross or microscopic test article-related lesions were observed in the selected F2 pups.

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.

 

Supporting evidence on toxicity to reproduction is further obtained in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, which was performed similar to OECD 422. This study was conducted as dose range finding study to select dose levels for the above mentioned extended one-generation reproduction toxicity study (EOGRTS, report number 480-1-04-26106). As only 8 animals were included in each test group, resulting in less than 8 pregnant females for the hazard assessment, the study is considered only as supporting study. Briefly, the test substance was given through diet to male and female Wistar rats at dietary concentrations of 0, 150 (25.52 – 25.77 mg/kg bw/day), 500 (86.63 – 93.98 mg/kg bw/day), 750 (126.20 - 137.3 mg/kg bw/da) and 1000 (195.31 – 210.07 mg/kg bw/day) ppm, during pre-mating, mating, gestation, and the lactation period. Males were treated for 50 days (two weeks before mating, during the mating (two weeks) and until the day before scheduled sacrifice). Females were treated two weeks before mating, the variable time to conception, the duration of pregnancy and twenty-one days after delivery. First filial generation (F1) selected pups were treated from postnatal day (PND) 21 to 35. The mean percent test substance recovery obtained for test diet was within the acceptance level of ± 20% of the nominal concentration demonstrating that the exposure concentrations were as intended in the study plan and the %CV was less than 20, suggesting that the test substance was homogeneously distributed in the diet.

The following results were obtained:

Parental (P0) generation:

No mortality, morbidity, and clinical signs of toxicity were observed in male and female rats up to the 1000 ppm group. The following parameters were found to be comparable with that of the control group; 1. Oestrous cycle length and the number of normal oestrous cycle 2. Pre-coital interval, fertility, mating, gestation and parturition indices, percentage of pregnant rats, duration of the gestation 3. Mean number and percent of pre- and postnatal losses 4. Mean number of implants. Statistically, the mean body weight gain, food consumption, and food efficiency of male rats of the 500, 750, and 1000 ppm groups were significantly lower as compared with that of the control group. The mean body weight was also lower in male rats of the 1000 ppm dose group. Statistically, the mean body weight (premating (PM), gestation day (GD) and lactation day (LD)), body weight gain (PM and GD), food consumption (PM, GD, LD), and food efficiency (PM) of female rats of the 750 and 1000 ppm groups were significantly lower when compared with that of the control group. Further, the mean body weight (GD), body weight gain (PM and GD), and food efficiency (PM) of female rats of the 500 ppm were statistically significantly lower, when compared with that of the control group. Statistically, the mean body weight gain and food efficiency (PM) of female rats of the 150 ppm dose group were significantly lower when compared with that of the control group. In addition, a statistically significant decrease in red cell mass (RBC, haemoglobin, and haematocrit) led to a significant increase in MCV and MCH of female rats of the 150, 500, 750 and 1000 ppm groups. A similar effect was also found in RBC, MCV and MCH of male rats of the 1000 ppm dose group. Statistically significant increase was noted in female reticulocyte count at 750 and 1000 ppm and decrease was noted in female basophil count at 500, 750, and 1000 ppm groups. These effects were considered as a treatment-related adverse effect of the test substance on haematopoietic cells. A treatment-related significant decrease was noted in total protein, albumin and globulin of female rats of the 500, 750, and 1000 ppm dose groups. Further, a statistically significantly decrease in globulin was also noted in female rats of the 150 ppm dose group. Additionally, a statistically significant decrease was observed in LDH, and an increase was noted in ALP of female rats of the 1000 ppm dose group. These alterations were considered as related to test substance treatment. No treatment-related effects were noted in urinalysis parameters of male and female rats. Test substance treatment led to a statistically significant decrease in the terminal body weight of male rats of the 1000 ppm and female rats of the 750 and 1000 ppm dose groups.

Statistically significant decreases were observed in the absolute weight of male reproductive organs (testes, seminal vesicles with coagulating glands and Cowper's gland) at 1000 ppm. Statistically significant decrease in Cowper's gland and LABC was also noted in 150, 500, and 750 ppm groups. Decrease without statistical significance was noted in the absolute weight of LABC in the 1000 ppm dose group. These effects were considered as related to the test substance treatment. Treatment-related statistically significant decrease was noted in absolute brain weight of male rats of the 1000 ppm dose group. In the female rats, a treatment-related statistically significant increase was noted in the relative weight of the liver in all the test substance treated groups. Similarly, treatment-related statistically significant increase was noted in absolute, and relative weights of the spleen in all the test substance treated groups.

First filial generation (F1) pups:

PND 0 – 21 (F1 pups)

The mean body weight and body weight gain of pups were statistically significantly lower in the 750 and 1000 ppm dose groups when compared with that of the control group. Mortality and survival indices of F1 pups were comparable with that of the control group. No treatment-related mortality, morbidity and clinical signs were observed in F1 pups. The mean live birth index, count (litter size), male sex ratio, anogenital distance, nipples retention in the male F1 pups were comparable with that of the control group.

PND 21 – 35 (F1 juveniles)

The mean body weight and body weight gain were statistically significantly lower due to the decreased food consumption of juvenile rats of the 750 and 1000 ppm groups. The overall body weight gain of male and female rats was decreased 14.21% and 11.36% respectively, in 500 ppm dose group. These effects were considered as treatment-related systemic adverse effect of the test substance. No treatment-related effect was observed in food efficiency of male and female rats up to the 1000 ppm group. The absolute weight of the brain, thymus, kidneys (male and female) and spleen (females) was statistically significantly decreased in rats of the 1000 ppm group. Similar effects were also observed in male (brain, thymus, and kidneys) and female (brain and thymus) rats of the 750 ppm and male (thymus) rats of the 500 ppm groups. Statistically significant increase was observed in the relative weight of liver in male and female rats of the 1000 ppm dose group and male rats of the 500 and 750 ppm dose groups. The relative weight of the brain was statistically significantly increased in male and female rats of the 1000 ppm dose group and male rats of the 750 ppm dose group. Statistically significant decrease was observed in the relative weight of thymus of female rats of the 1000 ppm dose group. These alterations were likely to be associated with a significant decrease observed in the terminal body weight.

 

In summary, treatment-related effects were observed on body weight, body weight gain, food consumption, food efficiency, haematology, clinical chemistry, and organ weight at 500, 750, and 1000 ppm. Some effects were also observed in the 150 ppm group and could be considered as treatment-related. No treatment-related effects were observed on reproduction and fertility up to the 1000 ppm dietary concentration. Treatment-related effects, especially lower body weights and body weight gain were observed on F1 pups (PND 0-21) at the 750 and 1000 ppm and F1 juveniles (PND 21-35) at the 500, 750 and 1000 ppm dietary concentrations. Mortality and survival indices of F1 pups were comparable with that of the control group. No treatment-related mortality, morbidity and clinical signs were observed in F1 pups. The mean live birth index, count (litter size), male sex ratio, anogenital distance, nipples retention were comparable with that of the control group. Further details on F1 pups and F1 juvenile offspring are discussed below under “Developmental toxicity”. 

Based on the results of this study, dietary concentrations of 0, 70, 200, and 550 ppm were selected for the subsequent EOGRT study.

 

Conclusion on reproductive toxicity

In the abovementioned studies, reproductive performance was unaffected by test article administration up to doses of 650 ppm. Neonatal toxicity was observed in the EOGRTS study at 200 ppm and in the 2-Generation study at 540 ppm, which was evident by lower pup body weights/weight gain, lower organ weights and/or reduced litter size at dose levels, which also caused maternal toxicity evident by inhibition of body weight gain and reduced food consumption. Therefore, the effects observed on pups can be attributed to maternal toxicity and considered a secondary effect. Thus, the data clearly suggest that, at the treatment levels studied, the test substance did not adversely affect the development of the offspring treated continuously in utero, via lactation and later via their diet. In conclusion, no classification or labelling according to the CLP Regulation 1272/2008 is required with regard to reproductive toxicity.

An assessment on endocrine disrupting potential is ongoing during the renewal (AIR) process under regulation(EC) No 1107/2009, a final outcome is currently not available.

