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REACH

2,2'-(2-methylpropylidene)bis[4,6-xylenol]

EC number: 251-394-8 | CAS number: 33145-10-7

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproduction/developmental toxicity screening test

Parental NOAEL: 10 mg/kg/day (based on clinical signs and behavioural changes observed in the functional observations tests)

Reproduction NOAEL: at least 30 mg/kg (the highest dose level tested in this study)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 June 2016 to 30 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Organisation of Economic Co-operation and Development (OECD) Guidelines for
Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2015, with the exception ofthe procedures described in the section litter size.

Deviations:

yes

Remarks:

See "Any other information" for details

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650

Version / remarks:

The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

yes

Remarks:

See "Any other information" for details

Principles of method if other than guideline:

In addition, the procedures described in the report essentially conform to the following guidelines:
• OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 2015.
• The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
• Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
• OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
• The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

GLP compliance:

yes

Limit test:

Justification for study design:

The purpose of the study was to evaluate the potential toxic effects of the test item when administered to rats for a minimum of 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development.
Parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.
This study should provide part of a rational basis for toxicological risk assessment in man.

Specific details on test material used for the study:

No further details specified in the study report.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Test system: Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and non-pregnant females and untreated animals were used at initiation of the study.
Rationale: This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies.
Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test system: Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and non-pregnant females and untreated animals were used at initiation of the study.
Source: F0 Charles River Deutschland, Sulzfeld, Germany.
Age at start pretest: Females: approximately 10-12 weeks.
Age at start F0-treatment: Males: approximately 10-12 weeks. Females: approximately 12-14 weeks.
Number of F0-animals: 48 females and 40 males.
At the end of the pretest phase, 40 females with at least two regular estrous cycles were selected at random and continued in the study. The remaining females were removed from the study.
Acclimatization F0: At least 5 days prior to start of pretest (females) or treatment (males).
Health inspection F0: At least upon receipt of the animals.
Randomization F0: Before initiation of pretest, by computer-generated random g algorithm according to body weight, with all animals within ± 20% of the sex mean.
Identification F0: During pretest (females only): by indelible marker, numbered 101 through 148 at random.
During treatment (males and females): by earmark and tattoo.
Parturition: The females were allowed to litter normally. Postnatal day (PND) 1 was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). Females that were littering were left undisturbed.
Number of pups: 446 pups.
Identification of pups: On PND 1, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.
When general hair growth blurred the identification, the pups were identified by tattoo on the feet.
Culling: To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (when possible) were selected. Blood samples were collected from two of the surplus pups. Selective elimination of pups, e.g. based upon body weight, or anogenital distance was not done.
Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

Animal Husbandry
Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 room air changes/hour, and a 12-hour light/12-hour dark cycle: the photoperiod was between 07:00 and 19:00 hrs daily.
The light/dark cycle was interrupted for study related activities.
Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation
Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.

General: Sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
Water: Free access to tap-water. During motor activity measurements, animals did not have access to water for a maximum of 2 hours.
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

Vehicle: Propylene glycol, specific gravity 1.036 (Merck, Darmstadt, Germany).
Rationale for vehicle: Based on trial formulations performed at Charles River Den Bosch.
Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level.
Adjustment was made for specific gravity of the vehicle.
Appearance of formulations: Solution (Groups 2-4).
Storage conditions: At room temperature.

Details on mating procedure:

Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
Detection of mating was not confirmed for animal nos. 41 and 53 which did deliver live offspring. The mating date of these animals was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dose formulations taken on two occasions during the treatment phase, i.e. on 08 Augustus (week 1) and on 06 September 2016 (week 5 of study), were analysed according to a validated method (Test Facility Study no. 512752, ABL Project 16101).
Samples taken in week 1 were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
Samples taken in week 5 were from the Group 2 formulation only and were analysed for determination of stability (over 5 hours at room temperature) and confirmation of accuracy, homogeneity, because the concentration of 0.6 mg test item/ml in this Group 2 formulation was outside the range of 1 – 200 mg/mL checked in the validated method.

