[2-(1-ethoxyethoxy)ethyl]benzene

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive toxicity - oral (OECD TG 422): NOAEL ≥377 mg/kg bw/day (highest dose tested)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08-08-2016 to 03-10-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Remarks:

endpoint 7.5.1

Reason / purpose for cross-reference:

reference to same study

Remarks:

endpoint 7.8.2

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

28 July 2015

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han IGS

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 9 weeks (males) or 10 weeks (females)
- Weight at study initiation: Mean body weight just before the start of treatment was 330 grams for males and 224 grams for females.
- Housing: The rats were housed in makrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. During the pre-treatment and pre-mating period the animals were housed four rats to a cage (separated by sex). For mating, one male and one female were housed together. Mated females were housed individually in cages, which were placed in another cage rack.
- Diet: The rats receive a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England), ad libitum
- Water: Tap-water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 45-65; slightly below 45 (minimum 43.3) and slightly above 65 (maximum 67.1) for short periods of time
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

The diets were mixed in a mechanical blender. Fresh batches of the experimental diets were prepared on 4 and 31 August 2016 and stored in a freezer (<-18°C) in plastic bags in portions sufficient for three to four days. The diets were given as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The food in the cans was replaced twice per week with fresh portions from the freezer.

Details on mating procedure:

Following a 2-week pre-mating period, each female was caged with one male from the same dose group. Animals were caged together for maximally one week until mating occurred of at least 10 animals per group. Mating pairs were clearly identified. Every consecutive morning during the mating period, vaginal smears were made for determination of the presence of sperm. The day on which sperm was detected in the vaginal smear was considered as gestation day (GD) 0. Upon evidence of copulation the females were caged individually for the birth and rearing of their pups. After the first week of the mating period, all females were successfully mated with their assigned male. Based on this number of mating pairs, the mating period was finalized after one week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

From both batches of diets prepared in the study (4 and 31 August 2016) samples were taken and stored in a freezer (≤ -18°C). The content of the test substance at each dietary level was determined in the batches prepared on 4 and 31 August 2016. The homogeneity of the test substance in the experimental diets was assessed in the first batch (4 August 2016), by analysing five samples (taken at different locations in the feed container) of each test diet in duplicate. One sample of the control diet was analysed. To demonstrate the stability of the test substance under experimental conditions, samples of the batch of test diets prepared on 4 August 2016 (low-dose diet, mid-dose diet and high-dose diet) were analyzed at t=0 and analyzed after storage in the animal room (in an open container) for 4 days, and after storage in a freezer (< -18 °C) for at least 5 weeks.

The concentration of the test substance in diet was determined using the following procedure: the test substance was extracted from diet using dichloromethane under shaking and sonication. After dilution with dichloromethane and centrifugation the diluted extracts were analyzed using Gas Chromatography – Mass Spectrometry (GC-MS). Undecane was used as internal standard. The content of the test substance in diet was considered to be “close to intended” if the mean measured concentration was between 90 and 110% of the intended concentration.
Before analysis of study samples, the analytical method was validated by analyzing three spiked samples per dose level, to conform to the following criteria:
- Linearity: the correlation coefficient of the calibration curve should be greater than or equal to 0.996.
- Recovery: the mean recovery of the test substance from diet should be between 80% and 110% at each of the dose levels of the study.
- Repeatability: the relative standard deviation in the percentage recovery, when the recovery test is performed three times at each of the dose levels to be used in the toxicity study, should be less than 10%.
With respect to specificity: signals should be corrected in case the signal obtained for blank samples was ≥ 5% of the signal obtained for low-dose samples.

The homogeneity of the test substance was assessed in the batch of diets, prepared for the study on 04 August 2016. Five samples of each test diet, taken at different locations in the diet container, and 1 sample of the control diet were analyzed in duplicate. For each concentration level, a one-way analysis of variance (Anova) was performed using the sample location (1-5) as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the mean concentrations differ significantly at the five locations in the diet container). The test substance was considered to be homogeneously distributed in the diet if p ≥ 0.01 and/or if the relative standard deviation (RSD) between the mean concentrations at the five locations was ≤ 5%.

The stability of the test substance in diet was assessed in the batch of diets, prepared for the study on 04 August 2016 (4 days animal room and 5 weeks at < -18°C). One sample of each test diet and one sample of the control diet were analyzed in duplicate. For each concentration level, a one-way analysis of variance (Anova) was performed using time as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the mean concentrations differ significantly before and after storage). The test substance was considered to be stable in diet if p ≥ 0.01 and/or if the mean concentration after storage was within 90-110% of the mean concentration at t = 0.

Duration of treatment / exposure:

Male animals were fed the experimental diets containing the test substance during a 2-week pre-mating period, during mating and until sacrifice after at least 28 days of exposure. The female animals were fed the experimental diets during a 2-week pre-mating period, during mating, gestation and lactation up to the day of sacrifice (at, or shortly after, day 13 of lactation).

Frequency of treatment:

Continuously

Dose / conc.:

750 mg/kg diet

Remarks:

Equivalent to at least 39 and 47 mg/kg bw/day for males and females, respectively

Dose / conc.:

2 250 mg/kg diet

Remarks:

Equivalent to at least 117 and 139 mg/kg bw/day for males and females, respectively

Dose / conc.:

7 500 mg/kg diet

Remarks:

Equivalent to at least 377 and 480 mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

12

Control animals:

yes, plain diet

Details on study design:

Dose selection rationale: The dose levels were selected in consultation with the sponsor and are based on the results of a 3-weeks dose-range finding study with the test substance in rats.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS
Each animal was observed daily in the morning hours by cage-side observations. All cages were checked again in the afternoon for dead or moribund animals. All abnormalities, signs of ill health or reactions to treatment were recorded.

DETAILED CLINICAL OBSERVATIONS
Detailed clinical examinations were conducted in all rats of all groups in the experimental room outside the home cage prior to the first exposure and then once weekly up to and including week 4 (males) or 8 (females). During the last week of gestation (week 6 of the study) and during the first week of lactation (week 7 of the study) detailed clinical observations were not performed in order not to disturb the pregnant females or litters. Detailed clinical examinations were performed as part of the Functional Observational Battery test in the selected male and female rats in week 4 and 8 of the study respectively. Signs noted included, but were not limited to changes in skin and fur, piloerection, changes in the eyes, gait (including posture), and presence of clonic or tonic movements, stereotypies and bizarre behaviour.

BODY WEIGHT
Body weights of male and female animals were recorded just before the start of the treatment (to enable randomization) and at the start of the study (day 0). Subsequently males were weighed weekly until sacrifice. Females were weighed once per week during the pre-mating and mating period. Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation and on day 0, 4, 7 and 13 of lactation. The animals were weighed on their scheduled necropsy date in order to calculate the organ to body weight ratios.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption: Food consumption was measured per cage for the same periods as the body weight are measured, except during the mating period when food intake was not registered. The results are expressed in g per animal per day.
- Intake of test substance: The intake of the test substance per kg body weight per day was calculated from the nominal dietary concentrations, the food consumption and the body weight for males and females separately.