Effects on developmental toxicity

Description of key information

Key, report number ZIR 15/24/891371, developmental toxicity (rat): 

NOAEL (maternal general toxicity): 4 mg/kg bw/day

LOAEL (maternal general toxicity): 16 mg/kg bw/day

NOAEL (maternal developmental toxicity): 64 mg/kg bw/day (highest dose level tested)

NOAEL (pup developmental toxicity): 4 mg/kg bw/day

LOAEL (pup developmental toxicity): 16 mg/kg bw/day

Additional study on diaphragmatic thinning, report number PFX 002/033719, developmental toxicity (rat):

NOAEL (maternal general toxicity): 8 mg/kg bw/day

LOAEL (maternal general toxicity): 16 mg/kg bw/day

NOAEL (pup developmental toxicity): 16 mg/kg bw/day

LOAEL (pup developmental toxicity): 64 mg/kg bw/day

Conclusion: an overall NOAEL of 16 mg/kg bw/day is considered appropriate for developmental toxicity, linked to maternal toxicity observed at the same dose

Key, report number 4913-508/2, developmental toxicity (rabbit): 

NOAEL (maternal general toxicity): 3 mg/kg bw/day

LOAEL (maternal general toxicity): 7.5 mg/kg bw/day

NOAEL (maternal developmental toxicity): 7.5 mg/kg bw/day

LOAEL (maternal developmental toxicity): 15 mg/kg bw/day

NOAEL (pup developmental toxicity): 7.5 mg/kg bw/day

LOAEL (pup developmental toxicity): 15 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Jun 1989 - 11 Jun 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 2018

Deviations:

yes

Remarks:

the treatment was restricted to the period of organogenesis and did not cover the complete gestation period, only 4 days of acclimation period, continued under "Principles of method if other than guideline"

Principles of method if other than guideline:

Further deviations to the current guideline (adopted in 2018): anogenital distance not measured, gravid uterus weights not shown, no historical control data provided and parameters related to the detection of endocrine disrupting potential (thyroid weights, thyroid histopathology and thyroid hormones) not examined as they were not required at the time the study was conducted, no special attention paid to reproductive tract of foetusses.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan, USA
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: 161 - 218 g
- Housing: 5 per cage in suspended galvanised metal cages
- Diet: Labsure Laboratory Animal Diet No. 1, ad libitum
- Water: tap water, ad libitum
- Acclimation period: Pregnant females arrived at the laboratory 4 days before beginning of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 52 - 60
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Jun 1989 To: 04 Jul 1989

Route of administration:

oral: gavage

Vehicle:

other: 1% Methylcellulose (MC)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test article were prepared on a daily basis. The highest required concentration was prepared by suspending a weighed quantity of the test article in the vehicle. The low concentrations were prepared by serial dilution. The dose volume was 0.1 mL/100 g.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken for analysis of achieved concentration on the second day of dosing. Results showed that achieved concentration was within 9% of nominal concentration.
At nominal concentrations of 1.0 and 80 mg/mL, the results indicate that the test substance produces a homogeneous suspension in 1% MC formulations. Analysis of formulation showed that homogeneity was maintained by magnetic stirring for 1 h and that the formulation could be successfully resupended 4 h after preparation during storage at ambient temperature and at 24 h after storage at 4 °C.
Further, chemical stability of the test substance in 1% MC formulations was confirmed during storage at ambient temperature in the dark for 4 h and at 4 °C for 24 h.

Details on mating procedure:

No data.- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy

Duration of treatment / exposure:

Day 6 - 15 post mating

Frequency of treatment:

Daily

Duration of test:

Day 20 of gestation

Dose / conc.:

1 mg/kg bw/day (nominal)

Dose / conc.:

4 mg/kg bw/day (nominal)

Dose / conc.:

16 mg/kg bw/day (nominal)

Dose / conc.:

64 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on study design:

Time-mated females were purchased, the first batch consisting of 78 animals followed by the second batch of 52 animals one day later. The first batch is referred to as batch A and the second batch as batch B.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: signs of reaction to treatment

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed initially (Day 1 of pregnancy for group A and Day 2 for group A and B) and on Day 3, 6, 8, 10, 12 14, 16, 18 and 20.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
Food consumption was measured from weigh day to weigh day.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was measured daily from Day 2 to Day 20 of pregnancy inclusive.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Uterus and ovaries
On Day 20 of pregnancy the animals were sacrificed, dissected and examined for congenital abnormalities and macroscopic pathological changes in maternal organs. Abnormal tissues were preserved. The ovaries and uteri were examined immediately.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- Plasma: No

Fetal examinations:

External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: no

Statistics:

Analyses were performed on litter data. Tests were two-tailed.
- Litter data: Basic sample unit was the litter, and due to the preponderance of non-normal distributions, non-parametric analyses proved most consistent.
Mean values of litter size, pre- and post implantation loss, litter weight, mean pup weight, sex ratios and the incidence of anomalous offspring were analysed by the Kruskal-Wallis test. Intergroup comparisons were made by the non-parametric equivalent of the ‘t’ test together with the Jonckheere test for an ordered series of treatments.
Where 75% of the values for a given variable consisted of one value, a Fisher’s exact test was used.
- Water, food and bodyweight data: Analysis of variance, followed by Williams’ test were used for assessing Intergroup differences in absolute values for mean water and food consumption and bodyweight during gestation.

Indices:

In assessing litter parameters, pre-implantation loss was calculated as a percentage as:
((No. of corpora lutea - no. of implantations) x 100)/ No. of corpora lutea

Post implantation loss was calculated as:
((No. of implantations - no. of live young) x 100)/ No. of implantations

Historical control data:

Historical control data were not provided.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment at 64 mg/kg bw/day was associated with slight post-dosing salivation and hair loss. At 16 mg/kg bw/day, two animals showed slight post-dosing salivation. There were no obvious treatment-related signs at 4 or 1 mg/kg bw/day.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was an initial slight body weight loss from Day 6 to Day 8 in 13/23 and 20/24 dams at 64 and 16 mg/kg bw/day, respectively, against a combined incidence of 0/69 in the control, 1 and 4 mg/kg bw/day dose group. Body weight values of dams adjusted for body weight at Day 6 were statistically significantly lower than the controls through the study period at 16 and 64 mg/kg bw/day.
Body weight gain at 64 mg/kg bw/day was slightly retarded to Day 16 and parity with controls was not regained by termination. At 16 mg/kg bw/day body weight gain to Day 16 paralleled controls. Body weight gain at 1 and 4 mg/kg bw/day was similar to controls.
For details, please refer to attachment 1 under "Overall remarks, attachments" and to table 1 under "Any other information on results incl. tables".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Treatment at 16 and 64 mg/kg bw/day was associated with a dosage-related decrease in mean food consumption throughout the dosing period; differences from controls attained statistical significance. At the end of treatment appetite returned and was similar to controls. Treatment at 1 and 4 mg/kg bw/day did not noticeably affect mean food consumption.
For details, please refer to attachment 2 under "Overall remarks, attachments" and to table 1 under "Any other information on results incl. tables".

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Treatment at 16 and 64 mg/kg bw/day was associated with a dose-related increased water consumption throughout the dosing period. Differences from controls attained statistical significance; during the post-dosing period (Days 16 to 19) mean consumption was similar to controls. At 4 mg/kg bw/day mean water consumption during the dosing period was marginally higher than the control, but differences were not statistically significant.
For details, please refer to attachment 2 under "Overall remarks, attachments" and to table 1 under "Any other information on results incl. tables".

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No obvious treatment-related macroscopic changes were detected at terminal autopsy.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

not specified

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Although slight intergroup variation in mean pre- and post-implantation losses were observed when compared to the control group, no statistically significance was obtained and no clear dose-dependency was observed. The implantation rate was coparable among all groups.
For details, please refer to attachment 3 under "Overall remarks, attachments"

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There was no instance of total resorption during the study.