Duration of treatment / exposure:

Males were treated for 31 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females that delivered were treated for 49 - 56 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy.
Females which failed to deliver healthy offspring were treated for 40-42 days.
When littering was observed at the time of dosing, these females, i.e. no. 41, 46, 47 (Group 1), 52, 57 (Group 2), 63, 64, 65 (Group 3) and 79 (Group 4) were not dosed. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Details on study schedule:

Pups were not treated directly but could have potentially been exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.

Dose / conc.:

3 mg/kg bw/day (actual dose received)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

No. of animals per sex per dose:

One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose levels
Based on the results of a 10-day dose range finder, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 3, 10 and 30 mg/kg.

Positive control:

Not required for this study.

Parental animals: Observations and examinations:

Mortality / Viability: At least twice daily.
Clinical signs: At least once daily from start of treatment onwards up to the day prior to necropsy detailed clinical observations were made for all animals, at least 1.5 hours (± 30 min) after treatment (on the peak period of anticipated effects after treatment). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Functional Observations: The following functional observations tests were performed on each individual animal of the selected 5 animals/sex/group:
-hearing ability (HEARING), pupillary reflex (PUPIL L/R), and static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
-fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
-locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA).
Total movements and ambulations are reported.
Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.
The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13). These tests were performed starting after the observation for clinical signs (incl. arena observation, if applicable) at 1.5 hours (±30 min) after treatment.

Body weights: Males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation was used to aid in confirmation of pregnancy. Cage debris of pregnant females were examined for evidence of premature delivery.
Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Clinical Laboratory Investigations (F0-Generation only)
Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m.
The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids. The following parameters were determined:

Haematology
The following haematology parameters were determined in blood prepared with K3-EDTA as an anti-coagulant, using the ADVIA® 2120i Hematology System (Siemens Healthcare Diagnostics B.V., Den Haag, The Netherlands):
White blood cells (WBC); Red blood cells; Reticulocytes; Red blood cell distribution width (RDW); Haemoglobin: Haematocrit; Mean corpuscular volume (MCV); Mean corpuscular haemoglobin (MCH); Mean corpuscular haemoglobin concentration (MCHC); Platelets
Differential leucocyte count: Neutrophils; Lymphocytes; Monocytes; Eosinophils; Basophils.

The following clotting parameters were determined in plasma prepared with citrate as anti-coagulant, using the STA Compact® (Diagnostica Stago S.A.S., Asnières, France):
Prothrombin Time (PT); Activated Partial Thromboplastin Time (APTT)

Clinical Biochemistry
The following clinical biochemistry parameters were determined using the AU400 (Beckman Coulter Nederland B.V., Woerden, The Netherlands). All parameters were determined in plasma, except for bile acids which were determined in serum:
Alanine aminotransferase (ALAT); Aspartate aminotransferase (ASAT); Alkaline Phosphatase (ALP); Total protein; Albumin; Total Bilirubin; Bile acids; Urea; Creatinine; Glucose; Cholesterol; Sodium; Potassium; Chloride; Calcium; Inorganic Phosphate (Inorg. Phos)

Blood Sampling for Thyroid Hormone Analysis
F0-generation, males and females:
End of study from all animals at planned necropsy; This included females on Day 14-16 of lactation, all females that failed to deliver healthy pups and all males after at least 4 weeks of treatment (including all males that failed to sire).
Blood samples were collected under anaesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples (0.9 mL) were drawn from the retroorbital sinus and collected into serum tubes (Greiner Bio-One GmbH, Kremsmünster, Austria).
After clotting and centrifugation, serum was used as listed below.
Males: 1 aliquot of 150 μL serum was used for measurement of thyroxine (T4), and the remaining volume of serum was kept for possible measurement of thyroid-stimulating hormone (TSH).
Females: The serum was stored for possible measurement of thyroxine (T4) and/or thyroidstimulating hormone (TSH).
Serum samples were stored at ≤-75°C. Under these storage conditions, the samples are stable for 6 months. Any samples remaining after 6 months will be discarded.
In general:
Total T4 was measured in serum using the Immulite 1000® (Siemens Healthcare Diagnostics B.V., Den Haag, The Netherlands):
Thyroxine (T4)

Oestrous cyclicity (parental animals):

Estrous cycle determination: Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period.
During pretest, this was done for 48 females.
On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.

