Benzoic acid, 2,3,4,5-tetrachloro-6-cyano-, methyl ester, reaction products with p-phenylenediamine and sodium methoxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Screening study for reproductive / developmental toxicity: according to OECD 422, GLP, rat, oral NOAEL for reproductive performance and fertility 1000 mg/kg bw/d (male and female)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Jun 2020 - 10 Mar 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

July 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2020

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Basis for dose level selection: The dose levels were selected based on a previous range-finding study and at the request of the Sponsor.
- Route of administration: The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals: The rat is the preferred animal species for reproduction studies according to the various test guidelines and the Wistar strain was selected. This Wistar rat strain (Crl:WI(Han)) was selected since extensive historical control data were available on these Wistar rats.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Not specified
- Lot/batch number of test material: 0018514410
- Purity, including information on contaminants, isomers, etc.: 99.0 g/100 g (project 19L00260)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (room temperature)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The stability of the test substance in 0.5% Sodium carboxymethyl cellulose in deionized water over a period of at least 7 days at room temperature was given. As the mixtures were stored no longer than this time period, the stability was guaranteed. The homogeneous distribution of the test substance in 0.5% Sodium carboxymethyl cellulose in deionized water was demonstrated.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final concentration of a dissolved solid, stock liquid or gel: Please, see section "Doses/concentrations"

FORM AS APPLIED IN THE TEST: Suspension in vehicle

OTHER SPECIFICS
- Physical state/ appearance: Solid/ orange

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: About 11 - 12 weeks (males); About 10 weeks (females)
- Weight at study initiation: 374.6 g (mean males); 219.6 g (mean females)
- Fasting period before study: No
- Housing: Up to 5 animals per sex and cage during pretreatment; up to 2 animals per sex and cage during premating; up to 1 animal per sex and cage with exception during mating (1 male/1 female per cage) and rearing up to PND 13 (1 dam with her litter); for motor activity (MA) measurements the animals were housed individually.
- Diet (e.g. ad libitum): Mouse and rat maintenance diet “GLP” (ad libitum)
- Water (e.g. ad libitum): water bottles (ad libitum)
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY:
- The supplier assayed the food used in the study for chemical and microbiological contaminants. On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable.
- The drinking water is regularly assayed for chemical contaminants as well as for the presence of microorganisms by a contract laboratory. On the basis of the analytical findings the drinking water was found to be suitable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours (light from 06.00-18.00 h, dark from 18.00-06.00 h)

IN-LIFE DATES: From: 16 Jun 2020 To: 09 Sep 2020 (Sacrifice of parental females)

Route of administration:

oral: gavage

Vehicle:

other: 0.5% Sodium carboxymethyl cellulose in deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the preparation of the administration suspensions the test substance was weighed in a graduated flask depending on the dose group, topped up with 0.5% Sodium carboxymethyl cellulose in deionized water and intensely mixed with magnetic stirrer until it was completely homogenized. From 14 Aug 2020 onwards the test substance preparations were homogenized with an Ultraturrax instead of a magnetic stirrer. The portions of the test substance preparations for the daily administration were stored at room temperature. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: 0, 0.67, 2.00, 6.67 g/100mL
- Amount of vehicle (if gavage): 15 mL/kg bw/d
- Purity: not specified

Details on mating procedure:

- Time point: Two weeks after the beginning of treatment, the surviving males and females from the same test group were mated
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight, from about 16.00 h until 06.30 - 09.00 h of the following morning (for a maximum of 2 weeks)
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day (GD) 0
- After successful mating each pregnant female was caged (how): 1 animal per cage provided with nesting material (cellulose wadding) toward the end of gestation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration control analysis was assessed by UV/VIS spectrospcopy.
The samples for concentration control analysis, which were taken at the start of the administration period (premating), were not measured in time directly after sampling as expected. Therefore, these samples were discarded without measurement.
Therefore, an additional sample set were taken during gestation on 03 Aug 2020 and send to the Analytical Laboratory for analysis in time. This sample set demonstrated equivocal results with values outside of the desired range of the test facility. It was assumed that these results were due to the sampling process and did not represent the actual test substance preparations.
Therefore two further sample sets, taken during the lactation period on 14 Aug 2020 and 18 Aug 2020 were sent to the Analytical Laboratory and analyzed in time.The results of the sample sets during the lactation period demonstrated the correctness of the concentrations of the test substance in 0.5% Sodium carboxymethyl cellulose in deionized water because all samples were clearly found to be in the range of 90 - 110%. The mean of all three mean values of the high-dose group (including the questionable analysis during gestation) is close to the desired range of the test facility (mean: 86%). Therefore, in general, the concentration control analyses of the samples were assessed to be in an acceptable range.

Duration of treatment / exposure:

The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals. The treatment lasted up to one day prior to sacrifice.
- males: 31 days
- females: 62 days

Frequency of treatment:

Daily at the same time in the morning

Dose / conc.:

100 mg/kg bw/day

Remarks:

low-dose level (Test group 1)

Dose / conc.:

300 mg/kg bw/day

Remarks:

mid-dose level (Test group 2)

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

high-dose level (Test group 3)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on a previous range-finding study and at the request of the Sponsor.
- Fasting period before blood sampling for clinical biochemistry: About 16 to 20 hours

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. All animals were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration. A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
- Parameters examined: Abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning). During the mating period all females with negative evidence of sperm were weighed on mating days 1, 7 and 14. Females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13. Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males (for the calculation of the administration volume, body weight data of these individuals are reported in the individual tables). Females without litter and after weaning (PND 13) were weighed once a week.

FOOD CONSUMPTION: Yes
- Time schedule: Once a week (except for the 2nd premating week (male parental animals) and during the mating period (male and female parental animals). Food consumption of the females with evidence of sperm was determined for GD 0-7, 7- 14 and 14-20. Food consumption of the females which gave birth to a litter was determined for PND 1-4, 4-7, 7-10 and 10-13.

WATER CONSUMPTION: Yes
- Time schedule: Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

OTHER:

BEHAVIOUR: Yes
- At the end of the administration period starting at about 10.00h (FOB) and from 14:00 h onwards (MA).
- Dose groups that were examined: The first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups.
- Battery of functions tested: Motor activity (MA); The functional observational battery (FOB) started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The observations were performed at random.
- Home cage observations: The animals were observed in their closed home cages; during this period, any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Impairment of gait, Other findings
- Open field observations: The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined: Behavior on removal from the cage, Fur, Skin, Salivation, Nasal discharge, Eyes/pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/stereotypes, Gait abnormalities, Activity/arousal level, Feces (consistency/color) excreted during examination (two minutes), Urine excreted within 2 minutes (amount/color), Rearing within 2 minutes, Other findings
- Sensory motor tests/ reflexes: The animals were then removed from the open field and subjected to following sensory motor or reflex tests: Reaction to an object being moved towards the face (approach response), Touch sensitivity (touch response), Vision (visual placing response), Pupillary reflex, Pinna reflex, Audition (auditory startle response), Coordination of movements (righting response), Behavior during handling, Vocalization, Pain perception (tail pinch), Grip strength of forelimbs, Grip strength of hindlimbs, Landing foot-splay test, Other findings
- Motor activity assessment: The examinations were performed using the TSE Labmaster System. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals of 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.

Oestrous cyclicity (parental animals):

Estrous cycle determination prior to treatment was performed in a pool of up to 50 non-randomized female animals. Only animals with regular estrous cycle were selected for randomization before the start of the treatment period.
In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality (between PNDs 1-4, 5-7 and 8-13), presence of gross anomalies, clinical symptoms (daily), weight gain (on PND 1, 4, 7 and 13), presence of nipples/areolae in male pups (on PND 13), anogenital distance (AGD on PND 1)

GROSS EXAMINATION OF DEAD PUPS: Yes
- All culled pups, including stillborn pups and those that died during their rearing period, were subjected to a macroscopic (external and visceral) examination. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were further evaluated on a case-by-case basis (e.g., histopathological evaluation or special staining), depending on the findings noted.

Postmortem examinations (parental animals):

SACRIFICE
All parental animals were sacrificed by decapitation under isoflurane anesthesia.

GROSS NECROPSY
The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Organ weights:
All animals: final body weight, epididymides ovaries, prostate (ventral and dorsolateral part together, fixed), seminal vesicles with coagulating glands (fixed), testes, thyroid glands (with parathyroid glands) (fixed), uterus with cervix
5 animals per sex/test group: adrenal glands (fixed), brain, heart, kidneys, liver, spleen, thymus (fixed)
- Organ/tissue fixation: The following organs or tissues of all parental animals were fixed in in 4% neutral buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, esophagus, epididymides (modified Davidson’s solution), extraorbital lacrimal glands, eyes with optic nerve (modified Davidson’s solution), femur with knee joint, heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, liver, lungs, lymph nodes (axillary and mesenteric), mammary gland (male and female), nose (nasal cavity), ovaries (modified Davidson’s solution), oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate, rectum, salivary glands (mandibular and sublingual), sciatic nerve, seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testes (modified Davidson’s solution), thymus, thyroid glands, trachea, urinary bladder, uterus, vagina
- Histopathology: Please, see table 3 in section "Any other information on materials and methods incl. tables")

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals:5 males and 5 females
- Parameters checked in table 1 were examined (Please, see section "Any other information on materials and methods incl. tables")

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination (males); at PND 14 (females)
- Animals fasted: Yes
- How many animals: 5 males and 5 females
- Parameters checked in table 2 were examined (Please, see section "Any other information on materials and methods incl. tables")

PLASMA/SERUM HORMONES: Yes
- Time of blood sample collection and number of animals: Blood samples from all males at termination were assessed for serum levels for thyroid hormones (T4 and TSH).
- Animals fasted: Yes (adults)
- Parameters checked: Total thyroxine (T4), Thyroid stimulating hormone (TSH)

Postmortem examinations (offspring):

SACRIFICE
- On PND 4, as a result of standardization, surplus pups were sacrificed under isoflurane anesthesia by decapitation. After sacrifice, these pups were examined externally, eviscerated and their organs were assessed macroscopically.
- On PND 13, all remaining pups were sacrificed under isoflurane anesthesia by decapitation and examined macroscopically for external and visceral findings. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Laboratory Pathology and were archived without further processing.