Early or late resorptions:

no effects observed

Description (incidence and severity):

No significant difference in early or late resorption was observed in any of the treatment groups compared to the control.
For details, please refer to attachment 3 under "Overall remarks, attachments"

Dead fetuses:

no effects observed

Description (incidence and severity):

No statistically significant difference in the number of death foetuses was observed in any of the treatment groups compared to the control.
For details, please refer to attachment 3 under "Overall remarks, attachments"

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No statistically significant changes in the number of pregnant rats were observed in any of the treatment groups compared to the control. In the control, 4, 16 and 64 mg/kg bw/day dose groups were 2, 1, 3, 2 and 1 rats non-pregnant out of 25 females, respectively.
For details, please refer to attachment 3 under "Overall remarks, attachments"

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

4 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: main study (ZIR 15/24/89137)

Key result

Dose descriptor:

LOAEL

Remarks:

maternal general toxicity

Effect level:

16 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

water consumption and compound intake

Remarks on result:

other: main study (ZIR 15/24/89137)

Key result

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

64 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

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

Remarks on result:

other: main study (ZIR 15/24/89137)

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

8 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: supplementary study (PFX 002/033719)

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

16 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Remarks on result:

other: supplementary study (PFX 002/033719)

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean fetal body weight showed a dose-dependent reduction and reached statistical significance at 64 mg/kg bw/day when compared to the control group, which was reflected in the low mean litter weight. Therefore, the effect was considered to be related to the treatment with the test substance, but represents a secondary effect due to maternal toxicity observed at the highest dose tested.
For details, please refer to attachment 3 under "Overall remarks, attachments" and to table 2 under "Any other information on results incl. tables".

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No difference in the number of live offspring were observed in any of the treatment groups when compared to the control group.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was similar among both control and treated animals.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

The slight intergroup variation in litter size was not considered treatment-related. Mean litter size at 64 mg/kg bw/day was identical to the control value. However, the litter weight was statistically significantly decreased in the high-dose group when compared to the control group. In the other treatment groups, no statistically significant difference compared to the control was observed. The reduction in litter weight in the high-dose group was considered to be related to the treatment with the test substance, but represents a secondary effect due to maternal toxicity observed at the highest dose tested.
For details, please refer to attachment 3 under "Overall remarks, attachments" and to table 2 under "Any other information on results incl. tables".

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations:
Observed external malformations included microphthalmia observed in 1/255 foetusses in the low-dose group and 1/272 foetusses in the high-dose group, both which are considered incidental.
For details, please refer to attachment 4 under "Overall remarks, attachments".

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations and anomalies:
No treatment-related skeletal malformations were observed during the study. The incidence of malformed foetuses was 4, 2, 1, 1 and 4 in control, 1, 4, 16 and 64 mg/kg bw/day dose groups.
The incidence of foetuses with skeletal anomalies was similar in both control and treated groups.

Variants:
The marginally higher incidence of foetuses at 64 mg/kg bw/day with unossified sternebrae was probably related to the lower foetal weight. Differences in percentage incidence were not statistically significant. Treatment at 1, 4 and 16 mg/kg bw/day was not considered to affect the occurrence of skeletal variants. The incidences of skeletal variants were not statistically significantly different from the control.

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

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Malformations and anomalies:
Visceral malformations observed included cardiac, situs inversus and anals atresia, diaphragmatic hernia and umbilicial hernia. These malformations only occured in one fetus and were considered incidental.
The incidence of foetuses with visceral anomalies was marginally higher at 16 and 64 mg/kg bw/day compared with controls. This increase was not dose-related but 5/133 and 5/134 foetuses at 16 and 64 mg/kg bw/d respectively showed thinning of the diaphragm/protrusion of the liver. Although a high proportion of foetuses at the two top doses showed the same visceral anomaly the relationship to treatment is not clear as no foetuses at these doses showed diaphragmatic hernia.
For details on visceral malformations, please refer to attachment 4 under "Overall remarks, attachments" and to table 2 under "Any other information on results incl. tables".

Effects of the test substance on the development of diaphragm in foetuses were further investigated in an supplementary study, which can be found under "Any other information on results incl. tables".

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: main study (ZIR 15/24/891371 Part I)

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Effect level:

16 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

visceral malformations

Remarks on result:

other: main study (ZIR 15/24/891371 Part I)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

16 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects at this dose level

Remarks on result:

other: supplementary study (PFX 002/033719)

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Effect level:

64 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

visceral malformations

Remarks on result:

other: supplementary study (PFX 002/033719)

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: visceral/soft tissue: thinning of the diaphragm

Description (incidence and severity):

the incidence of diaphragmatic thinning was lower in the offspring than in foetus; no incidence of development into diaphragmatic hernia was observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

16 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Table 1         Maternal toxic effects
Parameter	0 mg/kg	1 mg/kg	4 mg/kg	16 mg/kg	64 mg/kg	Dose-response +/–
Number of dams examined	25	25	25	25	25
Mean water consumption (Day 6-15)			(↑)	↑**	↑**	–
Mean food consumption (Day 6-15)				↓**	↓**	+
Bodyweight loss with retarded weight gain	No	No	No	Yes**	Yes**	+
**p 0.01 Williams' test
Table 2            Embryo toxic effects
Parameter	0 mg/kg	1 mg/kg	4 mg/kg	16 mg/kg	64 mg/kg	Dose-response +/–
Number of foetuses examined	126	125	123	133	134
Litter weight					↓*	–
Mean foetal weight					↓***	+
Incidence of foetuses with visceral anomalies	8.3%	4.9%	6.9%	14.9%	12.2%	–
Thinning of diaphragm (no. of foetus affected)			1	5	5	–
*    p 0.05 ***p 0.001 Jonckheere statistic

 

Supplementary study to report ZIR 15/24/891371: Investigation of the incidence of diaphragmatic thinning and diaphragmagic hernia in the fetuses and offspring of CD rats treated by oral gavage during organogenesis (supplementary study to report)

An additional study was conducted with the objective to investigate the influence of the test substance on the development of the diaphragm in the fetus, with specific reference to diaphragmatic thinning and diaphragmatic hernia recorded in fetuses in an earlier study (1990). In order to monitor the subsequent progression of this particular abnormality postnatally, similar observations were made on culled offspring at Day 4 of age and on weanling offspring from females allowed to litter. Treatment of the parental females was restricted to the period of organogenesis (Days 6 - 15 post coitum) to match the period of treatment in the original study. The test substance was administered during the organogenesis phase (Days 6 - 15) of pregnancy to rats. Three groups of 44 mated female rats received the test item by oral gavage at dosages 0, 8, 16 or 64 mg/kg bw/day from Days 6 - 15 after mating. Twenty two females in each group were sacrificed on Day 20 after mating for fetal examination and the remaining females were allowed to litter to permit examination of offspring on Day 4 and 21 of age.
Clinical signs, body weight and food consumption were monitored. Adult females were examined macroscopically at necropsy on Day 20 after mating or after weaning. Fetuses were examined macroscopically at necropsy and subsequently by detailed examination of the diaphragm after fixation. Offspring received detailed examinations of the diaphragm at either Day 4 or Day 21 of age.
Females receiving 64 mg/kg bw/day of the test substance showed reduced activity, piloerection, reduced body temperature and partially closed eyelids approximately 6 h after dose administration on the first day of treatment only and sporadic incidences of increased salivation and rales were recorded at later stages through the dosing period. There were no deaths. There was marked body weight loss in the early treatment period and a marked reduction in food intake. Food intake and body weight gain remained low throughout treatment and animals were much lighter than controls at necropsy on Day 20 of gestation. Females allowed to litter continued to recover body weight relative to controls during the lactation period. Live litter size was slightly low at Day 20 post coitum, but this was not confirmed in animals allowed to litter. Litter weight and fetal weight were low at Day 20 post coitum, but the females that were allowed to litter showed an increase in gestation length by approximately 24 h so that offspring weights at Day 1 post partum were similar to control values. Survival and growth of the offspring was unaffected by treatment administered to the dams during the organogenesis phase of pregnancy. The incidence of diaphragmatic thinning with protrusion of the liver into the thorax in the region of the central ligament (30%) was approximately twice the incidence seen in controls (17%). The incidence of this anomaly in offspring at Day 4 or 21 of age was about 9%, whereas the observed control incidence was 1 or 3% at Day 4 and Day 21, respectively. There were no cases of diaphragmatic hernia but the liver and diaphragm tended to become fused together where there was protrusion of the liver through a thin portion of the diaphragm.
At 16 mg/kg bw/day, approximately 25% of females were cold 6 h after dosing on the first day of treatment, but there were minimal other clinical signs of reaction to dosing. One female was killed for humane reasons on Day 11 of gestation because of marked respiratory distress and reduced activity.
Animals lost weight during the first few days of treatment, overall weight gain and food consumption during treatment was low but the animals recovered between the end of treatment at Day 15 and necropsy at Day 20 of gestation. Body weight of females at the start of lactation remained low, but there was a degree of recovery towards the end of lactation. Live litter size was slightly low at Day 20 post-coitum, but this was not confirmed in animals allowed to litter. The incidence of diaphragmatic thinning with protrusion of the liver was similar to control at all examination stages and declined with increasing age of the offspring.
At 8 mg/kg bw/day there were no significant clinical signs, although food intake during early treatment and body weight gain throughout treatment were slightly low compared to control. Litter and fetal parameters at Day 20 of gestation were unaffected by treatment. There was a minor extension of gestation length but post partum litter parameters for females allowed to give birth were unaffected by maternal treatment. The incidence of diaphragmatic thinning with protrusion of the liver was similar to control at all examination stages and declined with increasing age of the offspring. There was a single incidence of diaphragmatic hernia at Day 21 of age but in the absence of cases at higher levels this was considered to be unrelated to treatment. For detailed litter data with respect to diaphragmatic anomalies, please refer to attachment 5 under "Overall remarks, attachments".
In the subsequent oral teratogenicity study for the investigation of the incidence of diaphragmatic thinning and diaphragmatic hernia in the foetuses and offspring of rats, the NOAEL for maternal toxicity was 8 mg/kg bw/day, based on decreased body weight and food consumption and clinical signs. The NOAEL for developmental toxicity was 16 mg/kg bw/day based on increased incidence of the thinning on the diaphragm at 64 mg/kg bw/day. The incidence was lower in the offspring than in foetus and there was no incidence of development into diaphragmatic hernia.