Sperm parameters (parental animals):

Not specified

Litter observations:

Each litter was examined to determine the following, when practically possible:
Mortality / Viability: The numbers of live and dead pups were determined on PND 1 and daily thereafter.
Clinical signs: At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check are presented in the respective tables.
Body weights: Live pups were weighed on PND 1, 4, 7 and 13.
Sex: Sex was determined for all pups on PND 1 and 14. Sex ratio (% male pups / % female pups) was calculated per group.
Anogenital distance: Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
Areola/nipple retention: On PND 13, all males in each litter were examined for the number of areola/nipples.

Blood Sampling for Thyroid Hormone Analysis
F1-generation, PND 4 pups: From 2 surplus pups per litter at culling (when possible). Blood samples from the 2 pups per litter were collected into one serum tube. If only 1 surplus pup per litter was available at culling, as much as possible blood was collected from this single pup.
Blood samples were collected by decapitation, between 7.00 and 10.30 a.m.. Blood samples from the 2 pups per litter (0.4 mL in total) were collected into one serum tube (Greiner Bio-One GmbH, Kremsmünster, Austria) for possible future measurement of thyroxine (T4).
After clotting and centrifugation, serum samples were stored at ≤-75°C. Under these storage conditions, the samples are stable for 6 months. Any samples remaining after 6 months will be discarded.
F1-generation, PND 13-15 pups: From 2 pups per litter (when possible from one male and one female) at planned necropsy.
As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands), between 7.00 and 10.30 a.m.. Blood was collected into serum tubes.
After clotting and centrifugation, serum from each sample was divided into 2 aliquots: 150 μL serum for measurement of thyroxine (T4) and the remaining volume of serum for measurement of thyroid-stimulating hormone (TSH).
Serum samples were stored at ≤-75°C. Under these storage conditions, the samples are stable for 6 months. Any samples remaining after 6 months will be discarded.
In general:
Total T4 was measured in serum using the Immulite 1000® (Siemens Healthcare Diagnostics B.V., Den Haag, The Netherlands):
Thyroxine (T4)

Postmortem examinations (parental animals):

F0-generation - Termination
The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available.
All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.
Necropsy was conducted on the following days:
Condition Day of necropsy
Males Following completion of the mating period (a minimum of 28 days of dose administration).
Females which delivered PND 14-16.
Females which failed to deliver Post-coitum Days 25-27 (females with evidence of mating)
(nos. 62 and 67)

F0-generation – Macroscopic Examination
After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
The number of former implantation sites were recorded for all paired females.
Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
Selected 5 animals/sex/group (see Allocation):
Identification marks: not processed; Adrenal glands; (Aorta); Brain - cerebellum, mid-brain, cortex (7-levels); Caecum; Cervix; Clitoral gland; Colon; Coagulation gland; (Cowper’s gland); Duodenum; Epididymides; Eyes (with optic nerve (if detectable) and Harderian gland); Mammary gland area (males and females); Femur including joint; (Glans penis); (Levator ani plus bulbocavernosus muscle complex (LABC)); Heart; Ileum; Jejunum; Kidneys; (Lacrimal gland, exorbital); (Larynx); Liver; Lung, infused with formalin; Lymph nodes - mandibular, mesenteric; (Nasopharynx); (Esophagus); Ovaries; (Pancreas); Peyer's patches [jejunum, ileum] if detectable; Pituitary gland; Preputial gland; Prostate gland; Rectum; (Salivary glands - mandibular, sublingual); Sciatic nerve; Seminal vesicles; Skeletal muscle; (Skin); Spinal cord -cervical, midthoracic, lumbar; Spleen; Sternum with bone marrow; Stomach; Testes; Thymus; Thyroid including parathyroid if detectable; (Tongue); Trachea; Urinary bladder; Uterus; Vagina; All gross lesions
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