HAEMATOLOGY
Prior to sacrifice, all animals were fasted overnight (water was freely available). At sacrifice, blood was taken was taken from the abdominal aorta of 5 rats/sex/group whilst under CO2/O2 anaesthesia. For prothrombin time citrate was used as anticoagulant. For the other parameters EDTA was used as anticoagulant. In each sample the following determinations were carried out: Haemoglobin (Hb), packed cell volume (PCV), red blood cell count (RBC), reticulocytes, total white blood cell count (WBC), differential white blood cell counts (neutrophils, lymphocytes, eosinophils, basophils, monocytes), prothrombin time, thrombocyte count, mean corpuscular volume (MCV; calculated), mean corpuscular haemoglobin (MCV; calculated), mean corpuscular haemoglobin concentration (MCHC; calculated)

CLINICAL CHEMISTRY
Prior to sacrifice, all animals were fasted overnight (water was freely available). At sacrifice, blood was taken from the abdominal aorta of 5 rats/sex/group whilst under CO2/O2 anaesthesia. Blood was collected in tubes filled with heparin (used an anticoagulant) and plasma was prepared by centrifugation. In each plasma sample the following determinations were carried out: Alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin (calculated), urea, creatinine, glucose (fasting), bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl), inorganic phosphate (PO4), bile acids.

HORMONE DETERMINATIONS
DDuring necropsy blood was taken from the aorta under CO2/O2 anaesthesia from all male and female adult animals for determination of TSH and T4 hormone levels in serum samples. This blood was collected to determine T4 and TSH hormone levels in serum samples. Samples were stored in a freezer (≤-18°C) until analysis.nalysis were performed with commercially available ELISA kits of Cloud-Clone Corp (kit CEA463Ra for TSH and kit CEA452Ge for T4, respectively). The ELISA was performed according to a validated method based on the manufacturer’s protocol.

NEUROBEHAVIOURAL EXAMINATION
Functional Observational Battery (FOB) tests and Motor Activity Assessment (MAA) were performed in 5 rats/sex/group shortly prior to sacrifice of the male and female rats (week 4 and week 8 of the study respectively). FOB tests and MAA were performed in male rats with the lowest identification numbers in each cage, and in females with a litter. On the morning of testing (at least one hour prior to the start of the observations) the selected animals were placed individually in macrolon cages in a waiting area in an examination room. After testing the animals were returned to the experimental room.
- FOB: The FOB used in the laboratory is adapted from the WHO/IPCS Functional Observational Battery that was used in the Collaborative Study on Neurotoxicity Assessment sponsored by the International Programme on Chemical Safety of the World Health Organization. Details on the conduct of observations included in this battery and operational definitions of the different scores for each item are given in the FOB-manual entitled "Functional Observational Battery. Operational Definitions" (Lammers, 2000). The FOB is a series of non-invasive observational and interactive measures designed to assess the neurobehavioral and functional integrity of the rat. First, measurements were carried out in the cage. The rat’s posture, palpebral closure and the possible presence of clonic and tonic convulsions were recorded. Then the rat was removed from the cage and the ease of removal and handling were rated. Palpebral closure and any lacrimation or salivation were also rated, and the presence or absence of piloerection and vocalizations was recorded. In addition, other signs, such as changes in skin and fur, exophthalmus, crustiness around the eyes, bite marks on the tail or paws, missing toe nails or emaciation (shallow stomach, protruding spinal vertebrae) were recorded. The rat was then placed in an open arena (77 l x 55 w x 7 h cm) and observed for 3 minutes. Rears (both supported and unsupported) were counted. At the same time, gait characteristics were recorded and ranked, the ease with which the rat locomoted was ranked, and arousal was assessed and recorded. Further, the occurrence of clonic and/or tonic convulsions, stereotypies and bizarre behavior was recorded. At the end of the observation period, the number of faecal boluses and urine pools were recorded. Following this observation period, reflex testing was conducted. Reflex testing consisted of recording the rat's responses to the approach of a pencil, a touch of a pencil to the rump, a click stimulus, tail pinch, and the constriction of the pupil to light. Aerial righting was rated next. Forelimb and hindlimb gripstrength were measured. Three valid determinations (from a maximum of five attempts) were taken for each gripstrength measure. The rectal temperature was taken with the rat restrained by hand. Finally, the hindlimb feet were painted lightly and landing foot splay was measured.
- Motor activity: Motor activity was assessed following FOB testing. Changes in spontaneous motor activity were assessed using an automated quantitative microprocessor-based video image analysis system. Rats were placed individually in open roofed cages measuring 48.8 l x 44.7 w x 50 h cm on the insides and equipped with a video camera suspended above the test cage. The position of the rat was continuously monitored throughout the test session. Spontaneous motor activity was expressed as the total distance run in a 30 minute test period. In addition, habituation of activity was evaluated. To this end, each session was divided into 5 time blocks of 6 minutes each. Motor activity tests were recorded on DVD, in order to enable re-analysis of motor activity tests should that be necessary for technical reasons. However, re-analysis was not necessary. Therefore, recordings will be removed from the study dossier after submission of the final report. Squads of up to eight animals were monitored simultaneously. Dose groups were evenly distributed for motor activity test cage and for time as much as possible. Motor activity testing of a squad was conducted immediately after functional observations for that squad had finished.

Oestrous cyclicity (parental animals):

Vaginal smears to evaluate the oestrus cycle length and normality were made daily from the start of the pre-treatment period, during the pre-mating period and until confirmation of mating. An additional vaginal smear was made at the day of sacrifice. Smears were stained and examined in all females.

Litter observations:

PARTURATION AND LITTER EVALUATION
At the end of the gestation period (GD 21), females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded. To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.

LITTER SIZE, SEXES AND WEIGHT
The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups were evaluated on days 0, 4, 7 and 13 of lactation. The pups were individually weighed on days 0, 4, 7 and 13 of lactation. Mean pup weight was calculated per sex and for both sexes combined per dose group.

ANOGENITAL DISTANCE IN PUPS
At lactation day 4 the anogenital distance (AGD) was measured of each pup before culling of the litter. The AGD is reported as mean per litter and corrected by the cube root of body weight.

CULLING AND BLOOD COLLECTION FOR HORMONE ANALYSIS
On lactation day 4 the litter size was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four per sex per litter. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment was applied. Preference was to retain four male pups in order to have sufficient male pups for nipple retention determinations on lactation day 13. If a litter had no surplus pups, no blood was collected for hormone determinations. Blood samples were collected from the surplus pups per litter at culling on lactation day 4 (blood of all culled pups of each litter was pooled, PN 4 pups were sacrificed by decapitation for blood collection) and from two pups per litter at sacrifice on or shortly after lactation day 13 (individual, blood was collected from the heart whilst under CO2/O2 anaesthesia). The blood samples were analysed for T4 and TSH hormone levels. Analysis were performed with commercially available ELISA kits of Cloud-Clone Corp (kit CEA463Ra for TSH and kit CEA452Ge for T4, respectively). The ELISA was performed according to a validated method based on the manufacturer’s protocol.

NIPPLE RETENTION IN MALE PUPS
On postnatal day 13 all surviving male pups were examined for the presence of nipples and/or areolas.

SIGNS
Any abnormal behaviour of pups was recorded on day 0, 4, 7 and 13 of lactation.

Postmortem examinations (parental animals):

SACRIFICE
Prior to sacrifice, all adult male and female animals were fasted overnight (water was freely available). All surviving male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anesthesia at necropsy and then examined grossly for pathological changes. All males were sacrificed after the mating period on day 29 of the study. The dams were sacrificed, after overnight fasting, on day 14 of lactation.