PLASMA/SERUM HORMONES: Yes
- Time of blood sample collection and number of animals: PND 13 (one male and one female pup per litter)
- Parameters checked: Total thyroxine (T4), Thyroid stimulating hormone (TSH)

Statistics:

- Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days, anogenital distance and index: Simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided).

- Male and female mating and fertility indices, females mated, females delivering, gestation index, females with stillborn pups, females with all stillborn pups: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided).

- Mating days until day 0 p.c., % post-implantation loss, pups stillborn, % perinatal loss, nipple development, implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, survival Index: Pairwise comparison of the dose group with the control group using the WILCOXON test (one-sided) with BONFERRONI-HOLM adjustment.

- % live male day x, % live female day x: Comparison of the dose group with the control group was performed using the WILCOXON test (two-sided).

- Number of cycles and cycle length, rearing, grip strength of fore limbs and hind limbs, landing foot-splay test, motor activity, organ weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was ≤0.05, a pair-wise comparison of the dose groups with the control group was performed using the WILCOXON test (two-sided).

- Blood parameters: For parameters with bidirectional changes, non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was ≤0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided). For parameters with unidirectional changes, pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided).

Reproductive indices:

The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.

Male reproduction data:
- Male mating index (%) = (number of males with confirmed mating* / number of males placed with females) x 100
*defined by a female with vaginal sperm or with implants in utero

- Male fertility index (%) = (number of males proving their fertility* / number of males placed with females) x 100
* defined by a female with implants in utero

Female reproduction and delivery data:
- Female mating index (%) = (number of females mated* / number of females placed with males) x 100
* defined as the number of females with vaginal sperm or with implants in utero

- Female fertility index (%) = (number of females pregnant* / number of females mated**) x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

- Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant*) x 100
* defined as the number of females with implants in utero

- Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100

- Postimplantation loss (%) = [(number of implantations – number of pups delivered) / number of implantations] x 100

Offspring viability indices:

- Viability index (%) = (number of live pups on day 4* after birth / number of live pups on the day of birth) x 100
* before standardization of litters (i.e. before culling)

- Survival index (%) = (number of live pups on day 13 after birth / number of live pups on day 4* after birth) x 100
* after standardization of litters (i.e. after culling)

- Sex ratio (%) = (number of live male or female pups on PND 0 and 13 / number of live male and female pups on PND 0 and 13) x 100

- Anogenital index = (anogenital distance [mm] / cubic root of pup weight [g])

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Discolored feces, orange, were observed in all cages of male and female animals of test group 3 (1000 mg/kg bw/d) and in all cages of males and five cages of females of test group 2 (300 mg/kg bw/d). This finding was considered to be related to treatment but not adverse. The test substance is used as a pigment and the color of feces is the same color as the test substance.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse findings were observed on mean body weight and body weight change values in male and female animals in any test group. The following findings were assessed to be not treatment-related since they occurred without a relation to dose. Mean body weight was significantly reduced in female animals of test group 2 (300 mg/kg bw/d) on PND 7. Body weight change values were significantly decreased in female animals of test group 1 (100 mg/kg bw/d) during pre-mating days 0 – 7 as well as 0 - 13.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes in food consumption were observed in male and female animals of test groups 1 to 3 (100, 300 and 1000 mg/kg bw/d, respectively). The significantly lower mean value in female animals of test group 2 (300 mg/kg bw/d) on PND 4-7 was assessed as spontaneous in nature and not related to treatment since there was no relation to dose.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed. At the end of the administration period, in males of test groups 1 and 3 (100 and 1000 mg/kg bw/d) sodium levels were significantly decreased but the values were within the historical control range (males, sodium 140.1-145.8 mmol/L). Therefore, this change was regarded as incidental and not treatment related.

Endocrine findings:

no effects observed

Description (incidence and severity):

In parental males (test groups 1, 2 and 3; 100, 300 and 1000 mg/kg bw/d) no treatment-related alterations of T4 and TSH levels were observed.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Motor activity measurement: Regarding the overall motor activity and single intervals, no test substance-related deviations were noted for male and female animals of any test group. Comparing the single intervals with the control groups, significantly increased values were measured for male animals of test group 2 (300 mg/kg bw/d) at interval 4 and for male animals of test group 1 (100 mg/kg bw/d) at intervals 4 and 6. These differences were regarded to be incidental and not related to treatment as these intervals occurred without relation to dose and the overall motor activity was not affected.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All histopathological findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls. In high dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals. The female animal, which was not pregnant as well as the male mating partner did not show relevant histopathological findings.

Histopathological findings: neoplastic:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data revealed regular cycles in the rearing F1 female animals of all test groups including the control. The mean estrous cycle duration in test groups 0 to 3 was between 3.9 and 4.0 days.
In high dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

- The male mating index calculated after the mating period to produce F1 litter was 100% in all test groups. Fertility was proven in nearly all of the F0 parental males of test groups 0, 1 and 3 (0, 100 and 1000 mg/kg bw/d) within the scheduled mating interval to produce F1 litter. Only male animal of test group 2 (300 mg/kg bw/d), which was mated with a female of test group 2 (300 mg/kg bw/d), did not generate pregnancy. The apparently infertile male rat did not show relevant gross lesions. Thus, the male fertility index was 100% in test groups 0, 1 as well as test group 3 and 90% in test group 2. This reflects the normal range of biological variation inherent in the strain of rats used for this study.
- The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) was 1.9 days for test group 0 (control), 2.9 days for test group 1 (100 mg/kg bw/d) and 2.8 days for test groups 2 and 3 (300 and 1000 mg/kg bw/d) without any relation to dose. All sperm positive females of test groups 0 to 3 (control; 100, 300 and 1000 mg/kg bw/d) delivered pups with the exception of one female animal of test group 2, which did not deliver pups and showed no implantation sites. Thus, the female fertility index was 100% in test groups 0, 1 and 3, 90% in test group 2 without showing any relation to dose. The non-pregnant females had no relevant gross lesions or microscopic findings. The mean duration of gestation was 22.0 days in test group 0 (control) and 3 (1000 mg/kg bw/d), 22.1 days in test group 1 (100 mg/kg bw/d) and 22.4 days in test group 2 (300 mg/kg bw/d). The latter one showed statistical significance. However, the mean value of test group 2 is well within the range of the historical control data (GD range of 21.8 – 22.6 days). Furthermore, the finding was not related to dose. Therefore, it was assessed as incidental and not related to treatment. The gestation index was 100% in all test groups 0 – 3. Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (13.8 / 13.9 / 13.7 and 12.4 implants/dam in test groups 0 - 3, respectively). The postimplantation loss was 7.0% in test group 0 (control), 6.4% in test group 1 (100 mg/kg bw/d), 14.7% in test group 2 (300 mg/kg bw/d) and 6.3% in test group 3 (1000 mg/kg bw/d). The mean number of F1 pups delivered per dam remained unaffected (12.8 / 13.0 / 11.9 and 11.6 pups/dam in test groups 0 - 3, respectively). The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100 / 99.2 / 100 and 99.1% in test groups 0 - 3, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups.

Dose descriptor:

NOAEL

Remarks:

for general systemic toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related adverse effects observed up to and including the highest dose level of 1000 mg/kg bw/d.

Dose descriptor:

NOAEL

Remarks:

for reproduction and fertility

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related adverse effects observed up to and including the highest dose level of 1000 mg/kg bw/d.

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 mean number of delivered F1 pups per dam and the rates of liveborn, stillborn, found dead and cannibalized F1 pups were evenly distributed among the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
The viability index indicating pup survival during lactation (PND 0 – 4) was 100% in test groups 0 (control), 98.5% in test group 1 (100 mg/kg bw/d), 92.9% in test group 2 (300 mg/kg bw/d) and 99.0% in test group 3 (1000 mg/kg bw/d). The survival index indicating pup survival during lactation (PND 4 – 13) was 100% in test groups 0 to 2 (control, 100 and 300 mg/kg bw/d) and 96.2% in test group 3 (1000 mg/kg bw/d). Thus, the test substance did not influence pup survival in any of the treated groups.
Concerning mortality, in test group 3, one pup was stillborn, three pups were cannibalized and one pup was found dead. In test group 2, two pups were cannibalized, one pup was stillborn and one pup was found dead. In test group 1, two pups were cannibalized and one pup was stillborn.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean pup body weights and mean pup body weight change values of pups in test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) were comparable to the control group. Mean pup body weight change values were significantly decreased in male animals and male and female animals combined of test group 2 (300 mg/kg bw/d) from PND 4 to 7. These findings were assessed as being incidental and spontaneous in nature since there was no relation to dose. Runts were evenly distributed between all test group, i.e. 1 male and 2 female runts in test group 0 (control), 1 male and female runt in test group 1 (100 mg/kg bw/d) and test group 2 (300 mg/kg bw/d) and 1 male runt in test group 3 (1000 mg/kg bw/d). There was no relation to dose.

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

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One pup of test group 1 showed a spontaneous finding at gross necropsy: situs inversus in spleen. This finding occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, it was not considered to be associated to the treatment.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

In test groups 0, 1, 2 and 3 (control, 100, 300 and 1000 mg/kg bw/d, respectively), the sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups. Slight differences were regarded to be spontaneous in nature.