All remaining animals, males that failed to sire, females which failed to deliver and females with total litter loss:
Cervix; Clitoral gland; Coagulation gland; Cowper’s glands; Epididymides; Glans penis; Levator ani plus bulbocavernosus muscle complex (LABC); Mammary gland area (males and females); Ovaries; Preputial gland; Prostate gland; Seminal vesicles; Testes; Thyroid including parathyroid if detectable; Uterus; Vagina; All gross lesions; Identification marks: not processed
Reproductive organs were only examined by the pathologist for males that failed to sire and all females that failed to deliver healthy pups. Female mammary gland area was not examined by the pathologist as there were no females with total litter loss.

F0-generation – Organ Weights
The following organ weights and terminal body weight were recorded from the following animals:
Selected 5 animals/sex/group (see Allocation):
Adrenal glands; Brain; Cowper’s glands; Epididymides; Glans penis; Heart; Kidneys; Levator ani plus bulbocavernosus muscle complex (LABC); Liver; Ovaries; Prostate; Seminal vesicles including coagulating glands; Spleen; Testes; Thymus; Thyroid (including parathyroid if detectable); Uterus (including cervix).

All remaining animals:
Cowper’s glands; Epididymides; Glans penis; Levator ani plus bulbocavernosus muscle complex (LABC); Testes; Thyroid
Absolute organ weights and organ to body weight ratios are reported.

F0-generation - Histotechnology
All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

F0-generation – Histopathology
The following slides were examined by a pathologist:
-The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4 (see Allocation).
-Additional slides of the testes of the selected 5 males (see Allocation) of Groups 1 and 4 and all males that failed to sire to examine staging of spermatogenesis.
-All gross lesions of all animals (all dose groups).
-Thyroid gland and liver of all selected 5 males of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
-The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups:

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
A peer review on the histopathology data was performed by a second pathologist.

Postmortem examinations (offspring):

F1-generation Pups - Termination
All remaining pups (PND 7-15) were sacrificed using Euthasol® 20% (AST Farma B.V., Oudewater, The Netherlands) by intraperitoneal (ip) injection.
On PND 4 (at culling), from 2 pups per litter, blood samples (0.4 mL in total) were collected by decapitation between 7.00 and 10.30 a.m. Blood samples from the 2 pups per litter were collected into one serum tube.
On PND 13-15, from 2 pups per litter (when possible from one male and one female) blood samples were collected at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands), between 7.00 and 10.30 a.m., followed by exsanguination. Blood was collected into serum tubes.
Necropsy was conducted on the following days:
Condition Day of necropsy
Culling PND 4.
Terminal sacrifice PND 13-15.
Spontaneous deaths As soon as possible after death and always within 24 hours.

F1-generation pups - Macroscopic Examination
All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded.
At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling (see also section 5.10 and 5.12.1), was preserved in 10% buffered formalin.
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk.

Statistics:

The following statistical methods were used to analyse the data:
-If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
-The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
-The Fisher Exact-test was applied to frequency data.
-The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:
Mating index (%)
(Number of females mated/ Number of females paired) x 100

Fertility index (%)
(Number of pregnant females/ Number of females paired) x 100

Conception index (%)
(Number of pregnant females/ Number of females mated) x 100

Gestation index (%)
(Number of females bearing live pups/ Number of pregnant females) x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Post-implantation survival index (%)
(Total number of offspring born/ Total number of uterine implantation sites) x 100

Live birth index (%)
(Number of live offspring on Day 1 after littering/Total number of offspring born) x 100

Percentage live males at First Litter Check (%)
(Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100

Percentage live females at First Litter Check (%)
(Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100

Viability index (%)
(Number of live offspring on Day 4 before culling/Number of live offspring on Day 1 after littering) x 100