GROSS NECROPSY
At the day of necropsy, a vaginal smear was taken from each female for determination of the estrus cycle. At scheduled necropsy, the following organs, indicated below by an asterix (*), of the parent animals were weighed (paired organs together) as soon as possible after dissection to avoid drying. Samples of the following tissues and organs of the parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes, epididymides and ovaries, which were preserved in Bouin's fixative:
- Male reproductive organs for all males: Epididymides (paired)*, prostate (dorsolateral and ventral)*, seminal vesicles and coagulation glands (paired)*, testes*, Levator ani plus bulbocavernosus muscle complex Cowper’s glands and glans penis*.
- Female reproductive organs for all females: ovaries (paired)*, uterus including cervix*, vagina.
- All gross lesions were examined all males and females.
- The following organs of five parent animals/sex/group (surviving males with the lowest identification numbers in each cage; females with a litter were selected) were preserved: adrenal glands (paired)*, bone marrow (femur), brain* (including sections of cerebrum, cerebellum, medulla/pons), eye, heart*, kidney (paired)*, liver*, lungs with trachea and larynx (paired)*, mammary glands, mesenterial and axillary lymph nodes, peripheral nerve (sciatic or tibial), pituitary, small and large intestines (including Peyer’s patches), spinal cord, spleen*, stomach, thymus*, thyroid gland* (all males and females), urinary bladder.

HISTOPATHOLOGY
Tissues for microscopic examination were embedded in paraffin wax, sectioned, and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS haematoxylin. Microscopic examination was performed on the preserved organs of all animals of the control (group 1) and high-dose group (group 4). Furthermore, organs showing gross lesions of animals of all groups were microscopically examined.

Postmortem examinations (offspring):

Grossly malformed pups were sacrificed and examined. A necropsy was also performed on stillborn pups and pups that died during the study and macroscopic observations of these pups were recorded. At necropsy of the dams and litters, at or shortly after day 13 of lactation, pups were examined externally for gross abnormalities and killed by decapitation (except for the two pups per litter of which blood was collected for hormone analysis. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

Statistics:

Tests were generally two-sided tests, taken as significant if the probability was p<0.05 (*) or p<0.01 (**). Non-mated females excluded from mean data tables presenting data from the gestation and lactation periods.
- Continuous data: a decision tree for continuous data
- Dichotomous data: a decision tree for dichotomous data
- Functional observational battery: one-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
- Motor activity data: total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks (each session 5 time blocks of 6 minutes each).
- Clinical pathology data (haematology, clinical chemistry): ‘Generalized Anova/Ancova Test’ ), ‘Automatic’ data transformation method. This test is an automatic decision tree consisting of:
(1) Data preprocessing tests. First, normality of data distribution (Shapiro-Wilks test) and homogeneity of variances (Levene test) are checked. If any of these checks fail (p<0.05) they are repeated using Log transformation. If checks on log-transformed data fail, data are rank-transformed
(2) A group test assessing whether or not group means are all equal (parametric for untransformed or log-transformed data: one-way analysis of variance [Anova]; nonparametric for rank transformed data: Kruskal-Wallis test).
(3) Post-hoc analysis. If the group test shows significant (p<0.05) non-homogeneity of group means, pairwise comparisons with the control group are conducted by Dunnett’s multiple comparison test (parametric after Anova, non-parametric after Kruskal-Wallis; significance levels 0.01 and 0.05).
- Incidences of histopathological changes: Fisher’s exact probability test.

Reproductive indices:

- mating days until Day 0 pc = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
- female mating index = (number of females inseminated/number of females placed with males) x 100
- male mating index = (number of males placed with females /number of females inseminated) x 100
- female fertility index = number of pregnant females*100/number of inseminated females
- male fertility index = number of males with pregnant females*100/number of males placed with females
- gestation index = (number of females with live pups / number of females pregnant) x 100
- prenatal loss = (Total number of Implantations - Total number of pups delivered) x 100 / Total number of Implantation Sites
- perinatal loss = (Total number of pups delivered - Total number of alive pups delivered) x 100 / Total number of pups delivered.
- live birth index = (number of pups born alive/number of pups born) x 100
- sex ratio day 0 and 13 = [(number of live male or female pups on day 0 or 13/ number of live pups on day 0 or 13] x 100

Offspring viability indices:

- viability index day 0 - 4 = (number of pup surviving 4 days/number of liveborn on day 0) x100
- viability index day 4 - 13 = (number of pup surviving 13 days/number of liveborn on day 4) x100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related clinical signs during the premating period, mating period, post-mating period, gestation period or the lactation period. Home-cage observations and body temperature in female animals of the treatment-groups revealed a slightly increased activity. These observations were considered chance-findings since no effects in home-cage observations were observed in male animals and no clinical observations were observed outside the home cage.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on body weights and body weight changes of male and/or female animals during the pre-mating period, post-mating period, gestation period and the lactation period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no treatment-related effects on food consumption of male and/or female animals during the pre-mating period, post-mating period, gestation period and the lactation period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In male animals no differences in red blood cell and coagulation parameters were observed among the control and the treatment groups.
In female animals the number of red blood cells was statistically significantly higher in the low-dose group than in the control group. In the mid-dose group, the difference in the number of red blood cells did not reach the level of statistical significance whereas no difference was observed in the high-dose group. Due to these differences in the number of red blood cells and very slight differences in Haemoglobin and Packed Cell Volume, statistically significant decreases were observed on the Mean Corpuscular Volume (MCV – effects observed in the low- and mid-dose groups) and on Mean Corpuscular Haemoglobin (MCH – effects observed in all treatment groups). Since the differences observed were not related to dose, they were considered as chance-findings and not related to treatment.
No differences were observed on total and differential white blood cell parameters in male and female animals of the control and treatment groups, except for a statistically significantly higher number of neutrophils in the blood of female animals of the low-dose group. Since not dose-related, the difference in the number of neutrophils in the low-dose female animals was not considered as related to treatment.
In conclusion, there were no treatment-related changes in red blood cell and coagulation parameters and in total and differential white blood cell counts.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In male animals, no differences were observed in clinical chemistry parameters among the control and treatment groups.
In female animals, the concentration of creatinine in the low-dose group was statistically significantly higher than in control animals and the concentrations of PO4 and Ca were statistically significantly lower in the mid-dose and high-dose group, respectively, as compared to the control group. Since no dose-relationship could be established for creatinine and PO4 and because the concentration of Ca in the high-dose group was well within historical-control ranges, these findings were considered chance-findings and not related to treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Clinical observations outside the home cage, Functional Observation Battery (FOB) and motor activity assessment did not indicate any neurotoxic potential of the test substance in rats.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examinations revealed no treatment-related abnormalities. The histopathological findings were considered unremarkable and part of the background pathology of rats of this strain and age.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

No statistically significant effects were observed on TSH hormone and, except for a statistically significant increase of concentration of T4 in the blood of male parental animals of the high-dose group, on T4 hormone. The increased concentration of T4 as observed in the male parental animals of the high-dose group was considered a chance finding and of no toxicological significance since in male parental animals no effects were observed on any related parameter (e.g. body weight/growth, neurobehavioral performance, thyroid organ weight, microscopy of the thyroid) and no effects were observed in pups on PN day 4 and PN day 13.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No statistically significant effects were observed on the estrus cycle during the 2 weeks pretreatment and during the pre-mating phase, up to and including the day the animals were mated. In line with the microscopic examination of vagina and uterus, no remarkable differences were observed among the groups.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

- Fertility: The results of mating were not affected by treatment. In each group 12 females were placed with males for mating and all females were mated. The mean time to mating was comparable among the groups. Male and female mating and fertility indices were 100% for all groups. Pregnancy was not affected by treatment, all mated females were pregnant, the mean duration of gestation was comparable among the groups and the gestation index was 100% in all groups.
- Reproductive performance: All females delivered live born pups, there were no females with only stillborn pups. The number of litters with stillborn pups and the total number of stillborn within these litters (between brackets) accounted 2 (7), 1 (1), 0 (0), 1(1) for the control, low-, mid- and high-dose groups, respectively and in the mid-dose group there was one litter with one pup (pup 14 of dam 71) that was born alive (lungs were distended, see also but that was found dead at first inspection of the litters. The number of implantation sites and number of pups delivered were comparable among the groups. Consequently, no statistically significant differences were observed on prenatal loss between the control and treatment groups. The incidences of live- and stillborn pups and perinatal loss indices were also comparable among the groups (the number of still born pups and perinatal loss was highest in the control group).