Conclusions:

The current study was not performed under GLP conditions, but similar to guideline 414 (adopted in 2018). Deviations to the current OECD guideline included that the treatment was restricted to the period of organogenesis and did not cover the complete gestation, only 4 days of acclimation period, anogenital distance was not measured, gravid uterus weights were not noted, no historical control data were provided and parameters related to the detection of endocrine disrupting potential (thyroid weights, thyroid histopathology and thyroid hormons) were not examined as there were not required at the time the study was conducted, no special attention paid to reproductive tract of foetusses.
In conclusion, treatment with the test substance at 16 and 64 mg/kg bw/day exhibited effects on foetal and litter weight, but there was no indication of any teratogenic effect. There was no effect of treatment, maternal or foetal, at 4 mg/kg bw/day and below.
In the present oral teratogenicity study in rats, the NOAEL for maternal toxicity was 4 mg/kg bw/day based on decreased body weight and food and water consumption at 16 mg/kg bw/day and above. The NOAEL for maternal developmental toxicity was 64 mg/kg bw/day as no adverse effect were observed up to the highest dose level tested. Foetuses showed diaphragmatic thinning in the mid- and high-dose groups. Further, reduced body weight and litter weights at the highest dose tested was observed, which is considered secondary due to maternal toxicity. However, the NOAEL for developmental effects (foetuses) was set to 4 mg/kg bw/day. No teratogenic effect was observed.

An additional study (GLP-compliant) was conducted with the objective to investigate the influence of the test substance on the development of the diaphragm in the fetus, with specific reference to diaphragmatic thinning and diaphragmatic hernia recorded in fetuses in the previous study. In order to monitor the subsequent progression of this particular abnormality postnatally, similar observations were made on culled offspring at Day 4 of age and on weanling offspring from females allowed to litter. Treatment of the parental females was restricted to the period of organogenesis (Days 6 - 15 post coitum) to match the period of treatment in the original study. Female rats received the test item by oral gavage at dosages 0, 8, 16 or 64 mg/kg bw/day from Days 6 - 15 after mating. Twenty two females in each group were sacrificed on Day 20 after mating for fetal examination and the remaining females were allowed to litter to permit examination of offspring on Day 4 and 21 of age.
The majority of the changes in the diaphragm were small, but with the fixation process these shape changes were apparent when the foetus was sectioned. There was a clear indication in the current study that the incidence of diaphragmatic thinning with protrusion of the liver was increased in foetuses of females, which had been treated with 64 mg/kg bw/day during organogenesis. This would be consistent with the findings from the original study. There was no evidence, however, of any increase in the incidence of diaphragmatic thinning at 8 and 16 mg/kg bw/day, despite the detailed examinations and the fact that the numbers of fetuses examined was almost three times greater than in the original study and post partum offspring were also examined. The incidence of diaphragmatic changes was lower in the offspring than in the fetus and although this was still apparent at weaning there was no evidence that diaphragmatic thinning developed into diaphragmatic hernia after birth or that growth or survival of the offspring “at risk” was compromised. However, the observerd increasing incidences of diaphragmatic changes were related to systemic maternal toxicity, as treatment of pregnant rats with the test substance at levels of 16 or 64 mg/kg bw/day has marked effects on the mothers (dose-related reductions in food intake and body weight loss/reduced gain during treatment). Fetal weight was also reduced at 64 mg/kg bw/day.
In conclusion, the results of the detailed examinations of the fetuses/offspring in the current study clearly show that it is possible to detect high frequencies of cases where the diaphragm appears thin and the liver protrudes slightly through the diaphragm causing a bulge to appear in the cranial surface of the liver. There was an incidence of approximately 30% in fetuses from dams treated at 64 mg/kg bw/day, with about 14 - 17% incidence in controls and groups treated with 8 and 16 mg/kg bw/day. The NOAEL for maternal toxicity was 8 mg/kg bw/day, based on decreased body weight and food consumption and clinical signs. The NOAEL for developmental toxicity was 16 mg/kg bw/day, based on increased incidence of the thinning on the diaphragm at 64 mg/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 May 1985 - 04 Jul 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 2018

Deviations:

yes

Remarks:

treatment was restricted to the period of organogenesis and did not cover the complete gestation, housing conditions slightly different from guideline, continued under "Principles of method if other than guideline"

Principles of method if other than guideline:

mid-dose group was inappropriate with only 16 animals and a maternal mortality rate over 10%, anogenital distance not measured and parameters related to the detection of endocrine disrupting potential (thyroid weights, thyroid histopathology and thyroid hormones) not examined as there were not required at the time the study was conducted, no special attention paid to reproductive tract of foetusses.

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Ranch Rabbits Ltd.
- Age at study initiation: 16 - 28 weeks old at mating
- Weight at study initiation: 3.00 - 4.38 kg
- Housing: Individually housed in anodised steel grid floor cages.
- Diet: SQC Standard Rabbit Diet (Special Diets Services Ltd., Witham, UK), ad libitum
- Water: Filtered mains water, ad libitum
- Acclimation period: at least 32 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 - 22
- Humidity (%): 40 - 70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 10/14

IN-LIFE DATES: From: 30 May 1985 To: 02 Aug 1985

Route of administration:

oral: gavage

Vehicle:

other: 1% aqueous methyl cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was prepared daily and stored at ambient temperature until required for dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each dose formulation prepared on the first and last days of the dosing period were analysed. The method of verification of the concentration of the test article formulations was determined by their zinc concentration. This was in turn determined by atomic absorption spectroscopy. Zinc concentration was measured in Weeks 1 and 4. The results for the nominal concentration of 1.5, 3.75 and 7.5 mg/mL were all within 98 - 112% of nominal concentration. Thus, the result indicates that the formulations were adequately prepared and stable for the time used in the study.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: 5 days
- Proof of pregnancy: After
- Any other deviations from standard protocol: Does that successfully completed coitus were injected intravenously via the marginal ear vein with 10 I.U. of chorionic gonadotrophs to ensure ovulation. The day of mating was designated Day 0 of gestation.