In male and female pups at PND 13 (test groups 1, 2 and 3; 100, 300 and 1000 mg/kg bw/d), no
treatment-related alterations of T4 and TSH levels were observed.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Remarks:

for developmental toxicity

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related adverse effects observed up to and including the highest dose level of 1000 mg/kg bw/d.

Critical effects observed:

no

Reproductive effects observed:

no

Table 1: Summary Mating Report

		Test Group 0/ F 0 mg/kg bw/d		Test Group 1/ F 100 mg/kg bw/d	Test Group 2/ F 300 mg/kg bw/d	Test Group 3/ F 1000 mg/kg bw/d
No. of females mated	N	10		10	10	10
- Inseminated	N	10	f-	10	10	10
Female mating index	%	100.0		100.0	100.0	100.0
-- Pregnant	N	10	f-	10	9	10
Female fertility index	%	100.0		100.0	90.0	100.0
No. of males mated	N	10		10	10	10
- With inseminated females	N	10	f-	10	10	10
Male mating index	%	100.0		100.0	100.0	100.0
- With pregnant females	N	10	f-	10	9	10
Male fertility index	%	100.0		100.0	90.0	100.0
Females with defined Day 0 pc	N	10		10	10	10
Mating days until Day 0 pc	Mean	1.9	x+	2.9	2.8	2.8
	S.d.	0.9		1.1	1.2	1.2
	N	10		10	10	10
Days 0 To 4	N	10		10	9	10
	%	100.0		100.0	90.0	100.0
Days 5 To 9	N	0		0	1	0
	%	0.0		0.0	10.0	0.0
Days 10 To 14	N	0		0	0	0
	%	0.0		0.0	0.0	0.0

 

 

Table 2: Summary Litter Report

			Test Group 0/ F 0 mg/kg bw/d	Test Group 1/ F 100 mg/kg bw/d	Test Group 2/ F 300 mg/kg bw/d	Test Group 3/ F 1000 mg/kg bw/d
	Total Number of Pregnant Females	N	10	10	9	10
	Total number of litters	N	10	10	9	10
	Litters with liveborn pups	N	10  f-	10	9	10
		%	100.0	100.0	100.0	100.0
	Litters with stillborn pups	N	0  f+	1	0	1
		%	0.0	10.0	0.0	10.0
	Litters with all pups stillborn	N	0  f+	0	0	0
		%	0.0	0.0	0.0	0.0
	Pups delivered	N	128	130	107	116
		Mean	12.8  x-	13.0	11.9	11.6
		S.d.	1.0	1.9	4.7	1.5
		N	10	10	9	10
	Pups liveborn	N	128	129	107	115
		%	100.0	99.2	100.0	99.1
		Mean	12.8  x-	12.9	11.9	11.5
		S.d.	1.0	1.9	4.7	1.6
		N	10	10	9	10
Pups stillborn		N	0	1	0	1
		%	0.0	0.8	0.0	0.9
		Mean	0.0  x+	0.1	0.0	0.1
		S.d.	0.0	0.3	0.0	0.3
		N	10	10	9	10
Perinatal Loss		Mean%	0.0  x+	0.7	0.0	0.9
		S.d.	0.0	2.3	0.0	2.9
		N	10	10	9	10
	cannibalized [pup] / Dead	N	0	2	2	3
		%	0.0	1.5	1.9	2.6
	sacrificed scheduled [pup] / Dead	N	80	80	63	77
		%	62.5	61.5	58.9	66.4
	culled / Dead	N	48	47	40	34
		%	37.5	36.2	37.4	29.3
	Pups surviving days 0 To 4	N	128	127	104	114
	Viability Index	Mean%	100.0  x-	98.5	92.9	99.0
		S.d.	0.0	4.9	16.4	3.2
		N	10	10	9	10
	Pups surviving days 4 To 13	N	80	80	64	77
	Survival Index	Mean%	100.0  x-	100.0	100.0	96.2
		S.d.	0.0	0.0	0.0	11.9
		N	10	10	9	10

 

 Table 3: Summary of Pup Necropsy Observation

				Test Group 0/ M 0 mg/kg bw/d	Test Group 1/ M 100 mg/kg bw/d	Test Group 2/ M 300 mg/kg bw/d	Test Group 3/ M 1000 mg/kg bw/d
Sex: male
day 0 -> 13 With unscheduled data	Animals examined		N	67	60	59	52
	Animals with signs		N	0	1	1	2
	General		N	0	1	1	2
	not assessed		N	0	0	1	2
	Situs inversus		N	0	1	0	0
	Normal NAD		N	67	59	58	50
Sex: female
day 0 -> 13 With unscheduled data		Animals examined	N	60	70	48	64
		Animals with signs	N	0	2	1	1
		General not assessed	N	0	2	1	1
		Normal NAD	N	60	68	47	63

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

f=FISHER-EXACT; x=WILCOX

Conclusions:

Under the conditions of this OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test, the oral administration of the test substance by gavage to male and female Wistar rats revealed no signs of systemic toxicity up to the highest tested dose level of 1000 mg/kg bw/d. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for reproductive performance and fertility was set to 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was set to 1000 mg/kg bw/d.

Executive summary:

The test substance was administered daily as a suspension to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at dose levels of 0 mg/kg bw/d (control; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). 0.5% Sodium carboxymethyl cellulose in deionized water served as vehicle, control animals were dosed daily with the vehicle only. The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals. After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females.

Regarding clinical examinations, no treatment-related, adverse findings were observed up to a dose level of 1000 mg/kg bw/d. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. Concerning pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. All findings occurred either individually or  were biologically equally distributed over control and treatment groups. They  were considered to be incidental or spontaneous in origin and without any relation to treatment.

Concerning fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups during the entire study. Mating behavior, conception, implantation and parturition were not affected. Concerning developmental toxicity, no treatment-related, adverse findings were observed. Pup status, viability, survival and growth  remained unaffected by the test substance treatment. All other findings occurred either individually or were biologically equally distributed over control and  treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

According to OECD TG 422 and GLP, Klimisch 1.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a GLP-conform study according to OECD guideline 422, the test substance was administered daily as a suspension to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at dose levels of 0 mg/kg bw/d (control; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). 0.5% Sodium carboxymethyl cellulose in deionized water served as vehicle, control animals were dosed daily with the vehicle only. The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

Regarding clinical examinations, no treatment-related, adverse findings were observed up to a dose level of 1000 mg/kg bw/d. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. Concerning pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. All findings occurred either individually or  were biologically equally distributed over control and treatment groups. They  were considered to be incidental or spontaneous in origin and without any relation to treatment.

Concerning fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups during the entire study. Mating behavior, conception, implantation and parturition were not affected. Concerning developmental toxicity, no treatment-related, adverse findings were observed. Pup status, viability, survival and growth  remained unaffected by the test substance treatment. All other findings occurred either individually or were biologically equally distributed over control and  treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effects on developmental toxicity

Description of key information

- Screening study for reproductive / developmental toxicity: according to OECD 422, GLP, rat, NOAEL for developmental toxicity 1000 mg/kg bw/d

- Prenatal developmental toxicity study: according to OECD TG 414, GLP is currently ongoing as requested by an ECHA compliance check

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Remarks:

Screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Jun 2020 - 10 Mar 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

July 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD 422

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2020

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Not specified
- Lot/batch number of test material: 0018514410
- Purity, including information on contaminants, isomers, etc.: 99.0 g/100 g (project 19L00260)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (room temperature)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The stability of the test substance in 0.5% Sodium carboxymethyl cellulose in deionized water over a period of at least 7 days at room temperature was given. As the mixtures were stored no longer than this time period, the stability was guaranteed. The homogeneous distribution of the test substance in 0.5% Sodium carboxymethyl cellulose in deionized water was demonstrated.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final concentration of a dissolved solid, stock liquid or gel: Please, see section "Doses/concentrations"

FORM AS APPLIED IN THE TEST: Suspension in vehicle

OTHER SPECIFICS
- Physical state/ appearance: Solid/ orange

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: About 11 - 12 weeks (males); About 10 weeks (females)
- Weight at study initiation: 374.6 g (mean males); 219.6 g (mean females)
- Fasting period before study: No
- Housing: Up to 5 animals per sex and cage during pretreatment; up to 2 animals per sex and cage during premating; up to 1 animal per sex and cage with exception during mating (1 male/1 female per cage) and rearing up to PND 13 (1 dam with her litter); for motor activity (MA) measurements the animals were housed individually.
- Diet (e.g. ad libitum): Mouse and rat maintenance diet “GLP” (ad libitum)
- Water (e.g. ad libitum): water bottles (ad libitum)
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY:
- The supplier assayed the food used in the study for chemical and microbiological contaminants. On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable.
- The drinking water is regularly assayed for chemical contaminants as well as for the presence of microorganisms by a contract laboratory. On the basis of the analytical findings the drinking water was found to be suitable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours (light from 06.00-18.00 h, dark from 18.00-06.00 h)

IN-LIFE DATES: From: 16 Jun 2020 To: 09 Sep 2020 (Sacrifice of parental females)

Route of administration:

oral: gavage

Vehicle:

other: 0.5% Sodium carboxymethyl cellulose in deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the preparation of the administration suspensions the test substance was weighed in a graduated flask depending on the dose group, topped up with 0.5% Sodium carboxymethyl cellulose in deionized water and intensely mixed with magnetic stirrer until it was completely homogenized. From 14 Aug 2020 onwards the test substance preparations were homogenized with an Ultraturrax instead of a magnetic stirrer. The portions of the test substance preparations for the daily administration were stored at room temperature. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: 0, 0.67, 2.00, 6.67 g/100mL
- Amount of vehicle (if gavage): 15 mL/kg bw/d
- Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration control analysis was assessed by UV/VIS spectrospcopy.
The samples for concentration control analysis, which were taken at the start of the administration period (premating), were not measured in time directly after sampling as expected. Therefore, these samples were discarded without measurement.
Therefore, an additional sample set were taken during gestation on 03 Aug 2020 and send to the Analytical Laboratory for analysis in time. This sample set demonstrated equivocal results with values outside of the desired range of the test facility. It was assumed that these results were due to the sampling process and did not represent the actual test substance preparations.
Therefore two further sample sets, taken during the lactation period on 14 Aug 2020 and 18 Aug 2020 were sent to the Analytical Laboratory and analyzed in time.The results of the sample sets during the lactation period demonstrated the correctness of the concentrations of the test substance in 0.5% Sodium carboxymethyl cellulose in deionized water because all samples were clearly found to be in the range of 90 - 110%. The mean of all three mean values of the high-dose group (including the questionable analysis during gestation) is close to the desired range of the test facility (mean: 86%). Therefore, in general, the concentration control analyses of the samples were assessed to be in an acceptable range.