Key result

Dose descriptor:

NOAEL

Effect level:

>= 377 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: corresponding to NOAEL ≥ 7500 mg/kg diet

Remarks on result:

other: based on the highest concentration tested

Dose descriptor:

NOAEL

Effect level:

>= 480 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: corresponding to NOAEL ≥ 7500 mg/kg diet

Remarks on result:

other: based on the highest concentration tested

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related signs in pups during the lactation period.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of stillborn pups or pups found dead at first litter inspection after birth accounted 7, 1, 1 and 1 for the control, low-, mid- and high-dose groups, respectively whereas the number of number of pups that died or were missing between days 0-4 accounted 2, 4, 2 and 0 for the control, low-, mid- and high dose, respectively. After culling on day 4, no pups were lost.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related differences in mean pup weights between the test groups and the controls on day 0, 4, 7 and 13 of lactation.

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 examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Pup thyroid weight: There were no effects of the weight of the thyroid of male and female pups on post-natal day 13.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic examinations of stillborn pups and pups that died did not reveal treatment-related effects. Macroscopic observations af pups at necropsy on postnatal day 13 revealed no abnormalities.

Histopathological findings:

no effects observed

Description (incidence and severity):

Microscopic examination revealed no treatment-related abnormalities.

Other effects:

no effects observed

Description (incidence and severity):

- Sex ratio: Sex ratio on day 0 and day 13 was comparable among the groups.
- Pup anogenital distance: There were no treatment related effects on the absolute (expressed in mm) and corrected (expressed in mm/g) anogenital distance of male and female pups.
- Nipple retention: There were no effects on nipple retention of male pups on post-natal day 13.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 480 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Corresponding to NOAEL ≥ 7500 mg/kg diet

Remarks on result:

other: based on the highest concentration tested

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

	Mean (range) substance intake (mg test substance/kg body weight/day)
	Low dose	Mid dose	High dose
Males premating	44.70 (43-46)	129.93 (126-134)	418.13 (400-436)
Males post mating	39.15^a)	116.57^a)	377.48^a)
Premating females	47.75 (47-49)	141.37 (139-144)	482.45 (480-485)
Gestation females	49.96 (49-51)	146.74 (141-151)	499.14 (483-521)
Lactation females	104.80 (80-125)	315.35 (237-381)	1036.97 (755-1298)

^a)No range presented

Test substance analysis:

- The relative standard deviation between the mean concentrations at three different locations was < 5% for all dose levels and/or p was ≥ 0.01. Therefore the test substance was considered to be homogeneously distributed in the diets.

- Upon storage in the animal room for 4 days and after storage at < -18 °C °C for 5 weeks, the relative difference in test substance concentration was ≤ 10% at all dose levels. Therefore, the test substance was considered to be stable in the diets under the experimental conditions.

- The concentration of the test substance was close to intended (90-110%) for all diets at all dose levels, except for the mid-dose level of the diets prepared on 31 August 2016 (concentration was 13% lower than intended).

Conclusions:

Based on the absence of effects on fertility in a study with Hyacinth body, the NOAEL for fertility was established at ≥7500 mg test substance per kg diet (corresponding to a dose of ≥377 mg/kg bw/day for males and ≥480 mg/kg bw/day for females).

Executive summary:

A reproduction/ developmental toxicity screening with Hyacinth body was performed according to OECD TG 422 and GLP in groups of 12 male and 12 female Wistar rats. The test substance was administered at constant concentrations in the diet at levels of 0 (control), 750, 2250 and 7500 mg/kg diet during a pre-mating period of 2 weeks and during mating, gestation until day 13 of lactation. The doses were based on a dose-range finding study and the anticipated formation of phenyl ethyl acid. In the dose-range finding study, after dietary administration of 0, 750, 2250 and 7500 mg Hyacinth body per kg diet to male and pregnant female rats for up to 21 consecutive days, statistically significant effects on food consumption and body weight changes were observed in high-dose female animals. The dosing period in the main study, especially for the female animals, was much longer than the 3-weeks dosing period of the DRF-study. Therefore, using the same concentrations, also in the main study effects were expected and higher concentrations were expected to result in profound toxic effects. Therefore, for the main study the same dose levels were chosen as in the DRF study. These dietary levels provided a mean daily test substance intake of at least 39, 117 and 377 mg/kg bw/day in males of the low-, mid- and high-dose group, respectively. In females, the mean test substance intake was at least 47, 139 and 480 mg/kg bw/day in the low-, mid- and high-dose group, respectively. Female animals and pups were sacrificed at or shortly after day 13 of lactation. Male animals were sacrificed after the mating period. Although the content of the diet of the mid-dose group of the second batch of diets prepared was slightly (13%) lower than intended, in general, the concentration of the test substance was close to intended concentration (90 -110%) and the content, homogeneity and stability of the test substance in the carrier were confirmed by analysis.

Repeated dose effects, clinical signs:

There was no treatment-related mortality. Daily clinical observations did not reveal any treatment-related clinical signs. Neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. There were no differences in body weights, body weight changes and food consumption during the pre-mating period, during the post-mating period in males, or in dams during the gestation period and the lactation period. Haematology and clinical chemistry conducted in 5 rats/sex/group at sacrifice did not reveal any treatment-related effects. No adverse effects on T4 and TSH hormones were observed in male and female parental animals, in culled pups on postnatal day 4 and in male and female pups on postnatal day 13. Organ weights of the adult male and female animals were comparable among the control and treatment groups. Macroscopic and microscopic examinations of adult animals did not reveal any treatment-related abnormalities.

Fertility:

There were also no effects of the test substance on male and female fertility, estrus cyclicity and reproductive performance.

Developmental toxicity:

In the pups, no treatment related effects were observed in observational studies, in any of the measured parameters.

In conclusion, no systemic effects were observed up to the highest dose. Since there were no systemic adverse effects and no effects on fertility parameters, reproductive performance and developmental parameters, the NOAEL for systemic toxicity and for reproduction is ≥7500 mg/kg diet (the highest concentration tested; equivalent to a mean test substance intake of at least 377 mg/kg bw/day in males and 480 mg/kg bw/day in females).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

377 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD 422 guideline study in compliance with GLP, available as unpublished report, no restrictions, fully adequate for assessment. The NOAEL is worst-case set at the highest achieved dose, as the maximum dose as proposed in the guideline was not tested.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A reproduction/ developmental toxicity screening with Hyacinth body was performed according to OECD TG 422 and GLP in groups of 12 male and 12 female Wistar rats. The test substance was administered at constant concentrations in the diet at levels of 0 (control), 750, 2250 and 7500 mg/kg diet during a pre-mating period of 2 weeks and during mating, gestation until day 13 of lactation. The doses were based on a dose-range finding study and the anticipated formation of phenyl ethyl acid. In the dose-range finding study, after dietary administration of 0, 750, 2250 and 7500 mg Hyacinth body per kg diet to male and pregnant female rats for up to 21 consecutive days, statistically significant effects on food consumption and body weight changes were observed in high-dose female animals. The dosing period in the main study, especially for the female animals, was much longer than the 3-weeks dosing period of the DRF-study. Therefore, using the same concentrations, also in the main study effects were expected and higher concentrations were expected to result in profound toxic effects. Therefore, for the main study the same dose levels were chosen as in the DRF study. These dietary levels provided a mean daily test substance intake of at least 39, 117 and 377 mg/kg bw/day in males of the low-, mid- and high-dose group, respectively. In females, the mean test substance intake was at least 47, 139 and 480 mg/kg bw/day in the low-, mid- and high-dose group, respectively. Female animals and pups were sacrificed at or shortly after day 13 of lactation. Male animals were sacrificed after the mating period. Although the content of the diet of the mid-dose group of the second batch of diets prepared was slightly (13%) lower than intended, in general, the concentration of the test substance was close to intended concentration (90 -110%) and the content, homogeneity and stability of the test substance in the carrier were confirmed by analysis.