Duration of treatment / exposure:

Day 7 - 19 post mating

Frequency of treatment:

Daily

Duration of test:

Day 28 of gestation

Dose / conc.:

3 mg/kg bw/day (nominal)

Dose / conc.:

7.5 mg/kg bw/day (nominal)

Dose / conc.:

15 mg/kg bw/day (nominal)

No. of animals per sex per dose:

16 females

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: moribiditiy and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily during gestation
All abnormalities of appearance, behaviour and other signs of reaction to treatment were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 0, 7, 8, 9, 10, 13, 16, 19, 23 and 28 of gestation

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
The amount of food (g) consumed by each animal was recorded from Days 0 - 3, 3 - 7, 7 - 10, 10 - 13, 13 - 16, 16 - 19, 19 - 23, 23 - 25 and 25 - 28 of gestation.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 28
Moribund animals were sacrificed with an intravenous injection of sodium pentobarbitone solution (200 mg/mL). Animals which died during the study were necropsied to try to establish the cause of death. Surviving animals were sacrificed, in random order, on Day 28 of gestation, dissected and examined macroscopically.
- Organs examined: Ovaries and uteri

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- Plasma: No

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
- Anogenital distance of all live rodent pups: no

- Soft tissue examinations: All foetuses were evaluated for visceral malformations/ variations. Evaluations were performed on the fresh foetal specimens shortly after removal from the uterus.
- Skeletal examinations: After the visceral inspection, the foetuses were eviscerated and processed for staining of the ossified skeletal structures using the Alizarin Red S staining procedure.

Statistics:

Continuous and semi-continuous responses and some discrete responses:
- Statistical evaluation was made using an analysis of variance technique for normally distributed errors or by non-parametric techniques for non-normally distributed errors.
Analysis of variance established the significance of the variability between all the groups to determine a treatment-related response. The standard deviation obtained from this analysis was used for ‘t’ tests between the control and treatment groups. Where necessary the data were suitably transformed before analysis.
Non-parametric testing was carried out using the Kruskal-Wallis test to determine a treatment-related response. Significant differences between control and treatment groups were determined using the Wilcoxon rank sum test.
All tests were carried out at 1 and 5% significance levels for a two-sided risk.

Discrete responses:
- Statistical analysis was carried out using Fisher’s two-sum randomisation (permutation) test with a Monte Carlo simulation for computation of significance levels. The litter was the experimental unit and a square root transformation was used for weighting the number of incidences and adjusting for different litter sizes.
Each treatment group was tested against the control at 1 and 5% significance for a one-sided risk.

Indices:

Percentage pre-implantation loss was calculated for each litter as:
(Number of corpora lutea - number of implantations / number of corpora lutea) x 100

Percentage post-implantation loss was calculated for each litter as:
(Number of implantations - number of live foetuses / number of implantations) x 100

Sex ratios were calculated as:
1 / (number of females / number of males)

Historical control data:

Historical control data is provided in the report. For detail, please refer to attachment 6 under "Overall remarks, attachments".

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical observations were limited to minor, commonly occurring signs (including fur staining, rhinorrhoea and lachrymation) and there was no indication of an effect of treatment on their intergroup distribution.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One animal (15 mg/kg bw/day) died on Day 23 of gestation and one animal (7.5 mg/kg bw/day) died on Day 13 of gestation. In addition, one animal in the control and 3 mg/kg bw/day group was sacrificed (on Day 14 and 15 of gestation, respectively) after showing weight loss and noisy respiration. As no treatment-related abnormalities were observed at necropsy of these animals, an association with treatment was considered unlikely.
For details, please refer to table 1 under "Any other information on results incl. tables".

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the high-dose group animals showed overall body weight loss from Day 9 of gestation to Day 13, followed by partial recovery. However, weight gain over the entire gestation period (Days 0 to 28) remained lower than in the control group. In the 15 mg/kg bw/day group, the body weight change from Days 7 - 19 was statistically significant lower compared to the control group.
In the mid-dose group, a slight reduction in body weight gain was observed from Days 8 - 16 of gestation, including a slight weight loss from Days 10 - 13. However, a compensatory body weight gain as the control group was observed from Day 16 onwards.
Weight gain in the low-dose group was comparable to controls throughout the gestation period.
For details, please refer to attachment 1 under "Overall remarks, attachments" and to table 1 under "Any other information on results incl. tables".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intake in the high-dose group was statistically significantly lower than in controls over the treatment period (Days 7 - 19). The reduction was most marked during Days 10 - 16 of gestation.
Animals in the mid-dose group also showed slightly reduced food intake for Days 10 - 16 but the reduction was not statistically significant and from Day 16 onwards food intake was slightly higher than in controls.
Food intake of animals in the low dose group was comparable to controls throughout gestation.
For details, please refer to attachment 2 under "Overall remarks, attachments" and to table 1 under "Any other information on results incl. tables".

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 examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean gravid uterus weight was decreased in a dose-dependent manner with 0.46, 0.44, 0.39 and 0.37 kg in the control, 3, 7.5 and 15 mg/kg bw/day groups, respectively.
The lower mean gravid uterus weight in animals treated with 15 mg/kg bw/day compared to the control reflected the lower litter weight observed in this group, while the lower mean gravid uterus weight in animals treated with 7.5 mg/kg bw/day when compared to the control was primarily due to the higher mean pre-implantation loss in this group.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy findings were limited to those commonly occurring in this strain of rabbit and there was no indication of an effect of treatment on their intergroup distribution.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

not specified

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Pre-implantation loss was higher than controls in animals treated with 7.5 mg/kg bw/day, but was considered unlikely to have been associated with treatment as implantation would be expected to occur before the start of dosing on Day 7 of gestation. No difference in terms of pre-impantation losses were observed in the mid- and high-dose groups compared to the control group.

Post-implantation loss was within the normal range in the low- and mid-dose groups, but was higher than the control and expected values in the high-dose group. The increase was partially due to a high incidence of late intrauterine deaths in one animal (which also showed severe body weight loss between Days 16 and 23 of gestation). When data from this animal were excluded, however, incidence remained higher than controls but equal to the highest value seen in control groups during the six most recent studies conducted in this laboratory. It was concluded therefore, that the possibility of an association between treatment at 15 mg/kg bw/day and an increased incidence of late intrauterine deaths cannot be dismissed.
For details, please refer to attachment 4 under "Overall remarks, attachments" and to table 1 under "Any other information on results incl. tables".

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

not specified

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All animals (16/16) were preganat in both the control and in the treatment groups.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

maternal general toxicity

Effect level:

3 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed at this dose level

Key result

Dose descriptor:

LOAEL

Remarks:

maternal general toxicity

Effect level:

7.5 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

7.5 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed at this dose level

Key result

Dose descriptor:

LOAEL

Remarks:

maternal developmental toxicity

Effect level:

15 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

pre and post implantation loss

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: post-implantation loss

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the high-dose group, mean foetal weight was lower than controls (6%). No changes were observed in the low- and mid-dose groups. Since the maternal body weight was also decreased in the high-dose group, the lower foetal weight can be regarded as treatment-related, but as secondary due to maternal toxicity.
For details, please refer to attachment 4 under "Overall remarks, attachments" and to table 1 under "Any other information on results incl. tables".

Reduction in number of live offspring:

not specified

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was some intergroup variation in sex ratio but all values were within the normal range.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Litter size was lower than control and expected values in animals treated with 7.5 and 15 mg/kg bw/day.
In the mid-dose group the reduced litter size was considered a result of the high pre-implantation loss (non-treatment related). In the high-dose group, the reduced litter size was considered a result of the high post-implantation loss.
Litter size in the low dose group was comparable to control values.
Litter weight generally reflected litter size in animals treated with the test substance, and was statistically significantly lower than controls in animals treated with 15 mg/kg bw/day.
Therefore, changes in litter size and litter weights is considered to be related to the treatment with the test substance, but as a secondary effect due to maternal toxicity.
For details, please refer to attachment 4 under "Overall remarks, attachments" and to table 1 under "Any other information on results incl. tables".

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no apparent effect of treatment on the incidence of foetuses showing major or minor external defects or variants. In the low-dose group, major external defects observed were microstomia, cleft palate brachygnathia, cyclopia and hydrocephalus in one animal and microphthalmia in another animal. No major external defects or variants were observed for the other treatment groups including the control group. Therefore, the described observations were considered to be not related to the treatment with the test substance.
For details, please refer to attachment 5 under "Overall remarks, attachments".