Details on mating procedure:

- Time point: Two weeks after the beginning of treatment, the surviving males and females from the same test group were mated
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight, from about 16.00 h until 06.30 - 09.00 h of the following morning (for a maximum of 2 weeks)
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day (GD) 0
- After successful mating each pregnant female was caged (how): 1 animal per cage provided with nesting material (cellulose wadding) toward the end of gestation.

Duration of treatment / exposure:

The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals. The treatment lasted up to one day prior to sacrifice.
- males: 31 days
- females: 62 days

Frequency of treatment:

Daily at the same time in the morning

Dose / conc.:

100 mg/kg bw/day

Remarks:

low-dose level (Test group 1)

Dose / conc.:

300 mg/kg bw/day

Remarks:

mid-dose level (Test group 2)

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

high-dose level (Test group 3)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on a previous range-finding study and at the request of the Sponsor.
- Fasting period before blood sampling for clinical biochemistry: About 16 to 20 hours

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. All animals were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration. A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
- Parameters examined: Abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning). During the mating period all females with negative evidence of sperm were weighed on mating days 1, 7 and 14. Females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13. Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males (for the calculation of the administration volume, body weight data of these individuals are reported in the individual tables). Females without litter and after weaning (PND 13) were weighed once a week.

FOOD CONSUMPTION: Yes
- Time schedule: Once a week (except for the 2nd premating week (male parental animals) and during the mating period (male and female parental animals). Food consumption of the females with evidence of sperm was determined for GD 0-7, 7- 14 and 14-20. Food consumption of the females which gave birth to a litter was determined for PND 1-4, 4-7, 7-10 and 10-13.

WATER CONSUMPTION: Yes
- Time schedule: Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on PND 14
- Organs examined:
- Organ weights: All animals: final body weight, epididymides ovaries, prostate (ventral and dorsolateral part together, fixed), seminal vesicles with coagulating glands (fixed), testes, thyroid glands (with parathyroid glands) (fixed), uterus with cervix; 5 animals per sex/test group: adrenal glands (fixed), brain, heart, kidneys, liver, spleen, thymus (fixed)
- Organ/tissue fixation: The following organs or tissues of all parental animals were fixed in in 4% neutral buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, esophagus, epididymides (modified Davidson’s solution), extraorbital lacrimal glands, eyes with optic nerve (modified Davidson’s solution), femur with knee joint, heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, liver, lungs, lymph nodes (axillary and mesenteric), mammary gland (male and female), nose (nasal cavity), ovaries (modified Davidson’s solution), oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate, rectum, salivary glands (mandibular and sublingual), sciatic nerve, seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testes (modified Davidson’s solution), thymus, thyroid glands, trachea, urinary bladder, uterus, vagina

OTHER:
BEHAVIOUR: Yes
- Battery of functions tested: Motor activity (MA); The functional observational battery (FOB) started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The observations were performed at random.

Ovaries and uterine content:

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

Blood sampling:

Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement.
- Plasma: Yes
- Serum: Yes
- Volume collected: Not specified

Fetal examinations:

- No fetal examination

- On PND 4, as a result of standardization, surplus pups were sacrificed under isoflurane anesthesia by decapitation. After sacrifice, these pups were examined externally, eviscerated and their organs were assessed macroscopically.
- On PND 13, all remaining pups were sacrificed under isoflurane anesthesia by decapitation and examined macroscopically for external and visceral findings. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Laboratory Pathology and were archived without further processing.

- External examinations: Yes ( All culled pups, including stillborn pups and those that died during their rearing period)
- Soft tissue examinations: Yes ( All culled pups, including stillborn pups and those that died during their rearing period)
- Skeletal examinations: No
- Head examinations: No
- Anogenital distance: On all live male, female and uncertain pups on day 1 after birth.

Statistics:

- Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days, anogenital distance and index: Simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided).

- Male and female mating and fertility indices, females mated, females delivering, gestation index, females with stillborn pups, females with all stillborn pups: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided).

- Mating days until day 0 p.c., % post-implantation loss, pups stillborn, % perinatal loss, nipple development, implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, survival Index: Pairwise comparison of the dose group with the control group using the WILCOXON test (one-sided) with BONFERRONI-HOLM adjustment.

- % live male day x, % live female day x: Comparison of the dose group with the control group was performed using the WILCOXON test (two-sided).

- Number of cycles and cycle length, rearing, grip strength of fore limbs and hind limbs, landing foot-splay test, motor activity, organ weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was ≤0.05, a pair-wise comparison of the dose groups with the control group was performed using the WILCOXON test (two-sided).

- Blood parameters: For parameters with bidirectional changes, non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was ≤0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided). For parameters with unidirectional changes, pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided).

Indices:

The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.

Male reproduction data:
- Male mating index (%) = (number of males with confirmed mating* / number of males placed with females) x 100
*defined by a female with vaginal sperm or with implants in utero

- Male fertility index (%) = (number of males proving their fertility* / number of males placed with females) x 100
* defined by a female with implants in utero

Female reproduction and delivery data:
- Female mating index (%) = (number of females mated* / number of females placed with males) x 100
* defined as the number of females with vaginal sperm or with implants in utero

- Female fertility index (%) = (number of females pregnant* / number of females mated**) x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

- Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant*) x 100
* defined as the number of females with implants in utero

- Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100

- Postimplantation loss (%) = [(number of implantations – number of pups delivered) / number of implantations] x 100

- Viability index (%) = (number of live pups on day 4* after birth / number of live pups on the day of birth) x 100
* before standardization of litters (i.e. before culling)

- Survival index (%) = (number of live pups on day 13 after birth / number of live pups on day 4* after birth) x 100
* after standardization of litters (i.e. after culling)

- Sex ratio (%) = (number of live male or female pups on PND 0 and 13 / number of live male and female pups on PND 0 and 13) x 100

- Anogenital index = (anogenital distance [mm] / cubic root of pup weight [g])

Historical control data:

Historical Control Data for Reproduction Toxicity, Clinical Pathology Testing and Pathology were provided.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Discolored feces, orange, were observed in all cages of male and female animals of test group 3 (1000 mg/kg bw/d) and in all cages of males and five cages of females of test group 2 (300 mg/kg bw/d). This finding was considered to be related to treatment but not adverse. The test substance is used as a pigment and the color of feces is the same color as the test substance.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse findings were observed on mean body weight and body weight change values in male and female animals in any test group. The following findings were assessed to be not treatment-related since they occurred without a relation to dose. Mean body weight was significantly reduced in female animals of test group 2 (300 mg/kg bw/d) on PND 7. Body weight change values were significantly decreased in female animals of test group 1 (100 mg/kg bw/d) during pre-mating days 0 – 7 as well as 0 - 13.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes in food consumption were observed in male and female animals of test groups 1 to 3 (100, 300 and 1000 mg/kg bw/d, respectively). The significantly lower mean value in female animals of test group 2 (300 mg/kg bw/d) on PND 4-7 was assessed as spontaneous in nature and not related to treatment since there was no relation to dose.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed. At the end of the administration period, in males of test groups 1 and 3 (100 and 1000 mg/kg bw/d) sodium levels were significantly decreased but the values were within the historical control range (males, sodium 140.1-145.8 mmol/L). Therefore, this change was regarded as incidental and not treatment related.

Endocrine findings:

no effects observed

Description (incidence and severity):

In parental males (test groups 1, 2 and 3; 100, 300 and 1000 mg/kg bw/d) no treatment-related alterations of T4 and TSH levels were observed.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Motor activity measurement: Regarding the overall motor activity and single intervals, no test substance-related deviations were noted for male and female animals of any test group. Comparing the single intervals with the control groups, significantly increased values were measured for male animals of test group 2 (300 mg/kg bw/d) at interval 4 and for male animals of test group 1 (100 mg/kg bw/d) at intervals 4 and 6. These differences were regarded to be incidental and not related to treatment as these intervals occurred without relation to dose and the overall motor activity was not affected.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

When compared to control group 0 (=100%), the mean absolute weight of the thyroid gland was significantly increased in male animals of test group 3 (=120%). All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
When compared to control group 0 (=100%), the mean relative weight of the liver was significantly decreased in female animals of test group 3 (=93%). All other mean relative weight parameters did not show significant differences when compared to the control group 0.