Repeated dose effects, clinical signs:

There was no treatment-related mortality. Daily clinical observations did not reveal any treatment-related clinical signs. Neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. There were no differences in body weights, body weight changes and food consumption during the pre-mating period, during the post-mating period in males, or in dams during the gestation period and the lactation period. Haematology and clinical chemistry conducted in 5 rats/sex/group at sacrifice did not reveal any treatment-related effects. No adverse effects on T4 and TSH hormones were observed in male and female parental animals, in culled pups on postnatal day 4 and in male and female pups on postnatal day 13. Organ weights of the adult male and female animals were comparable among the control and treatment groups. Macroscopic and microscopic examinations of adult animals did not reveal any treatment-related abnormalities.

Fertility:

There were also no effects of the test substance on male and female fertility, estrus cyclicity and reproductive performance.

Developmental toxicity:

In the pups, no treatment related effects were observed in observational studies, in any of the measured parameters.

In conclusion, no systemic effects were observed up to the highest dose. Since there were no systemic adverse effects and no effects on fertility parameters, reproductive performance and developmental parameters, the NOAEL for systemic toxicity and for reproduction is ≥7500 mg/kg diet (the highest concentration tested; equivalent to a mean test substance intake of at least 377 mg/kg bw/day in males and 480 mg/kg bw/day in females).

Effects on developmental toxicity

Description of key information

Developmental toxicity (OECD TG 422): Maternal NOAEL ≥480 mg/kg bw/day (highest dose tested)

Developmental toxicity (OECD TG 422): Developmental NOAEL ≥480 mg/kg bw/day (highest dose tested)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08-08-2016 to 03-10-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Remarks:

endpoint 7.5.1

Reason / purpose for cross-reference:

reference to same study

Remarks:

endpoint 7.8.1

Qualifier:

according to guideline

Guideline:

other: OECD guideline 422

Version / remarks:

28 July 2015

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han IGS

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 9 weeks (males) or 10 weeks (females)
- Weight at study initiation: Mean body weight just before the start of treatment was 330 grams for males and 224 grams for females.
- Housing: The rats were housed in makrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. During the pre-treatment and pre-mating period the animals were housed four rats to a cage (separated by sex). For mating, one male and one female were housed together. Mated females were housed individually in cages, which were placed in another cage rack.
- Diet: The rats receive a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England), ad libitum
- Water: Tap-water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 45-65; slightly below 45 (minimum 43.3) and slightly above 65 (maximum 67.1) for short periods of time
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh batches of the experimental diets were prepared on 4 and 31 August 2016 and stored in a freezer in plastic bags in portions sufficient for three to four days. The food in the cans was replaced twice per week with fresh portions from the freezer.
- Mixing appropriate amounts with (Type of food): The diets were mixed in a mechanical blender.
- Storage temperature of food: Freezer <-18°C
- Other: The diets were given as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

From both batches of diets prepared in the study (4 and 31 August 2016) samples were taken and stored in a freezer (≤ -18°C). The content of the test substance at each dietary level was determined in the batches prepared on 4 and 31 August 2016. The homogeneity of the test substance in the experimental diets was assessed in the first batch (4 August 2016), by analysing five samples (taken at different locations in the feed container) of each test diet in duplicate. One sample of the control diet was analysed. To demonstrate the stability of the test substance under experimental conditions, samples of the batch of test diets prepared on 4 August 2016 (low-dose diet, mid-dose diet and high-dose diet) were analyzed at t=0 and analyzed after storage in the animal room (in an open container) for 4 days, and after storage in a freezer (< -18 °C) for at least 5 weeks.

The concentration of the test substance in diet was determined using the following procedure: the test substance was extracted from diet using dichloromethane under shaking and sonication. After dilution with dichloromethane and centrifugation the diluted extracts were analyzed using Gas Chromatography – Mass Spectrometry (GC-MS). Undecane was used as internal standard. The content of the test substance in diet was considered to be “close to intended” if the mean measured concentration was between 90 and 110% of the intended concentration.
Before analysis of study samples, the analytical method was validated by analyzing three spiked samples per dose level, to conform to the following criteria:
- Linearity: the correlation coefficient of the calibration curve should be greater than or equal to 0.996.
- Recovery: the mean recovery of the test substance from diet should be between 80% and 110% at each of the dose levels of the study.
- Repeatability: the relative standard deviation in the percentage recovery, when the recovery test is performed three times at each of the dose levels to be used in the toxicity study, should be less than 10%.
With respect to specificity: signals should be corrected in case the signal obtained for blank samples was ≥ 5% of the signal obtained for low-dose samples.

The homogeneity of the test substance was assessed in the batch of diets, prepared for the study on 04 August 2016. Five samples of each test diet, taken at different locations in the diet container, and 1 sample of the control diet were analyzed in duplicate. For each concentration level, a one-way analysis of variance (Anova) was performed using the sample location (1-5) as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the mean concentrations differ significantly at the five locations in the diet container). The test substance was considered to be homogeneously distributed in the diet if p ≥ 0.01 and/or if the relative standard deviation (RSD) between the mean concentrations at the five locations was ≤ 5%.

The stability of the test substance in diet was assessed in the batch of diets, prepared for the study on 04 August 2016 (4 days animal room and 5 weeks at < -18°C). One sample of each test diet and one sample of the control diet were analyzed in duplicate. For each concentration level, a one-way analysis of variance (Anova) was performed using time as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the mean concentrations differ significantly before and after storage). The test substance was considered to be stable in diet if p ≥ 0.01 and/or if the mean concentration after storage was within 90-110% of the mean concentration at t = 0.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: each female was caged with one male from the same dose group, Mating pairs were clearly identified.
- Length of cohabitation: animals were caged together for maximally one week until mating occurred of at least 10 animals per group, upon evidence of copulation the females were caged individually for the birth and rearing of their pups
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol:

Duration of treatment / exposure:

Male animals were fed the experimental diets containing the test substance during a 2-week pre-mating period, during mating and until sacrifice after at least 28 days of exposure. The female animals were fed the experimental diets during a 2-week pre-mating period, during mating, gestation and lactation up to the day of sacrifice (at, or shortly after, day 13 of lactation).

Frequency of treatment:

Continously

Duration of test:

Until postnatal day 13

Dose / conc.:

750 mg/kg diet

Remarks:

Equivalent to at least 39 and 47 mg/kg bw/day for males and females, respectively

Dose / conc.:

2 250 mg/kg diet

Remarks:

Equivalent to at least 117 and 139 mg/kg bw/day for males and females, respectively

Dose / conc.:

7 500 mg/kg diet

Remarks:

Equivalent to at least 377 and 480 mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

12

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected in consultation with the sponsor and are based on the results of a 3-weeks dose-range finding study with the test substance in rats.