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no apparent effect of treatment on the incidence of foetuses showing major or minor skeletal defects or variants. However, observed major defects included multiple vertebral and rib defects in two animals of each the control, low-dose and hogh-dose group. In addition, in the low-dose group sternebral defects were noted in one animal and multiple vertebral defects in two animals. In the mid-dose group no major defects were observed, while in the high-dose group, two animals showed interpariezal absent.
The observed incidence of major skeletal anomalies was higher than expected values and may have been adversely influenced by 2 males, which sired 5 of the 8 foetuses showing major vertebral/rib defects. No effect of treatment was concluded, however, as these defects are known to occur spontaneously in this strain of rabbit and their intergroup distribution was not related to dose.
For details, please refer to attachment 5 under "Overall remarks, attachments".

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no apparent effect of treatment on the incidence of foetuses showing major or minor visceral defects or variants.
For details, please refer to attachment 5 under "Overall remarks, attachments".

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

7.5 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Effect level:

15 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

changes in litter size and weights

other: crown/rump length (reduced)

Remarks on result:

other: effects observed were considered secondary due to maternal toxicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

15 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Table 1                    Toxic effects in dams and foetuses

Parameter	0 mg/kg	3 mg/kg	7.5 mg/kg	15 mg/kg	Dose-response +/–
No. of dams:	16	16	16	16
Mortality	1		1	2	–
Body weight gain			↓ day 8-16	↓ entire gestation period	+
Food intake				↓ day 7-19 **	–
Post-implantation loss (%) *	8.6	4.1	7.2	16.9	–
Foetuses (no.) and litter:	128	140	103	107
Mean litter weight				↓ **	–
Mean foetal weight				↓	–
Litter size			↓	↓	–
Mean crown/rump length (mm)	98.7	98.5	99.2	96.5	–
*   Post implantation loss was higher in top dose than controls but equals the highest value seen in control groups during the six most recent studies.The increase is partially due to a high incidence of late intrauterine deaths in one animal. ** Statistically significantly different from control

Table 2:    Litter data
Dose	[mg/kg bw/d]		0	3	7.5	15
Mean gravid uterus weight (kg)			0.46	0.44	0.39	0.37
% Pre-implantation loss			8.5	8.2	19	6.5
No. of early intrauterine deaths			12	3**	4	5
Post-implantation loss (%)*			8.6	4.1	7.2	16.9
Foetus and litter
Mean litter weight (g)			318.2	319.1	271.5	247.9**
Mean foetal weight (g)			37.6	37.0	37.8	35.2
Mean crown/rump length (mm)			98.7	98.5	99.2	96.5
External and visceral defects: number of foetuses affected (%)
Major defects			0 (0)	2 (1.4)	0 (0)	0 (0)
Minor defects***			11 (8.6)	4 (2.9)	6 (5.8)	8 (7.5)
Variants			25(19.5)	17 (12.1)	14 (13.6)	13 (12.1)
Skeletal defects: number of foetuses affected (%)
Major defects			2 (1.6)	6 (4.3)	0 (0)	4 (3.7)
Minor defects***			76 (59.4)	82 (58.6)	60 (58.3)	57 (53.3)
Variants			123 (96.1)	114 (81.4)	97 (94.2)	97 (90.7)
* Post implantation loss was higher in top dose than controls but equals the highest value seen in control groups during the six most recent studies. The increase is partially due to a high incidence of late intrauterine deaths in | animal. ** statistically significantly different from control (analysis of variance, ‘t’ test ; Kruskal-Wallis, Wilcoxon’s tests) *** historical control data = 39.5%

Conclusions:

The current study was not performed under GLP conditions, but similar to OECD guideline 414 (adopted in 2018). Deviations to the current OECD guideline included that the treatment was restricted to the period of organogenesis and did not cover the complete gestation, the housing conditions were slightly different from guideline, the mid-dose group was inappropriate with only 16 animals and a maternal mortality over 10% (which does not invalidate the study), anogenital distance was not measured and parameters related to the detection of endocrine disrupting potential (thyroid weights, thyroid histopathology and thyroid hormones) were not examined as there were not required at the time the study was conducted, no special attention paid to reproductive tract of foetusses.
In conclusion, treatment with the test substance at 15 mg/kg bw/day via gavage elicited marked maternal toxicity in rabbits, an adverse effect on foetal weight and possibly on post-implantation loss, but there was no indication of any teratogenic effect. Slight reductions in maternal body weight gain and food intake were also observed at 7.5 mg/kg bw/day, but there were no adverse effects on embryonic or foetal development. There was no effect of treatment, maternal or foetal, at 3 mg/kg bw/day.
In the oral teratogenicity study in rabbits, the NOAEL for maternal toxicity was 3 mg/kg bw/d based on decreased body weight and food consumption and reduced foetal weights in the highest dose group. The NOAEL for developmental toxicity was 7.5 mg/kg bw/day. There was no teratogenic effect.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

7.5 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Developmental toxicity was assessed in two prenatal toxicity studies conducted in rats and rabbits. Both studies were performed similar to OECD guideline 414 under GLP conditions. Further, toxicity to reproduction was assessed in rats in an extended one-generation study and 2-generation study conducted according to OECD TG 443 and 416 under GLP conditions. The studies are considered of reliable quality and validity, fulfill the criteria of key studies and thus, are suitable for assessment of the present endpoint.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity studies via the oral route

Data on developmental toxicity following oral administration are available to characterize the potential to induce toxicity after in utero exposure in rodents and non-rodents. Two key studies each were conducted addressing developmental toxicity of the test substance in pregnant rats and rabbits after oral administration, which are both considered as key studies.

Rat

The first developmental toxicity study (Report number: ZIR 15/24/891371) was conducted under GLP conditions and similar to OECD guideline 414. The doses tested were based on a dose range finding study (Report number: ZIR 15/24/891371), which is briefly summarized in the following:

In this preliminary range finding study, the test substance was given to pregnant rats at dose levels of 0, 5, 20 and 80 mg/kg bw/day via gavage from Day 6 through to Day 15 of pregnancy. On Day 20 of pregnancy, the females were sacrificed and subjected to post mortem examinations. Treatment with the test substance was associated with post-dosing salivation on one or two days (only 80 mg/kg bw/day) and hair loss in occasional animals (20 and 80 mg/kg bw/day). An increase in water and decrease in food consumption was observed in all dose groups (only marginal at 5 mg/kg bw/day). Treatment at 80 mg/kg bw/day was associated with a slight body weight loss to Day 8 of pregnancy (thereafter gain paralleled controls), an increase in post-implantation loss and reduced litter and mean foetal weights. Mean litter size was not altered at 80 mg/kg bw/day although an increased post-implantation loss was observed. Since implantation rate was higher at this dose, the mean litter size remained comparable to controls. Treatment at 20 mg/kg bw/day was associated with a reduced weight gain to Day 8 of pregnancy and a slightly reduced litter and mean foetal weight. Treatment at 5 mg/kg bw/day was associated only with a slightly reduced weight gain to Day 8 of pregnancy, parity with control being regained by Day 20. At 5 mg/kg bw/day, no adverse effects on mean litter parameters were observed. No obvious structural abnormalities among foetuses at examination on Day 20 were observed in all dose groups. On the basis of these findings it was considered that 80 mg/kg bw/day was too high for the main embryotoxicity study. A high dosage of 64 mg/kg bw/day was chosen since it was considered this would elicit sufficient toxicity in terms of retarded body weight gain. 5 mg/kg bw/day was not a NOEL therefore a low dosage of 1 mg/kg bw/day and intermediate dosages of 4 and 16 mg/kg bw/day were chosen.