The significant absolute thyroid gland weight increase in males of test group 3 (24.3 mg) was within the historical control range (19.300 – 27.600 mg). The same was true for the significant decrease of the relative liver weight in females of test group 3 (2.392%) which was also within the historical control values (2.4 – 3.312%). No histopathological correlates were seen in both organs. Therefore, these weight changes were not considered treatment related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

An orange discoloration of the gastrointestinal content in the glandular stomach, jejunum, cecum, colon and rectum was noted in general in male and female animals of test groups 2 and 3. This color reflects the presence of the test substance which was also observed microscopically as gold-brown pigment particles in the lumen of these organs. These findings were not considered adverse. All other findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The female animal, which was not pregnant as well as the male mating partner did not show relevant gross lesions.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All histopathological findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls. In high dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals. The female animal, which was not pregnant as well as the male mating partner did not show relevant histopathological findings.

Histopathological findings: neoplastic:

not specified

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (13.8 / 13.9 / 13.7 and 12.4 implants/dam in test groups 0 - 3, respectively).
The postimplantation loss was 7.0% in test group 0 (control), 6.4% in test group 1 (100 mg/kg bw/d), 14.7% in test group 2 (300 mg/kg bw/d) and 6.3% in test group 3 (1000 mg/kg bw/d).

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Description (incidence and severity):

The mean number of F1 pups delivered per dam remained unaffected (12.8 / 13.0 / 11.9 and 11.6 pups/dam in test groups 0 - 3, respectively). The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100 / 99.2 / 100 and 99.1% in test groups 0 - 3, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups.

Changes in pregnancy duration:

effects observed, non-treatment-related

Description (incidence and severity):

The mean duration of gestation was 22.0 days in test group 0 (control) and 3 (1000 mg/kg
bw/d), 22.1 days in test group 1 (100 mg/kg bw/d) and 22.4 days in test group 2 (300 mg/kg
bw/d). The latter one showed statistical significance. However, the mean value of test group
2 is well within the range of the historical control data (Supplement, HCD, gestation days,
range of 21.8 – 22.6 days). Furthermore, the finding was not related to dose. Therefore, it
was assessed as incidental and not related to treatment.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

All sperm positive females of test groups 0 to 3 (control; 100, 300 and 1000 mg/kg bw/d) delivered pups with the exception of one female animal of test group 2, which did not deliver pups and showed no implantation sites. Thus, the female fertility index was 100% in test groups 0, 1 and 3, 90% in test group 2 without showing any relation to dose. The non-pregnant females had no relevant gross lesions or microscopic findings.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed up to and including the limit dose

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean pup body weights and mean pup body weight change values of pups in test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) were comparable to the control group. Mean pup body weight change values were significantly decreased in male animals and male and female animals combined of test group 2 (300 mg/kg bw/d) from PND 4 to 7. These findings were assessed as being incidental and spontaneous in nature since there was no relation to dose. Runts were evenly distributed between all test group, i.e. 1 male and 2 female runts in test group 0 (control), 1 male and female runt in test group 1 (100 mg/kg bw/d) and test group 2 (300 mg/kg bw/d) and 1 male runt in test group 3 (1000 mg/kg bw/d). There was no relation to dose

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

In test group 3, one pup was stillborn, three pups were cannibalized and one pup was found dead. In test group 2, two pups were cannibalized, one pup was stillborn and one pup was found dead. In test group 1, two pups were cannibalized and one pup was stillborn.
The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn, found dead and cannibalized F1 pups were evenly distributed among the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

In test groups 0, 1, 2 and 3 (control, 100, 300 and 1000 mg/kg bw/d, respectively), the sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups. Slight differences were regarded to be spontaneous in nature.

Changes in litter size and weights:

not specified

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

The viability index indicating pup survival during lactation (PND 0 – 4) was 100% in test groups 0 (control), 98.5% in test group 1 (100 mg/kg bw/d), 92.9% in test group 2 (300 mg/kg bw/d) and 99.0% in test group 3 (1000 mg/kg bw/d). The survival index indicating pup survival during lactation (PND 4 – 13) was 100% in test groups 0 to 2 (control, 100 and 300 mg/kg bw/d) and 96.2% in test group 3 (1000 mg/kg bw/d). Thus, the test substance did not influence pup survival in any of the treated groups.

External malformations:

no effects observed

Description (incidence and severity):

No test substance related findings could be observed in test groups 1 to 3 (100, 300 and
1000 mg/kg bw/d) when compared to the control group (test group 0).

Skeletal malformations:

not specified

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One pup of test group 1 showed a spontaneous finding at gross necropsy: situs inversus in spleen. This finding occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, it was not considered to be associated to the treatment.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to and including the limit dose

Abnormalities:

not specified

Developmental effects observed:

no

Table 1: Summary of female reproduction and delivery data

			Test Group 0/ F 0 mg/kg bw/d			Test Group 1/ F 100 mg/kg bw/d	Test Group 2/ F  300 mg/kg bw/d	Test Group 3/ F 1000 mg/kg bw/d
No. of females at start		N	10			10	10	10
No. of females mated		N	10			10	10	10
Females inseminated		N	10		f-	10	10	10
Female mating index		%	100.0			100.0	100.0	100.0
Females with defined Day 0 pc		N	10			10	10	10
Mating days until Day 0 pc		Mean	1.9		x+	2.9	2.8	2.8
		S.d.	0.9			1.1	1.2	1.2
		N	10			10	10	10
Days 0 To 4		N	10			10	9	10
		%	100.0			100.0	90.0	100.0
Days 5 To 9		N	0			0	1	0
		%	0.0			0.0	10.0	0.0
Days 10 To 14		N	0			0	0	0
		%	0.0			0.0	0.0	0.0
Females pregnant		N	10		f-	10	9	10
Female fertility index		%	100.0			100.0	90.0	100.0
	Gestation Index	%	100.0			100.0	100.0	100.0
	Gestation days	Mean	22.0		n	22.1	22.4 *	22.0
		S.d.	0.0			0.3	0.7	0.0
		N	10			10	9	10
	Implantation Sites	N	138			139	123	124
		Mean	13.8		x-	13.9	13.7	12.4
		S.d.	1.3			2.0	2.5	1.6
		N	10			10	9	10
	Postimplantation Loss	Mean%	7.0		X+	6.4	14.7	6.3
		S.d.	6.5			7.0	27.4	7.0
		N	10			10	9	10
	Females without delivery	N	0			0	1	0
	Females delivering	N	10		f-	10	9	10
		%	100.0			100.0	100.0	100.0

 

Table 2: Summary Litter Report

			Test Group 0/ F 0 mg/kg bw/d	Test Group 1/ F 100 mg/kg bw/d	Test Group 2/ F 300 mg/kg bw/d	Test Group 3/ F 1000 mg/kg bw/d
	Total Number of Pregnant Females	N	10	10	9	10
	Total number of litters	N	10	10	9	10
	Litters with liveborn pups	N	10  f-	10	9	10
		%	100.0	100.0	100.0	100.0
	Litters with stillborn pups	N	0  f+	1	0	1
		%	0.0	10.0	0.0	10.0
	Litters with all pups stillborn	N	0  f+	0	0	0
		%	0.0	0.0	0.0	0.0
	Pups delivered	N	128	130	107	116
		Mean	12.8  x-	13.0	11.9	11.6
		S.d.	1.0	1.9	4.7	1.5
		N	10	10	9	10
	Pups liveborn	N	128	129	107	115
		%	100.0	99.2	100.0	99.1
		Mean	12.8  x-	12.9	11.9	11.5
		S.d.	1.0	1.9	4.7	1.6
		N	10	10	9	10
Pups stillborn		N	0	1	0	1
		%	0.0	0.8	0.0	0.9
		Mean	0.0  x+	0.1	0.0	0.1
		S.d.	0.0	0.3	0.0	0.3
		N	10	10	9	10
Perinatal Loss		Mean%	0.0  x+	0.7	0.0	0.9
		S.d.	0.0	2.3	0.0	2.9
		N	10	10	9	10
	cannibalized [pup] / Dead	N	0	2	2	3
		%	0.0	1.5	1.9	2.6
	sacrificed scheduled [pup] / Dead	N	80	80	63	77
		%	62.5	61.5	58.9	66.4
	culled / Dead	N	48	47	40	34
		%	37.5	36.2	37.4	29.3
	Pups surviving days 0 To 4	N	128	127	104	114
	Viability Index	Mean%	100.0  x-	98.5	92.9	99.0
		S.d.	0.0	4.9	16.4	3.2
		N	10	10	9	10
	Pups surviving days 4 To 13	N	80	80	64	77
	Survival Index	Mean%	100.0  x-	100.0	100.0	96.2
		S.d.	0.0	0.0	0.0	11.9
		N	10	10	9	10

 

Table 3: Summary of Pup Necropsy Observation

				Test Group 0/ M 0 mg/kg bw/d	Test Group 1/ M 100 mg/kg bw/d	Test Group 2/ M 300 mg/kg bw/d	Test Group 3/ M 1000 mg/kg bw/d
Sex: male
day 0 -> 13 With unscheduled data	Animals examined		N	67	60	59	52
	Animals with signs		N	0	1	1	2
	General		N	0	1	1	2
	not assessed		N	0	0	1	2
	Situs inversus		N	0	1	0	0
	Normal NAD		N	67	59	58	50
Sex: female
day 0 -> 13 With unscheduled data		Animals examined	N	60	70	48	64
		Animals with signs	N	0	2	1	1
		General not assessed	N	0	2	1	1
		Normal NAD	N	60	68	47	63

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

f=FISHER-EXACT; x=WILCOX

Conclusions:

Under the conditions of this OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test, the oral administration of the test substance by gavage to male and female Wistar rats revealed no signs of systemic toxicity up to the highest tested dose level of 1000 mg/kg bw/d. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for reproductive performance and fertility was set to 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was set to 1000 mg/kg bw/d.