Maternal examinations:

CAGE SIDE OBSERVATIONS
Each animal was observed daily in the morning hours by cage-side observations. All cages were checked again in the afternoon for dead or moribund animals. All abnormalities, signs of ill health or reactions to treatment were recorded.

DETAILED CLINICAL OBSERVATIONS
Detailed clinical examinations were conducted in all rats of all groups in the experimental room outside the home cage prior to the first exposure and then once weekly up to and including week 8. During the last week of gestation (week 6 of the study) and during the first week of lactation (week 7 of the study) detailed clinical observations were not performed in order not to disturb the pregnant females or litters. Detailed clinical examinations were performed as part of the Functional Observational Battery test in the selected female rats in week 4 and 8 of the study respectively. Signs noted included, but were not limited to changes in skin and fur, piloerection, changes in the eyes, gait (including posture), and presence of clonic or tonic movements, stereotypies and bizarre behaviour.

BODY WEIGHT
Body weights were recorded just before the start of the treatment (to enable randomization) and at the start of the study (day 0). Subsequently females were weighed once per week during the pre-mating and mating period. Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation and on day 0, 4, 7 and 13 of lactation. The animals were weighed on their scheduled necropsy date in order to calculate the organ to body weight ratios.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption: Food consumption was measured per cage for the same periods as the body weight are measured, except during the mating period when food intake was not registered. The results are expressed in g per animal per day.
- Intake of test substance: The intake of the test substance per kg body weight per day was calculated from the nominal dietary concentrations, the food consumption and the body weight for males and females separately.

HAEMATOLOGY
Prior to sacrifice, all animals were fasted overnight (water was freely available). At sacrifice, blood was taken was taken from the abdominal aorta of 5 rats/group whilst under CO2/O2 anaesthesia. For prothrombin time citrate was used as anticoagulant. For the other parameters EDTA was used as anticoagulant. In each sample the following determinations were carried out: Haemoglobin (Hb), packed cell volume (PCV), red blood cell count (RBC), reticulocytes, total white blood cell count (WBC), differential white blood cell counts (neutrophils, lymphocytes, eosinophils, basophils, monocytes), prothrombin time, thrombocyte count, mean corpuscular volume (MCV; calculated), mean corpuscular haemoglobin (MCV; calculated), mean corpuscular haemoglobin concentration (MCHC; calculated)

CLINICAL CHEMISTRY
Prior to sacrifice, all animals were fasted overnight (water was freely available). At sacrifice, blood was taken from the abdominal aorta of 5 rats/group whilst under CO2/O2 anaesthesia. Blood was collected in tubes filled with heparin (used an anticoagulant) and plasma was prepared by centrifugation. In each plasma sample the following determinations were carried out: Alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin (calculated), urea, creatinine, glucose (fasting), bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl), inorganic phosphate (PO4), bile acids.

HORMONE DETERMINATIONS
During necropsy blood was taken from the aorta under CO2/O2 anaesthesia from all female adult animals for determination of TSH and T4 hormone levels in serum samples. In addition, blood samples were collected from the surplus pups per litter at culling on lactation day 4 (blood of all culled pups of each litter was pooled, PN 4 pups were sacrificed by decapitation for blood collection) and from two pups per litter at sacrifice on or shortly after lactation day 13 (individual, blood was collected from the heart whilst under CO2/O2 anaesthesia).The blood samples were analysed for T4 and TSH hormone levels. Analysis were performed with commercially available ELISA kits of Cloud-Clone Corp (kit CEA463Ra for TSH and kit CEA452Ge for T4, respectively). The ELISA was performed according to a validated method based on the manufacturer’s protocol.

NEUROBEHAVIOURAL EXAMINATION
Functional Observational Battery (FOB) tests and Motor Activity Assessment (MAA) were performed in 5 rats/sex/group shortly prior to sacrifice of female rats (week 8 of the study respectively). FOB tests and MAA were performed in females with a litter. On the morning of testing (at least one hour prior to the start of the observations) the selected animals were placed individually in macrolon cages in a waiting area in an examination room. After testing the animals were returned to the experimental room.
- FOB: The FOB used in the laboratory is adapted from the WHO/IPCS Functional Observational Battery that was used in the Collaborative Study on Neurotoxicity Assessment sponsored by the International Programme on Chemical Safety of the World Health Organization. Details on the conduct of observations included in this battery and operational definitions of the different scores for each item are given in the FOB-manual entitled "Functional Observational Battery. Operational Definitions" (Lammers, 2000). The FOB is a series of non-invasive observational and interactive measures designed to assess the neurobehavioral and functional integrity of the rat. First, measurements were carried out in the cage. The rat’s posture, palpebral closure and the possible presence of clonic and tonic convulsions were recorded. Then the rat was removed from the cage and the ease of removal and handling were rated. Palpebral closure and any lacrimation or salivation were also rated, and the presence or absence of piloerection and vocalizations was recorded. In addition, other signs, such as changes in skin and fur, exophthalmus, crustiness around the eyes, bite marks on the tail or paws, missing toe nails or emaciation (shallow stomach, protruding spinal vertebrae) were recorded. The rat was then placed in an open arena (77 l x 55 w x 7 h cm) and observed for 3 minutes. Rears (both supported and unsupported) were counted. At the same time, gait characteristics were recorded and ranked, the ease with which the rat locomoted was ranked, and arousal was assessed and recorded. Further, the occurrence of clonic and/or tonic convulsions, stereotypies and bizarre behavior was recorded. At the end of the observation period, the number of faecal boluses and urine pools were recorded. Following this observation period, reflex testing was conducted. Reflex testing consisted of recording the rat's responses to the approach of a pencil, a touch of a pencil to the rump, a click stimulus, tail pinch, and the constriction of the pupil to light. Aerial righting was rated next. Forelimb and hindlimb gripstrength were measured. Three valid determinations (from a maximum of five attempts) were taken for each gripstrength measure. The rectal temperature was taken with the rat restrained by hand. Finally, the hindlimb feet were painted lightly and landing foot splay was measured.
- Motor activity: Motor activity was assessed following FOB testing. Changes in spontaneous motor activity were assessed using an automated quantitative microprocessor-based video image analysis system. Rats were placed individually in open roofed cages measuring 48.8 l x 44.7 w x 50 h cm on the insides and equipped with a video camera suspended above the test cage. The position of the rat was continuously monitored throughout the test session. Spontaneous motor activity was expressed as the total distance run in a 30 minute test period. In addition, habituation of activity was evaluated. To this end, each session was divided into 5 time blocks of 6 minutes each. Motor activity tests were recorded on DVD, in order to enable re-analysis of motor activity tests should that be necessary for technical reasons. However, re-analysis was not necessary. Therefore, recordings will be removed from the study dossier after submission of the final report. Squads of up to eight animals were monitored simultaneously. Dose groups were evenly distributed for motor activity test cage and for time as much as possible. Motor activity testing of a squad was conducted immediately after functional observations for that squad had finished.