In the main developmental toxicity study with rats (Report number: ZIR 15/24/891371), daily doses of 0, 1, 4, 16 and 64 mg/kg bw/day were administered by oral gavage to females from Day 6 through to Day 15 of pregnancy. On Day 20 of pregnancy, the females were killed and subjected to post mortem examination. Treatment at 64 and 16 mg/kg bw/day was associated with post-dosing salivation in occasional adult animals. Further increased water consumption and decreased food consumption during the dosing period was observed. Dose-related body weight loss to Day 8 followed by retarded weight gain throughout the dosing period was as well observed. Treatment at 64 mg/kg bw/day was also associated with significantly lower mean foetal and litter weight. Treatment at 4 mg/kg bw/day was associated with a marginally increased water consumption during the dosing period. Treatment at 1 mg/kg bw/day did not produce any overt maternal response. Mean litter parameters as assessed by pre- and post-implantation loss, litter size and litter and mean foetal weight were unaffected by treatment at 1, 4 and 16 mg/kg bw/day. Embryofoetal development as assessed by the incidence of skeletal and visceral anomalies was unaffected by treatment. At 64 mg/kg bw/day the incidence of unossified sternebrae was marginally higher than the control and was probably related to the lower foetal weight observed in this dose group.

In conclusion, treatment with the test substance at 16 and 64 mg/kg bw/day via gavage elicited marked maternal toxicity in rats and an adverse effect on foetal and litter weight, but there was no indication of any teratogenic effect. There was no effect of treatment, maternal or foetal, at 4 mg/kg bw/day and below. The NOAEL for maternal general toxicity was 4 mg/kg bw/day based on decreased body weight and food and water consumption at 16 mg/kg bw/day and above. The NOAEL for maternal developmental toxicity was 64 mg/kg bw/day as no adverse effect were observed up to the highest dose level tested. Foetuses showed diaphragmatic thinning in the mid- and high-dose groups. Further, reduced body weight and litter weights at the highest dose tested was observed, which is considered secondary due to maternal toxicity. However, the NOAEL for developmental effects (foetuses) was set to 4 mg/kg bw/day. No teratogenic effect was observed. Regarding the incidence of diaphragmatic thinning a supplementary study was conducted, which is described in the following:

An additional GLP-compliant study (Report number PFX 002/033719) was conducted with the objective to investigate the influence of the test substance on the development of the diaphragm in the fetus, with specific reference to diaphragmatic thinning and diaphragmatic hernia recorded in fetuses in the previous study. In order to monitor the subsequent progression of this particular abnormality postnatally, similar observations were made on culled offspring at Day 4 of age and on weanling offspring from females allowed to litter. Treatment of the parental females was restricted to the period of organogenesis (Days 6 - 15 post coitum) to match the period of treatment in the original study. Female rats received the test item by oral gavage at dosages 0, 8, 16 or 64 mg/kg bw/day from Days 6 - 15 after mating. Twenty two females in each group were sacrificed on Day 20 after mating for fetal examination and the remaining females were allowed to litter to permit examination of offspring on Day 4 and 21 of age. The majority of the changes in the diaphragm were small, but with the fixation process these shape changes were apparent when the foetus was sectioned. There was a clear indication in the current study that the incidence of diaphragmatic thinning with protrusion of the liver was increased in foetuses of females, which had been treated with 64 mg/kg bw/day during organogenesis. This would be consistent with the findings from the original study. There was no evidence, however, of any increase in the incidence of diaphragmatic thinning at 8 and 16 mg/kg bw/day, despite the detailed examinations and the fact that the numbers of fetuses examined was almost three times greater than in the original study and post partum offspring were also examined. The incidence of diaphragmatic changes was lower in the offspring than in the fetus and although this was still apparent at weaning there was no evidence that diaphragmatic thinning developed into diaphragmatic hernia after birth or that growth or survival of the offspring “at risk” was compromised. The observed increasing incidences of diaphragmatic changes were related to systemic maternal toxicity, as treatment of pregnant rats with the test substance at levels of 16 or 64 mg/kg bw/day has marked effects on the mothers (dose-related reductions in food intake and body weight loss/reduced gain during treatment). Fetal weight was also reduced at 64 mg/kg bw/day. In conclusion, the results of the detailed examinations of the fetuses/offspring in the current study clearly show that it is possible to detect high frequencies of cases where the diaphragm appears thin and the liver protrudes slightly through the diaphragm causing a bulge to appear in the cranial surface of the liver. There was an incidence of approximately 30% in fetuses from dams treated at 64 mg/kg bw/day, with about 14 - 17% incidence in controls and groups treated with 8 and 16 mg/kg bw/day. The NOAEL for maternal general toxicity was 8 mg/kg bw/day, based on decreased body weight and food consumption and clinical signs. The NOAEL for developmental toxicity was 16 mg/kg bw/day, based on increased incidence of the thinning on the diaphragm at 64 mg/kg bw/day.

Overall, taking in to account that the numbers of fetuses examined in the follow-up study was almost three times greater than in the original study and that the experimental phase was extended also to post partum offspring, the follow-up study in regard to developmental toxicity (in regard to diaphragmatic changes) is considered to add a higher biological and toxicological significance to the hazard assessment. Thus, an overall NOAEL of 16 mg/kg bw/day is considered appropriate for developmental toxicity, linked to maternal toxicity observed at the same dose. As described above and as well stated by the EFSA in the related comment, diaphragm thinning with liver protrusion and skeletal anomalies were limited to, and only observed in doses inducing maternal toxicity. These effects are therefore to be judged as secondary effects, induced by maternal systemic toxicity. As Ziram was already evaluated during the first inclusion without a classification for reprotoxicity, a proposal for reprotoxic ity is still not warranted. It should also be noted that no similar finding an diaphragmatic thinning was reported neither in the older 2- Generation study nor in the most recent EOGRTS.

 

Supporting evidence on developmental toxicity can further be obtained in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, which was performed similar to OECD 422. This study was conducted as dose range finding study to select dose levels for the above mentioned extended one-generation reproduction toxicity study (EOGRTS, report number 615-1-04-24768). As only 8 animals were included in each test group, resulting in less than 8 pregnant females for the hazard assessment, the study is only considered as supporting study. Briefly, the test substance was given through diet to male and female Wistar rats at dietary concentrations of 0, 150 (25.52 – 25.77 mg/kg bw/day), 500 (86.63 – 93.98 mg/kg bw/day), 750 (126.20 - 137.3 mg/kg bw/da) and 1000 (195.31 – 210.07 mg/kg bw/day) ppm, during pre-mating, mating, gestation, and the lactation period. Males were treated for 50 days (two weeks before mating, during the mating (two weeks) and until the day before scheduled sacrifice). Females were treated two weeks before mating, the variable time to conception, the duration of pregnancy and twenty-one days after delivery. First filial generation (F1) selected pups were treated from postnatal day (PND) 21 to 35. The mean percent test substance recovery obtained for test diet was within the acceptance level of ± 20% of the nominal concentration demonstrating that the exposure concentrations were as intended in the study plan and the %CV was less than 20, suggesting that the test substance was homogeneously distributed in the diet.

In the parental (P0) generation, treatment-related effects were observed on body weight, body weight gain, food consumption, food efficiency, haematology, clinical chemistry, and organ weight at 500, 750, and 1000 ppm. Some effects were also observed in the 150 ppm group and could be considered as treatment-related. Further results observed in the parental generation are summarized under “Effects on fertility”.

In pups, the following effects were observed: 

PND 0 - 21 (F1 pups)

The mean body weight and body weight gain of pups were statistically significantly lower in 750 and 1000 ppm dose groups when compared with that of the control group. Mortality and survival indices of F1 pups were comparable with that of the control group. No treatment-related mortality, morbidity and clinical signs were observed in F1 pups. The mean live birth index, count (litter size), male sex ratio, anogenital distance, nipples retention in the male F1 pups were comparable with that of the control group.

PND 21 - 35 (F1 juveniles)

The mean body weight and body weight gain were statistically significantly lower due to the decreased food consumption of juvenile rats of the 750 and 1000 ppm groups. The overall body weight gain of male and female rats was decreased 14.21% and 11.36% respectively, in 500 ppm dose group. These effects were considered as treatment-related systemic adverse effect of the test substance. No treatment-related effect was observed in food efficiency of male and female rats up to the 1000 ppm group. The absolute weight of the brain, thymus, kidneys (male and female) and spleen (females) was statistically significantly decreased in rats of the 1000 ppm group. Similar effects were also observed in male (brain, thymus, and kidneys) and female (brain and thymus) rats of the 750 ppm and male (thymus) rats of the 500 ppm groups. A statistically significant increase was observed in the relative weight of liver in male and female rats of the 1000 ppm dose group and male rats of the 500 and 750 ppm dose groups. The relative weight of the brain was statistically significantly increased in male and female rats of the 1000 ppm dose group and male rats of the 750 ppm dose group. Statistically significant decrease was observed in the relative weight of thymus of female rats of the 1000 ppm dose group. These alterations were likely to be associated with a significant decrease observed in the terminal body weight. Systemic adverse effects were observed on body weight, body weight gain, food consumption, food efficiency, haematology (all treated groups), clinical chemistry, and organ weight at the 500, 750, and 1000 ppm levels.