Executive summary:

The test substance was administered daily as a suspension to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at dose levels of 0 mg/kg bw/d (control; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). 0.5% Sodium carboxymethyl cellulose in deionized water served as vehicle, control animals were dosed daily with the vehicle only. The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

Regarding clinical examinations, no treatment-related, adverse findings were observed up to a dose level of 1000 mg/kg bw/d. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. Concerning pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. All findings occurred either individually or  were biologically equally distributed over control and treatment groups. They  were considered to be incidental or spontaneous in origin and without any relation to treatment.

Concerning fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups during the entire study. Mating behavior, conception, implantation and parturition were not affected. Concerning developmental toxicity, no treatment-related, adverse findings were observed. Pup status, viability, survival and growth  remained unaffected by the test substance treatment. All other findings occurred either individually or were biologically equally distributed over control and  treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Reference 2

Endpoint:

developmental toxicity

Type of information:

other: ongoing experimental study

Adequacy of study:

key study

Study period:

27 May 2021 - September 2021 (planned end of experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

ECHA has issued a compliance check (Communication number: CCH-D-2114445795-37-01/D) requesting an OECD 414 study and the study is currently ongoing. The provided data are preliminary and are based on internal information from the study plan and a draft report. However, several analysis are still ongoing (e.g. examination of skeletal and visceral abnormalities, thyroid hormones). Therefore, no NOAEL values are provided.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

TEST MATERIAL
- Lot/batch number of test material: 0018514410
- Purity, including information on contaminants, isomers, etc.: 99.0 g/100 g
- Expiry date: 29 December 2027
- Appearance: Solid, orange

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: proven.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Suspensions of the test item, in 0.5 % CMC, was prepared using the following procedure:
1. The required amount of test item was weighed.
2. The required amount of vehicle was added.
3. The mixture was treated with a Silverson (medium head) for approximately 5 minutes.
4. The resulting suspension was left under magnetic stirring for at least 1 hour prior to dosing or analysis. The formulation was prepared daily or weekly, since the stability data supported the weekly preparation.


FORM AS APPLIED IN THE TEST: suspension in vehicle

Species:

rat

Strain:

Wistar

Remarks:

Hannover

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: 9 weeks (virgin females); 11 weeks (males)
- Weight at study initiation: 200-225 g (females); at least 350 g (males)
- Fasting period before study: not specified
- Housing: no more than 5 of one sex to a cage (before mating for all animals and after mating for males); one male with one female rat (during the mating period); individually (mated females)
- Diet (e.g. ad libitum): commercially available laboratory rodent diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approximately 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): approximately 15 - 20
- Photoperiod (hrs dark / hrs light): 12 hours

IN-LIFE DATES: From: 2021-05-27 (arrival) To: 2021-07-19 (start of necropsy)

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% in water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
1. The required amount of test item was weighed.
2. The required amount of vehicle was added.
3. The mixture was treated with a Silverson (medium head) for approximately 5 minutes.
4. The resulting suspension was left under magnetic stirring for at least 1 hour prior to dosing or analysis. The formulation was prepared daily or weekly.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis were performed in a separate study in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations. Samples of the formulations prepared during the current study (the first and the last week of treatment where possible) were analysed to check the homogeneity and concentration.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

From Day 6 through Day 19 post coitum.

Frequency of treatment:

Once daily

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

30 mg/kg bw/day

Dose / conc.:

10 mg/kg bw/day

No. of animals per sex per dose:

25 mated female rats/ dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels have been selected by the Sponsor.
- Rationale for animal assignment (if not random): On the day of allocation (Day 0 post coitum) all females were weighed and allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights.
- Fasting period before blood sampling for (rat) dam thyroid hormones: not specified.
- Time of day for (rat) dam blood sampling: The blood sampling was performed on the morning of the day of necropsy.

Maternal examinations:

CAGE SIDE OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were checked early in each working day and again in the afternoon for mortality. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. Severely debilitated animals were observed carefully. Clinical signs were recorded daily starting from allocation until sacrifice.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: Food consumption was measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 20 post coitum
- All animals, including those found dead, were euthanized by carbon dioxide inhalation and subjected to necropsy. All foetuses were sacrificed by intraperitoneal injection of Sodium Thiopental followed by hypothermia.
- Organs examined: From all females completing the scheduled test period, the thyroid and the brain were weighed, fixed and preserved in 10% neutral buffered formalin.


OTHER:
- Thyroid hormone determination (T3, T4 and TSH): all females, samples taken on day 20 post coitum, determination via immunoanalysis

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (not obtained from animals found dead or killed during the study)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number, sex and weight of all live foetuses; number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing); gross evaluation of placentae; Uteri or individual uterine horns without visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation.

Blood sampling:

- Plasma: Yes
- Serum: Yes
- Volume collected: approximately 1 mL
- Other: On Day 20 post coitum, blood samples for thyroid hormones determination were collected, randomizing (equalised) between treatment groups, from the sublingual vein of all females, under slight isoflurane anaesthesia. This procedure was performed within a short timeframe (e.g. two hours, if possible) on the morning of the day of necropsy. Samples were transferred into tubes containing no anticoagulant and centrifuged at room temperature. The serum obtained was divided in two aliquots (300µL in the aliquot A, the remaining in the aliquot B, if possible) and stored at -20°C, pending analysis.
- Immunoanalysis: Serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH))

Fetal examinations:

- External examinations: Yes: all live foetuses
- Visceral examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: Yes: all live foetuses on Day 20 post coitum

Structural deviations were classified as follows:
- Malformations: major abnormalities that are rare and/or affect the survival or health of the species under investigation.
- Anomalies: minor abnormalities that are detected relatively frequently.
- Variants: a change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis that would have otherwise followed a normal pattern of development.

Statistics:

For continuous variables the significance of the differences amongst group means were assessed by Dunnett's test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables were carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.

Indices:

- Corrected maternal body weight (Body weight on Day 20 post coitum minus gravid uterus weight)
- Corrected maternal body weight gain (Body weight on Day 20 post coitum minus gravid uterus weight minus maternal weight on Day 6 post coitum)
- Pre-implantation loss: [(no. of corpora lutea - no. of implantations) x 100] / no. of corpora lutea
- Post-implantation loss: [(no. of implantations - no. of live foetuses) x 100] / no. of implantations
- Total implantation loss: [(no. of corpora lutea - no. of live foetuses) x 100] / no. of corpora lutea
- Sex ratios of the foetuses were calculated as the percentage of males per litter.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No treatment related adverse clinical signs were described.

Hairloss was observed in one control female, in one mid- dose female and in 2 high dose females.
Considering the low incidence of hariloss and the presence of the sign also in a control animal the observation was deemed representative of normal background variability within the Wistar Han rat.
During the last days of gestation period, yellow faeces were observed in all high dose group females.
One low dose female showed a palpable mass on gestation Days 18-20. The presence of mass is considered spontaneous in origin.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Maternal body weight and body weight gain were unaffected by treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was comparable between groups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No changes in organ weights were seen between the controls and the treated females.
No changes in gravid uterus weight was observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examinations of thyroid
- No changes were seen between the controls and the treated females at macroscopic observations.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic examinations of thyroid
- No changes were seen between the controls and the treated females at microscopic observations.

Dead fetuses:

no effects observed

Description (incidence and severity):

The number of dams with live foetuses were 25 each in the control, low and high dose groups and 23 in the mid dose group.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All females were pregnant with the exception of 2 females in the mid- dose group. Unilateral implantation was observed in one control female.

Description (incidence and severity):

Thyroid hormones
- data not yet available (study currently ongoing)

Remarks on result:

other: The study is currently ongoing.

Abnormalities:

not specified

Description (incidence and severity):

The study is currently ongoing.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

Post-implantation losses and the mean number of fetuses per dam were not affected by treatment with the test item at all dose levels. In group 3, a statistically significant higher pre-implantation loss was noted. Since pre-implantation loss occurs before treatment start and in the absence of dose-dependency this difference was considered to be incidental.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratios were comparable between the control and the treated groups.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Anogenital distance was unaffected by treatment.

External malformations:

no effects observed

Description (incidence and severity):

No treatment related findings were observed.

Description (incidence and severity):

data not yet available (study currently ongoing)

Description (incidence and severity):

data not yet available (study currently ongoing)

Remarks on result:

other: The study is currently ongoing.

Abnormalities:

not specified

Description (incidence and severity):

The study is currently ongoing.

Developmental effects observed:

not specified

TAB. 1: SUMMARY OF REPRODUCTION DATA – GROUP DATA

 

Observations	Dose Levels [mg/kg/day]
	0	100	300	1000
No. Dams Inseminated	25	25	25	25
No. Dams That Conceived	25	25	23	25
Percent Dams Conceived	100.0	100.0	92.0	100.0
No. Dams Died During Study	0	0	0	0
No. Dams with Resorptions only	0	0	0	0
No. of Litters	25	25	23	25
Total No. Corpora Lutea	276	301	263	303
Mean No. Corpora Lutea/Pregnancy	11.0	12.0	11.4	12.1
Total No. Implantation	269	285	243	293
Mean No. Implants/Pregnancy	10.8	11.4	10.6	11.7
Total No. Live Fetuses	263	281	235	288
Mean No. Live Fetuses/Pregnancy	10.5	11.2	10.2	11.5
Total No. Dead Fetuses	0	0	0	0
Mean No. Dead Fetuses/Pregnancy	0.0	0.0	0.0	0.0
No. Dams with Resorptions and/or Dead Fetuses	6	2	5	4
Total No. Resorptions	6	4	8	5
Mean No. Resorptions/Pregnancy	0.2	0.2	0.3	0.2
Preimplantation Loss (%)(mean)	2.1	4.8	7.6	2.9
Post Implantation Loss (%)(mean)	3.6	1.7	3.3	1.6
Mean Foetal Wt. Live Fetuses	4.13	4.09	4.18	4.11

 

 