POST-MORTEM EXAMINATIONS
- Prior to sacrifice, all female animals were fasted overnight (water was freely available). All surviving female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anesthesia at necropsy and then examined grossly for pathological changes. All males were sacrificed after the mating period on day 29 of the study. The dams were sacrificed, after overnight fasting, on day 14 of lactation.
- Gross necropsy: At the day of necropsy, a vaginal smear was taken from each female for determination of the estrus cycle. At scheduled necropsy, the following organs, indicated below by an asterix (*), of the parent animals were weighed (paired organs together) as soon as possible after dissection to avoid drying. Samples of the following tissues and organs of the parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes, epididymides and ovaries, which were preserved in Bouin's fixative: Female reproductive organs for all females: ovaries (paired)*, uterus including cervix*, vagina. All gross lesions were examined all females. The following organs of five parent animals/group (surviving males with the lowest identification numbers in each cage; females with a litter were selected) were preserved: adrenal glands (paired)*, bone marrow (femur), brain* (including sections of cerebrum, cerebellum, medulla/pons), eye, heart*, kidney (paired)*, liver*, lungs with trachea and larynx (paired)*, mammary glands, mesenterial and axillary lymph nodes, peripheral nerve (sciatic or tibial), pituitary, small and large intestines (including Peyer’s patches), spinal cord, spleen*, stomach, thymus*, thyroid gland* (all females), urinary bladder.
- Histopathology: Tissues for microscopic examination were embedded in paraffin wax, sectioned, and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS haematoxylin. Microscopic examination was performed on the preserved organs of all animals of the control (group 1) and high-dose group (group 4). Furthermore, organs showing gross lesions of animals of all groups were microscopically examined.

Ovaries and uterine content:

Ovaries and uterus were examined. The number of implantation sites and resorptions were counted.

Fetal examinations:

PARTURATION AND LITTER EVALUATION
At the end of the gestation period (GD 21), females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded. To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.

LITTER SIZE, SEXES AND WEIGHT
The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups were evaluated on days 0, 4, 7 and 13 of lactation. The pups were individually weighed on days 0, 4, 7 and 13 of lactation. Mean pup weight was calculated per sex and for both sexes combined per dose group.

ANOGENITAL DISTANCE IN PUPS
At lactation day 4 the anogenital distance (AGD) was measured of each pup before culling of the litter. The AGD is reported as mean per litter and corrected by the cube root of body weight.

CULLING AND BLOOD COLLECTION FOR HORMONE ANALYSIS
On lactation day 4 the litter size was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four per sex per litter. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment was applied. Preference was to retain four male pups in order to have sufficient male pups for nipple retention determinations on lactation day 13. If a litter had no surplus pups, no blood was collected for hormone determinations. Blood samples were collected from the surplus pups per litter at culling on lactation day 4 (blood of all culled pups of each litter was pooled, PN 4 pups were sacrificed by decapitation for blood collection) and from two pups per litter at sacrifice on or shortly after lactation day 13 (individual, blood was collected from the heart whilst under CO2/O2 anaesthesia). The blood samples were analysed for T4 and TSH hormone levels. Analysis were performed with commercially available ELISA kits of Cloud-Clone Corp (kit CEA463Ra for TSH and kit CEA452Ge for T4, respectively). The ELISA was performed according to a validated method based on the manufacturer’s protocol.

NIPPLE RETENTION IN MALE PUPS
On postnatal day 13 all surviving male pups were examined for the presence of nipples and/or areolas.

SIGNS AND PATHOLOGY
Any abnormal behaviour of pups was recorded on day 0, 4, 7 and 13 of lactation. Grossly malformed pups were sacrificed and examined. A necropsy was also performed on stillborn pups and pups that died during the study and macroscopic observations of these pups were recorded. At necropsy of the dams and litters, at or shortly after day 13 of lactation, pups were examined externally for gross abnormalities and killed by decapitation (except for the two pups per litter of which blood was collected for hormone analysis. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

Statistics:

Tests were generally two-sided tests, taken as significant if the probability was p<0.05 (*) or p<0.01 (**). Non-mated females excluded from mean data tables presenting data from the gestation and lactation periods.
- Continuous data: a decision tree for continuous data
- Dichotomous data: a decision tree for dichotomous data
- Functional observational battery: one-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
- Motor activity data: total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks (each session 5 time blocks of 6 minutes each).
- Clinical pathology data (haematology, clinical chemistry): ‘Generalized Anova/Ancova Test’ ), ‘Automatic’ data transformation method. This test is an automatic decision tree consisting of:
(1) Data preprocessing tests. First, normality of data distribution (Shapiro-Wilks test) and homogeneity of variances (Levene test) are checked. If any of these checks fail (p<0.05) they are repeated using Log transformation. If checks on log-transformed data fail, data are rank-transformed
(2) A group test assessing whether or not group means are all equal (parametric for untransformed or log-transformed data: one-way analysis of variance [Anova]; nonparametric for rank transformed data: Kruskal-Wallis test).
(3) Post-hoc analysis. If the group test shows significant (p<0.05) non-homogeneity of group means, pairwise comparisons with the control group are conducted by Dunnett’s multiple comparison test (parametric after Anova, non-parametric after Kruskal-Wallis; significance levels 0.01 and 0.05).
- Incidences of histopathological changes: Fisher’s exact probability test.

Indices:

- mating days until Day 0 pc = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
- female mating index = (number of females inseminated/number of females placed with males) x 100
- male mating index = (number of males placed with females /number of females inseminated) x 100
- female fertility index = number of pregnant females*100/number of inseminated females
- male fertility index = number of males with pregnant females*100/number of males placed with females
- gestation index = (number of females with live pups / number of females pregnant) x 100
- prenatal loss = (Total number of Implantations - Total number of pups delivered) x 100 / Total number of Implantation Sites
- perinatal loss = (Total number of pups delivered - Total number of alive pups delivered) x 100 / Total number of pups delivered.
- live birth index = (number of pups born alive/number of pups born) x 100
- viability index day 0 - 4 = (number of pup surviving 4 days/number of liveborn on day 0) x100
- viability index day 4-13 = (number of pup surviving 13 days/number of liveborn on day 4) x100
- sex ratio day 0 and 13 = [(number of live male or female pups on day 0 or 13/ number of live pups on day 0 or 13] x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related clinical signs during the premating period, mating period, post-mating period, gestation period or the lactation period. Home-cage observations and body temperature in female animals of the treatment-groups revealed a slightly increased activity. These observations were considered chance-findings since no effects in home-cage observations were observed in male animals and no clinical observations were observed outside the home cage.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on body weights and body weight changes of female animals during the pre-mating period, post-mating period, gestation period and the lactation period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no treatment-related effects on food consumption of female animals during the pre-mating period, post-mating period, gestation period and the lactation period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In female animals the number of red blood cells was statistically significantly higher in the low-dose group than in the control group. In the mid-dose group, the difference in the number of red blood cells did not reach the level of statistical significance whereas no difference was observed in the high-dose group. Due to these differences in the number of red blood cells and very slight differences in Haemoglobin and Packed Cell Volume, statistically significant decreases were observed on the Mean Corpuscular Volume (MCV – effects observed in the low- and mid-dose groups) and on Mean Corpuscular Haemoglobin (MCH – effects observed in all treatment groups). Since the differences observed were not related to dose, they were considered as chance-findings and not related to treatment.
No differences were observed on total and differential white blood cell parameters in female animals of the control and treatment groups, except for a statistically significantly higher number of neutrophils in the blood of female animals of the low-dose group. Since not dose-related, the difference in the number of neutrophils in the low-dose female animals was not considered as related to treatment.
In conclusion, there were no treatment-related changes in red blood cell and coagulation parameters and in total and differential white blood cell counts.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In female animals, the concentration of creatinine in the low-dose group was statistically significantly higher than in control animals and the concentrations of PO4 and Ca were statistically significantly lower in the mid-dose and high-dose group, respectively, as compared to the control group. Since no dose-relationship could be established for creatinine and PO4 and because the concentration of Ca in the high-dose group was well within historical-control ranges, these findings were considered chance-findings and not related to treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Clinical observations outside the home cage, Functional Observation Battery (FOB) and motor activity assessment did not indicate any neurotoxic potential of the test substance in rats.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

In female animals, no significant differences were observed in the mean absolute and the mean relative organ weights between the control and treatment groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy revealed no treatment-related abnormalities. The findings were unremarkable and part of the background pathology of rats of this strain and age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examinations revealed no treatment-related abnormalities. The histopathological findings were considered unremarkable and part of the background pathology of rats of this strain and age.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No statistically significant effects were observed on TSH hormone and on T4 hormone.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The number of implantation sites and number of pups delivered were comparable among the groups. Consequently, no statistically significant differences were observed on prenatal loss between the control and treatment groups. The incidences of live- and stillborn pups and perinatal loss indices were also comparable among the groups (the number of still born pups and perinatal loss was highest in the control group).