In summary, treatment-related effects indicative for growth retardation effects were observed on F1 pups (PND 0-21) at the 750 and 1000 ppm and F1 juveniles (PND 21-35) at the 500, 750 and 1000 ppm dietary concentrations.

Based on the results of this study, dietary concentrations of 0, 70, 200, and 550 ppm were selected for the subsequent EOGRT study.

Rabbits

The developmental toxicity study on rabbits (Report number: 4913-508/2) was not performed under GLP conditions, but similar to guideline 414 (adopted in 2018). The doses tested were based on a dose range finding study (Report number: 4588-508/1), which is briefly summarized in the following

Three groups of 5 mated female rabbits were given the test substance orally, by gavage, from Day 7 to Day 19 of gestation at dose levels of 0 (control) 5, 10 or 20 mg/kg bw/day. At 20 mg/kg bw/day, 2 animals died and one aborted. An association with treatment was concluded as other toxic signs were previously observed in these animals. One incidental death was observed at 5 mg/kg bw/day and one was observed in the control group. There was no indication of an effect of treatment on clinical condition or necropsy findings of animals surviving to term. Marked reductions in weight gain and food intake, particularly at the start of treatment, were observed in animals treated at 20 mg/kg bw/day. Slight, temporary reductions in weight gain and food intake were also observed at 10 mg/kg bw/day. There was no effect of treatment on body weight gain or food intake at 5 mg/kg bw/day. Pregnancy incidence and the mean number of corpora lutea or implantations were within the normal range in all groups. At 20 mg/kg bw/day, post-implantation loss, litter size, litter weight and foetal weight were all lower as compared to the control group, possibly due to compound administration. There was no indication of any effect after treatment with 5 or 10 mg/kg bw/day in pregnant rabbits. In conclusion, because of the maternal toxicity observed at 20 mg/kg bw/day in this study, it was recommended that the high dose level for the main teratology study should be less than 20 mg/kg bw/day.

In the main developmental toxicity study with rabbits (Report number: 4913-508/2), three groups of 16 mated female rabbits received the test substance by gavage at dose levels of 3, 7.5 or 15 mg/kg bw/day from Days 7 to 19 of gestation. A similar group of rabbits received the vehicle only and served as the control group.  Two animals died during the study and two were killed in extremis, but these mortalities were considered incidental to treatment. There was no adverse effect of treatment on clinical condition of the surviving animals. Significant reductions in body weight gain and food intake were observed from the start of treatment at 15 mg/kg bw/day. Slight reductions in weight gain and food intake were also observed during the early part of the treatment period at 7.5 mg/kg bw/day. Body weight gain and food intake of animals treated at 3 mg/kg bw/day was comparable to controls throughout gestation. Maternal necropsy findings were not adversely affected by treatment. There was no effect of treatment on pregnancy incidence or the mean number of corpora lutea or implantations per doe. Post-implantation loss was slightly higher than control and expected values at 15 mg/kg bw/day, but there was no effect of treatment at 3 or 7.5 mg/kg bw/day. Litter size was slightly smaller than controls in the highest dose group, as a result of the increased post-implantation loss. There was no effect of treatment on litter size in low and mid dose group. Some variation in sex ratio was observed but no effect of treatment was concluded. Litter weight, foetal weight and crown/rump length were all reduced at 15 mg/kg bw/day. At 3 and 7.5 mg/kg bw/day litter weight generally reflected litter size and foetal size was comparable to controls. There was no apparent effect of treatment on the incidence of foetuses showing major or minor external/visceral or skeletal defects or variants. In conclusion, treatment with the test substance at 15 mg/kg bw/day via gavage elicited marked maternal toxicity in rabbits, an adverse effect on foetal weight and possibly on post-implantation loss, but there was no indication of any teratogenic effect. Slight reductions in maternal body weight gain and food intake were also observed at 7.5 mg/kg bw/day, but there were no adverse effects on embryonic or foetal development. There was no effect of treatment, maternal or foetal, at 3 mg/kg bw/day.

In the present oral teratogenicity study in rabbits, the NOAEL for maternal toxicity was 3 mg/kg bw/day based on decreased body weight and food consumption at 7.5 mg/kg bw/day. The NOAEL for maternal developmental toxicity was 7.5 mg/kg bw/day based late resorptions, uterus weight and post-implantation losses observed in the high-dose group. Foetuses showed reduced body weight, litter size and litter weights at the highest dose tested, which is considered secondary due to maternal toxicity. No teratogenic effect was observed, but the NOAEL for developmental effects (foetuses) was set to 7.5 mg/kg bw/day.

Conclusion on developmental toxicity

In the oral teratogenicity study in rats the NOAEL for maternal toxicity was 4 mg/kg bw/day based on decreased body weight and food consumption at 16 and 64 mg/kg bw/day. The overall NOAEL for developmental toxicity was 4 mg/kg bw/day taking into account the marginal increase in visceral anomalies (diaphragmatic thinning, but no hernia) observed in the higher dose groups. At 64 mg/kg bw/day reduced foetal and litter weight were also observed, but this was attributed to maternal toxicity and therefore considered a secondary effect. There was no teratogenic effect.

In the subsequent oral teratogenicity study for the investigation of the incidence of diaphragmatic thinning and diaphragmatic hernia in the foetuses and offspring of rats, the NOAEL for maternal toxicity was 8 mg/kg bw/day, based on decreased body weight and food consumption and clinical signs. The NOAEL for developmental toxicity was 16 mg/kg bw/day, based on increased incidence of the thinning on the diaphragm at 64 mg/kg bw/day. The incidence was lower in the offspring than in foetus and there was no incidence of development into diaphragmatic hernia. Overall, an overall NOAEL of 16 mg/kg bw/day is considered appropriate for developmental toxicity based on both developmental toxicity studies considering that a higher number of fetuses was examined in the follow-up study and that the experimental phase was extended also to post partum offspring. As described above and as well stated by the EFSA in the related comment, diaphragm thinning with liver protrusion and skeletal anomalies were limited to, and only observed in doses inducing maternal toxicity. These effects are therefore to be judged as secondary effects, induced by maternal systemic toxicity. As Ziram was already evaluated during the first inclusion without a classification for reprotoxicity, a proposal for reprotoxicity is still not warranted. It should also be noted that no similar finding on diaphragmatic thinning was reported neither in the older 2- Generation study nor in the most recent EOGRTS.

 

In the oral teratogenicity study in rabbits, the NOAEL for maternal toxicity was 3 mg/kg bw/day based on decreased body weight and food consumption and reduced foetal weights in the highest dose group. The NOAEL for developmental toxicity was 7.5 mg/kg bw/day based on reduced body weight, litter size and litter weights at the highest dose tested, which was considered secondary due to maternal toxicity. There was no teratogenic effect.

In conclusion, the observed reduction in pup weights, litter size and/or litter weights were considered secondary due to maternal toxicity. Although the observed diaphragmatic changes were still apparent at weaning at Day 21 post partum, there was no evidence that diaphragmatic thinning developed into diaphragmatic hernia after birth or that growth or survival of the offspring at risk was compromised. Concerns for this effect are therefore unfounded. Thus, in the absence of a teratogenic effect, the data suggested that, at the treatment levels studied, the test substance did not adversely affect the development of the offspring. Therefore, no classification or labelling according to the CLP Regulation 1272/2008 is required with regard to developmental toxicity.

An assessment on endocrine disrupting potential is ongoing during the renewal (AIR) process under regulation(EC) No 1107/2009, a final outcome is currently not available.

Justification for classification or non-classification

The available data on reproductive toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008. Thus, the test substance does not require classification for reproductive or developmental toxicity.