TAB. 2: CLINICAL SIGNS OF FEMALES – GROUP INCIDENCE

 

Group	0 mg/kg/day		100 mg/kg/day		300 mg/kg/day			1000 mg/kg/day
Observations	a	b	a	b	a	b	a		b
APPEARANCE
Hairloss	1	4.0	0	0.0	1	4.0	2		8.0
Presence of palpable mass	0	0.0	1	4.0	0	0.0	0		0.0
Coloured faeces, yellow	0	0.0	0	0.0	0	0.0	25		100.0

 

Key:         () = Number of animals alive at start of interval

a = Number of animals affected

b = Percent of animals with observation during interval

 

 

TAB. 3: BODY WEIGHT (g) OF PREGNANT FEMALES - GROUP MEAN DATA

 

Group(s)	Day of Phase
		0!	3"	6	9	12	15	18	20
1	(n)	25	25	25	25	25	25	25	25
	Mean	215.67	225.12	237.05	244.64	259.30	274.26	306.16	330.42
	SD	11.96	13.19	13.16	13.29	15.88	16.92	21.13	24.61
2	(n)	25	25	25	25	25	25	25	25
	Mean	212.70	220.04	231.39	239.71	252.47	268.15	302.95	327.30
	SD	14.19	17.38	16.13	17.66	17.02	19.15	20.56	25.22
3	(n)	23	23	23	23	23	23	23	23
	M n	216.02	226.23	236.17	243.73	257.54	274.63	308.58	333.25
	S	12.82	12.60	12.98	13.41	13.22	18.42	18.59	21.16
4	(n)	25	25	25	25	25	25	25	25
	Mean	215.65	6	5	4	7	3	6.98	3
	SD	17.29	8.54	9.24	9.35	0.93	2.21	5.26	7.77

 

Note: ! = Gestation phase; " = Dosing/Gestation phase

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is significantly different from control at p < 0.01

Statistical analysis: Dunnett`s test if group variances are homogeneous

Modified t test if group variances are inhomogeneous ($)

 

 

TAB. 4: BODY WEIGHT GAIN PER DAY° (g) OF PREGNANT FEMALES - GROUP MEAN DATA

 

Group(s)	Day of Phase
		3"	6	9	12	15	18	20
1	(n)	25	25	25	25	25	25	25
	Mean	225.12	237.05	244.64	259.30	274.26	306.16	330.42
	SD	13.19	13.16	13.29	15.88	16.92	21.13	24.61
2	(n)	25	25	25	25	25	25	25
	Mean	220.04	231.39	239.71	252.47	268.15	302.95	327.30
	SD	17.38	16.13	17.66	17.02	19.15	20.56	25.22
3	(n)	23	23	23	23	23	23	23
	M n	226.23	236.17	243.73	257.54	274.63	308.58	333.25
	S	12.60	12.98	13.41	13.22	18.42	18.59	21.16
4	(n)	25	25	25	25	25	25	25
	Mean	6	5	4	7	3	6.98	3
	SD	8.54	9.24	9.35	0.93	2.21	5.26	7.77

 

Note: Data for Dosing/Gestation phase

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is significantly different from control at p < 0.01

Statistical analysis: Dunnett`s test if group variances are homogeneous

Modified t test if group variances are inhomogeneous ($)

° = mean daily body weight gain over the previous period starting from gestation day 0

 

 

TAB. 5: FOOD CONSUMPTION° (g/animal/day) OF PREGNANT FEMALES – GROUP MEAN DATA

 

Group(s)	Day of Phase
		3	6	9	12	15	18	20
1	(n)	25	25	25	25	25	25	25
	Mean	8.55	0.90	1.02	2.54	3.82	5.84	6.04
	SD	3.66	2.02	2.67	2.69	1.75	2.60	2.03
2	(n)	25	25	25	25	25	25	25
	Mean	8.55	0.76	0.97	2.09	3.50	6.10	5.81
	SD	3.40	2.51	3.31	2.07	2.24	2.85	1.93
3	(n)	23	23	23	23	23	23	23
	M n	8.56	0.89	1.40	2.77	5.07	7.08	6.41
	S	2.74	2.32	2.27	2.50	3.57	3.15	2.31
4	(n)	25	25	25	25	25	25	25
	Mean	8.17	0.51	1.25	2.78	3.63	4.88	5.63
	SD	3.58	2.50	2.71	2.84	2.32	2.64	2.75

 

Note: Data for Dosing/Gestation phase

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is significantly different from control at p < 0.01

Statistical analysis: Dunnett`s test if group variances are homogeneous

Modified t test if group variances are inhomogeneous ($)

° = food consumed over the previous period starting from Day 0 post coitum

 

 

TAB. 6: TERMINAL BODY WEIGHT, GRAVID UTERUS WEIGHT, CORRECTED MATERNAL BODY WEIGHT AND CORRECTED MATERNAL BODY WEIGHT GAIN OF FEMALES - GROUP MEAN DATA

 

Group(s)		Terminal Body weight (g)	Gravid uterus weight (g)	Body weight Day 6 (g)	Corrected maternal body weight^ (g)	Corrected body weight gain# (g)
1	Mean	325.81	64.98	237.96	260.83	23.77
	SD	25.81	15.16	13.15	17.78	8.33
	(n)	25	25	25	25	25
2	Mean	323.78	69.04	231.40	254.74	23.34
	SD	23.03	12.22	16.13	16.84	7.86
	(n)	25	25	25	25	25
3	Mean	329.40	64.38	236.17	265.02	28.84
	SD	21.11	13.17	12.97	18.18	11.89
	(n)	23	23	23	23	23
4	Mean	327.70	66.29	235.85	261.41	25.56
	SD	26.98	15.89	19.24	32.34	22.56
	(n)	25	25	25	25	25

 

^ = Corrected maternal body weight at necropsy minus gravid uterus weight

# = Corrected maternal body weight at necropsy minus gravid uterus weight, minus body weight at day 6 of pregnancy

* = mean value of group is significantly different from control

Statistical analysis: Kruskall Wallis test

William’s test if group means are different from control at p < 0.05

 

 

TAB. 7: Thyroid hormone determination - F0 animals - Group data

 

Not yet available

 

 

TAB. 8: LITTER DATA AND SEX RATIOS OF FEMALES - GROUP MEAN DATA

 

Group(s)		Corpora Lutea	Implan-tations	Uterine Deaths			Viable young			% males	Implantation loss (%)			Litter Weight (g)	Mean Foetal Weight (g)
				Early	Late	Total	Total	Male	Female		Pre	Post	Total		Male	Female	combined
1	Mean	11.04	10.76	0.24	0.00	0.24	10.52	4.60	6.17	45.05	2.11	3.55	5.64	43.31	4.23	4.07	4.13
	SD	2.75	2.57	0.44	0.00	0.44	2.57	1.68	1.83	17.98	4.67	10.18	10.68	11.63	0.73	0.68	0.69
	(n)	25	25	25	25	25	25	25	24	25	25	25	25	25	25	24	25
2	Mean	12.04	11.40	0.16	0.00	0.16	11.24	5.64	5.60	50.64	4.78	1.69	6.40	45.52	4.19	4.00	4.09
	SD	2.26	1.96	0.55	0.00	0.55	2.19	1.96	2.22	15.65	6.96	5.85	8.82	8.87	0.59	0.57	0.56
	(n)	25	25	25	25	25	25	25	25	25	25	25	25	25	25	25	25
3	Mean	11.44	10.57	0.35	0.00	0.35	10.22	5.30	4.91	51.35	7.60*	3.34	10.91	42.74	4.30	4.07	4.18
	SD	1.70	1.85	0.89	0.00	0.89	2.04	2.20	1.91	17.25	10.45	8.78	11.79	11.02	0.68	0.61	0.62
	(n)	23	23	23	23	23	23	23	23	23	23	23	23	23	23	23	23
4	Mean	12.12	11.72	0.20	0.00	0.20	11.52	5.96	5.56	51.78	2.94	1.64	4.58	47.35	4.23	4.02	4.14
	SD	1.94	1.84	0.50	0.00	0.50	1.85	1.90	1.98	14.63	7.30	4.13	7.76	8.01	0.59	0.69	0.62
	(n)	25	25	25	25	25	25	25	25	25	25	25	25	25	25	25	25

 

* = mean value of group is significantly different from control

Statistical analysis: Kruskall Wallis test

William’s test if group means are different from control at p < 0.05

 

 

TAB. 9: ANOGENITAL DISTANCE ON DAY 20 POST COITUM – GROUP MEAN DATA, MALES

 

Parameter/units	Group	0 mg/kg/day	100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
ANOGENITAL DISTANCE (mm)	Mean	4.19	4.18	4.13	4.16
	SD	0.37	0.41	0.27	0.32
	(n)	25	25	23	25
Pup weight (g)	Mean	4.23	4.19	4.30	4.23
	SD	0.73	0.59	0.68	0.59
	(n)	25	25	23	25
ANOGENITAL DISTANCE ^ (NORMALISED) mm/g⅓	Mean	2.60	2.60	2.55	2.58
	SD	0.18	0.20	0.20	0.24
	(n)	25	25	23	25

 

^ = normalised for the cube root of the body weight collected on Day 20 post coitum (mm/g)

Statistical analysis: Kruskall Wallis test

T test if group mean differences are different from control at p < 0.05

* = mean value of group is significantly different from control

 

 

TAB. 10: ANOGENITAL DISTANCE ON DAY 20 POST COITUM – GROUP MEAN DATA, FEMALES

 

Parameter/units	Group	0 mg/kg/day	100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
ANOGENITAL DISTANCE (mm)	Mean	2.50	2.31	2.46	2.48
	SD	0.44	0.43	0.39	0.35
	(n)	24	25	23	25
Pup weight (g)	Mean	4.07	4.00	4.07	4.02
	SD	0.68	0.57	0.61	0.69
	(n)	24	25	23	25
ANOGENITAL DISTANCE ^ (NORMALISED) mm/g⅓	Mean	1.57	1.46	1.55	1.57
	SD	0.25	0.25	0.27	0.24
	(n)	24	25	23	25

 

^ = normalised for the cube root of the body weight collected on Day 20 post coitum (mm/g)

Statistical analysis: Kruskall Wallis test

T test if group mean differences are different from control at p < 0.05

* = mean value of group is significantly different from control

 

 

TAB. 11: ABSOLUTE ORGAN WEIGHTS (g) - GROUP MEAN DATA, FEMALES, BRAIN

 

Group	0 mg/kg/day	100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
(n)	25	25	25	25
Mean	1.828	1.810	1.806	1.810
SD	0.094	0.116	0.109	0.068
Group diff. at p < 0.05		0.067	0.067	0.067
Group diff. at p < 0.01		0.083	0.083	0.083

 

Data homogeneous by Bartlett's test (Dunnett's test)

Analysis of variance: F ratio = 0.26 Df = 3/ 96 F probability = 0.854

Note: a * indicates group mean is significantly different from control at level of significance shown.