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

All females delivered live born pups, there were no females with only stillborn pups. The number of litters with stillborn pups and the total number of stillborn within these litters (between brackets) accounted 2 (7), 1 (1), 0 (0), 1(1) for the control, low-, mid- and high-dose groups, respectively and in the mid-dose group there was one litter with one pup (pup 14 of dam 71) that was born alive (lungs were distended) but that was found dead at first inspection of the litters.
The incidences of live- and stillborn pups and perinatal loss indices were also comparable among the groups (the number of still born pups and perinatal loss was highest in the control group).

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Pregnancy was not affected by treatment, the mean duration of gestation was comparable among the groups.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The results of mating was not affected by treatment. In each group 12 females were placed with males for mating and all females were mated. The mean time to mating was comparable among the groups. Male and female mating and fertility indices were 100% for all groups.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 480 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: corresponding to NOAEL ≥7500 mg/kg diet

Remarks on result:

other: based on the highest concentration tested

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related differences in mean pup weights between the test groups and the controls on day 0, 4, 7 and 13 of lactation.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of pups delivered per litter and the incidences of live born pups were comparable among the groups. The number of stillborn or pups found dead at first litter inspection after birth accounted 7, 1, 1 and 1 for the control, low-, mid- and high-dose groups, respectively whereas the number of number of pups that died or were missing between days 0-4 accounted 2, 4, 2 and 0 for the control, low-, mid- and high dose, respectively.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio on day 0 and day 13 was comparable among the groups.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

The number of pups that died or were missing between days 0-4 accounted 2, 4, 2 and 0 for the control, low-, mid- and high dose, respectively. After culling on day 4, no pups were lost.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no treatment related effects on the absolute (expressed in mm) and corrected (expressed in mm/g) anogenital distance of male and female pups.
- There were no effects on nipple retention of male pups on post-natal day 13.
- Macroscopic examinations of stillborn pups and pups that died did not reveal treatment-related effects.
- Macroscopic observations af pups at necropsy on postnatal day 13 revealed no abnormalities.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Microscopic examination revealed no treatment-related abnormalities.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 480 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Corresponding to NOAEL ≥ 7500 mg/kg diet

Remarks on result:

other: based on the highest concentration tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

	Mean (range) substance intake (mg test substance/kg body weight/day)
	Low dose	Mid dose	High dose
Premating females	47.75 (47-49)	141.37 (139-144)	482.45 (480-485)
Gestation females	49.96 (49-51)	146.74 (141-151)	499.14 (483-521)
Lactation females	104.80 (80-125)	315.35 (237-381)	1036.97 (755-1298)

Test substance analysis:

- The relative standard deviation between the mean concentrations at three different locations was < 5% for all dose levels and/or p was ≥ 0.01. Therefore the test substance was considered to be homogeneously distributed in the diets.

- Upon storage in the animal room for 4 days and after storage at < -18 °C °C for 5 weeks, the relative difference in test substance concentration was ≤ 10% at all dose levels. Therefore, the test substance was considered to be stable in the diets under the experimental conditions.

- The concentration of the test substance was close to intended (90-110%) for all diets at all dose levels, except for the mid-dose level of the diets prepared on 31 August 2016 (concentration was 13% lower than intended).

Conclusions:

Based on the absence of effects on developmental parameters in the study with Hyacinth body, the NOAEL for developmental toxicity was established at ≥7500 mg test substance per kg diet (corresponding to a dose of ≥480 mg/kg bw/day for females).

Executive summary:

A reproduction/ developmental toxicity screening with Hyacinth body was performed according to OECD TG 422 and GLP in groups of 12 male and 12 female Wistar rats. The test substance was administered at constant concentrations in the diet at levels of 0 (control), 750, 2250 and 7500 mg/kg diet during a pre-mating period of 2 weeks and during mating, gestation until day 13 of lactation. The doses were based on a dose-range finding study and the anticipated formation of phenyl ethyl acid. In the dose-range finding study, after dietary administration of 0, 750, 2250 and 7500 mg Hyacinth body per kg diet to male and pregnant female rats for up to 21 consecutive days, statistically significant effects on food consumption and body weight changes were observed in high-dose female animals. The dosing period in the main study, especially for the female animals, was much longer than the 3-weeks dosing period of the DRF-study. Therefore, using the same concentrations, also in the main study effects were expected and higher concentrations were expected to result in profound toxic effects. Therefore, for the main study the same dose levels were chosen as in the DRF study. These dietary levels provided a mean daily test substance intake of at least 39, 117 and 377 mg/kg bw/day in males of the low-, mid- and high-dose group, respectively. In females, the mean test substance intake was at least 47, 139 and 480 mg/kg bw/day in the low-, mid- and high-dose group, respectively. Female animals and pups were sacrificed at or shortly after day 13 of lactation. Male animals were sacrificed after the mating period. Although the content of the diet of the mid-dose group of the second batch of diets prepared was slightly (13%) lower than intended, in general, the concentration of the test substance was close to intended concentration (90 -110%) and the content, homogeneity and stability of the test substance in the carrier were confirmed by analysis.

Repeated dose effects, clinical signs:

There was no treatment-related mortality. Daily clinical observations did not reveal any treatment-related clinical signs. Neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. There were no differences in body weights, body weight changes and food consumption during the pre-mating period, during the post-mating period in males, or in dams during the gestation period and the lactation period. Haematology and clinical chemistry conducted in 5 rats/sex/group at sacrifice did not reveal any treatment-related effects. No adverse effects on T4 and TSH hormones were observed in male and female parental animals, in culled pups on postnatal day 4 and in male and female pups on postnatal day 13. Organ weights of the adult male and female animals were comparable among the control and treatment groups. Macroscopic and microscopic examinations of adult animals did not reveal any treatment-related abnormalities.

Fertility:

There were also no effects of the test substance on male and female fertility, estrus cyclicity and reproductive performance.

Developmental toxicity:

In the pups, no treatment related effects were observed in observational studies, in any of the measured parameters.

In conclusion, no systemic effects were observed up to the highest dose. Since there were no systemic adverse effects and no effects on fertility parameters, reproductive performance and developmental parameters, the NOAEL for systemic toxicity and for reproduction is ≥7500 mg/kg diet (the highest concentration tested; equivalent to a mean test substance intake of at least 377 mg/kg bw/day in males and 480 mg/kg bw/day in females).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

480 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD 422 guideline study in compliance with GLP, available as unpublished report, no restrictions, fully adequate for assessment. The NOAEL is worst-case set at the highest achieved dose, as the maximum dose as proposed in the guideline was not tested.

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

See additional information in the Fertility section.

Justification for classification or non-classification

Based on the available data, Hyacinth body does not need to be classified for toxicity to reproduction in accordance with EU CLP (EC No. 1272/2008 and its amendments).