 

 

TAB. 12: ABSOLUTE ORGAN WEIGHTS (g) - GROUP MEAN DATA, FEMALES, THYROID

 

Group	0 mg/kg/day	100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
(n)	25	25	25	25
Mean	0.0223	0.0224	0.0244	0.0233
SD	0.0037	0.0041	0.0042	0.0039
Group diff. at p < 0.05		0.0027	0.0027	0.0027
Group diff. at p < 0.01		0.0034	0.0034	0.0034

 

Data homogeneous by Bartlett's test (Dunnett's test)

Analysis of variance: F ratio = 1.42 Df = 3/ 95 F probability = 0.240

Note: a * indicates group mean is significantly different from control at level of significance shown.

 

 

TAB. 13: ORGAN WEIGHTS° TO BRAIN WEIGHT - GROUP MEAN DATA, THYROID, FEMALES

 

Group	0 mg/kg/day	100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
(n)	25	25	25	25
Mean	1.220	1.238	1.352	1.288
SD	0.188	0.234	0.261	0.220
Group diff. at p < 0.05		0.153	0.155	0.153
Group diff. at p < 0.01		0.192	0.194	0.192

 

Data homogeneous by Bartlett's test (Dunnett's test)

Analysis of variance: F ratio = 1.65 Df = 3/ 95 F probability = 0.181

Note: a * indicates group mean is significantly different from control at level of significance shown.

° = expressed as % organ to brain weight ratio

 

 

TAB. 14: MACROSCOPIC OBSERVATIONS OF FEMALES – FINAL SACRIFICE - GROUP INCIDENCE, FEMALES

 

Group	0 mg/kg/day	100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
(n)	25	25	25	25
Whole animal      No abnormalities detected	23	24	22	23
Forelimbs      Hairloss	1	0	1	0
Skin      Subcutaneous mass(es)      Hairloss	0 1	1 0	0 1	0 2
Uterus      Not pregnant      Unilateral implantation	0 1	0 0	2 0	0 0

 

 

TAB. 15: EXTERNAL EXAMINATION OF FOETUSES - GROUP INCIDENCE

 

Group	0 mg/kg/day	100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
(n)	25	25	25	25
Thyroid      THYRO-GLOSSAL DUCT REMNANT      ECTOPIC THYMIC TISSUE	1 1	1 2	1 0	1 1

 

 

TAB. 16: MICROSCOPIC OBSERVATIONS – FINAL SACRIFICE - GROUP INCIDENCE

 

Group	Organ	Cat	Observations	No. Foetuses			No. Litters
				Observed	Affected	%	Observed	Affected	%
1	Forelimb	AN	Short	263	1	0.38	25	1	4.00
	Head	AN	Domed shape	263	1	0.38	25	1	4.00
	Hindlimb	AN	Short	263	1	0.38	25	1	4.00
	Mouth	AN	Abnormal shape	263	1	0.38	25	1	4.00
	Whole Foetus		No abnormalities detected	263	262	99.62	25	24	96.00
2	Whole Foetus		No abnormalities detected	281	281	100.00	25	25	100.00
3	Tail	AN	Short	235	1	0.43	23	1	4.35
	Whole Foetus		No abnormalities detected	235	234	99.57	23	23	100.00
4	Hindlimb	AN	Abnormal shape	288	2	0.69	25	2	8.00
	Whole Foetus		No abnormalities detected	288	286	99.31	25	25	100.00

 

 

TAB. 17: Fixed visceral examination of foetuses - Group incidence

 

Not yet available

 

 

TAB. 18: Skeletal examination of foetuses - Group incidence

 

Not yet available

 

 

 

Conclusions:

Study currently ongoing

Executive summary:

Preliminary summary (Study currently ongoing):

In a GLP-conform study according to OECD guideline 414, the effects of the test item on pregnant female Wistar Han rats and embryo-fetal development were assessed when administered orally by gavage once daily to mated female rats from Day 6 through to Day 19 post coitum, inclusive. Each group consisted of 25 mated female rats. Each group consisted of 25 mated female rats. The test material was administered once daily at dose levels of: 0 mg/kg bw/day (vehicle control); 100 mg/kg bw/day, 300 mg/kg bw/day and 1000 mg/kg bw/day. A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight  was used. Control animals were dosed with the vehicle alone (0.5 % carboxy- methylcellulose (CMC).

All females were sacrificed on Day 20 post coitum and the fetuses

were removed by Caesarean section.Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination. Thyroid hormone determination was performed. The brain and thyroid were weighed. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, gravid uterus weights, foetal weight and sex were recorded. All foetuses were examined for external abnormalities. The anogenital distance (AGD) in all live foetuses was recorded. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.

No mortality occurred during the study. All females were pregnant with the exception of 2 females in the mid- dose group. Unilateral implantation was observed in one control female. The number of dams with live foetuses were 25 each in the control, low and high dose groups and 23 in the mid dose group. No treatment related clinical signs were described. Yellow faeces were noted for all high dose females during the last days of gestation period. Maternal body weight and body weight gain were unaffected by treatment. Food consumption was comparable between groups. No changes in terminal body weight, gravid uterus weight or absolute weight gain was observed. No changes in organ weights were seen between the controls and the treated females. Litter data and sex rations was comparable between the control and the treated groups. Anogenital distance was unaffected by treatment. No changes were seen between the controls and the treated females at macroscopic or microscopic observations. Regarding the external examinations of foetuses, no treatment related abnormal findings were observed.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

According to OECD TG 422 and 414, GLP, Klimisch 1.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

In a GLP-conform study according to OECD guideline 422, the test substance was administered daily as a suspension to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at dose levels of 0 mg/kg bw/d (control; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). 0.5% Sodium carboxymethyl cellulose in deionized water served as vehicle, control animals were dosed daily with the vehicle only. The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

Regarding clinical examinations, no treatment-related, adverse findings were observed up to a dose level of 1000 mg/kg bw/d. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. Concerning pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. All findings occurred either individually or  were biologically equally distributed over control and treatment groups. They  were considered to be incidental or spontaneous in origin and without any relation to treatment.

Concerning fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups during the entire study. Mating behavior, conception, implantation and parturition were not affected. Concerning developmental toxicity, no treatment-related, adverse findings were observed. Pup status, viability, survival and growth  remained unaffected by the test substance treatment. All other findings occurred either individually or were biologically equally distributed over control and  treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

 

An OECD TG 414 study is currently ongoing as requested by an ECHA compliance check. Preliminary data from this study are given. Of note, several analysis are still ongoing (e.g. examination of skeletal and visceral abnormalities, thyroid hormones). Therefore, no NOAEL values are provided.

In a GLP-conform study according to OECD guideline 414, the effects of the test item on pregnant female Wistar Han rats and embryo-fetal development were assessed when administered orally by gavage once daily to mated female rats from Day 6 through to Day 19 post coitum, inclusive. Each group consisted of 25 mated female rats. The test material was administered once daily at dose levels of: 0 mg/kg bw/day (vehicle control); 100 mg/kg bw/day, 300 mg/kg bw/day and 1000 mg/kg bw/day. A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight  was used. Control animals were dosed with the vehicle alone (0.5 % carboxy- methylcellulose (CMC). All females were sacrificed on Day 20 post coitum and the fetuses were removed by Caesarean section.Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination. Thyroid hormone determination was performed. The brain and thyroid were weighed. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, gravid uterus weights, foetal weight and sex were recorded. All foetuses were examined for external abnormalities. The anogenital distance (AGD) in all live foetuses was recorded. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities. No mortality occurred during the study. All females were pregnant with the exception of 2 females in the mid- dose group. Unilateral implantation was observed in one control female. The number of dams with live foetuses were 25 each in the control, low and high dose groups and 23 in the mid dose group. No treatment related clinical signs were described. Yellow faeces were noted for all high dose females during the last days of gestation period. Maternal body weight and body weight gain were unaffected by treatment. Food consumption was comparable between groups. No changes in terminal body weight, gravid uterus weight or absolute weight gain was observed. No changes in organ weights were seen between the controls and the treated females. Litter data and sex rations was comparable between the control and the treated groups. Anogenital distance was unaffected by treatment. No changes were seen between the controls and the treated females at macroscopic or microscopic observations. Regarding the external examinations of foetuses, no treatment related abnormal findings were observed.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available screening study is reliable and suitable for classification purposes under Regulation 1272/2008. No adverse effects on fertility or development were observed in a screening study in rats (OECD 422).  As a result, the substance is not considered to be classified for fertility or developmental toxicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.