Lactation index (%)
(Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling) x 100

Group mean values were calculated from individual litter values.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Within one week after start of treatment, uncoordinated movements were observed in a few males and females treated at 30 mg/kg/day at 1-2 hours after dosing. The appearance of uncoordinated movements was slightly earlier in females than in males, i.e on day 4 and on day 6 after start of treatment, respectively. The number of males and females affected gradually increased during the treatment-period until the majority of animals showed uncoordinated movements after three weeks of treatment. The presence of this symptom was not consistent in most of the animals or was observed only incidentally. Moreover, in females the degree of uncoordinated movements varied between 1 and 2 (maximum possible grade 3) in several animals, whereas in males uncoordinated movements grade 1 only were observed.
In addition, in some females temporary behavioural changes, including lethargy, restlessness or fearfulness, were observed at the end of the post-coitum or early in the lactation phase.

Salivation was observed for a large part of the study immediately after treatment. However, on-line recording of the observation of salivation was performed from week 4 of treatment onwards. Salivation was observed immediately after dosing in a dose related manner among the animals of all test groups. Salivation was not observed in any of the animals at the observation performed at the time of peak effect 1-2 hours after dosing. The number of males per dose group exhibiting salivation was slightly higher than the number of females of the concurrent dose group. Salivation was also observed in a single male and female in the vehicle control group. Salivation was considered to be a physiological response to the formulation rather than a sign of systemic toxicity considering the nature of the effect and its time of occurrence (i.e. after dosing).

Piloerection was observed in one female of the mid- and one of the high-dose group for a short period shortly after delivery. Signs of discomfort in females at the time of delivery are occasionally observed and in this case, also based on the single occurrences, not related to treatment.

Incidental findings that were noted included, rales and scabs or alopecia in various locations.
These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study.
On one occasion (Day 13 of treatment), some fluid was observed in the oral cavity of female 55 during dosing, likely originated from the stomach and might have contained test formulation, stomach contents and/or saliva. At the incidence observed, these incidental findings were considered not to be signs of toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

For males and females, body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
The body weight gain in females treated at 30 mg/kg/day on day 4 post coitum (PC), when compared to day 0 PC, was slightly lower and reached a level of statistical significance when compared to the body weight gain in controls. Since body weight gain in these high dose females from day 7 PC onwards was similar to that in controls and the other test groups, the difference in body weight on day 4 PC was considered of no toxicological significance.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption before or after allowance for body weight was similar between treated and control animals.
In high dose females, the food consumption relative to body weight was slightly lower over the last few days of the post-coitum phase (days 17-20) and over the lactation phase, achieving a level of statistical significance at days 17-20 post-coitum when compared to that in controls. The lower values for relative food consumption were the result of slightly higher body weights in combination with a normal food consumption in high dose females. These changes in high dose females might be explained by the assumption that the test item had some nutritional value. However, no toxicological relevance was attached to these findings.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Haematological parameters of treated male and female rats were considered not to be affected by treatment.
In the absence of a treatment related distribution, the levels of statistical significance observed for the coagulation parameter PT in the low and mid dose males were considered not to be of toxicological relevance. Moreover, (minimal) shortening of the PT is considered to have little clinical relevance.
The statistically significant changes in mid dose females for monocytes and haematocrit were considered not to be toxicologically relevant as they occurred in the absence of a treatment related distribution. Moreover, the individual monocytes and haematocrit values in these females remained within the range considered normal for female rats of this age and strain.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical biochemistry parameters of treated male and female rats were considered not to be affected by treatment.
The statistically significant changes for chloride (mid dose males) and total protein (mid dose females), when compared to controls, were considered not to be toxicologically significant as they occurred in the absence of a treatment-related distribution. Moreover, no corroborative findings were observed in these animals and all values were within normal limits.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex, and static righting reflex were normal in all selected animals.
A dose-related decrease in grip strength of the fore limbs was observed in both males and females, achieving a level of statistical significance in the males treated at 30 mg/kg/day compared to the concurrent vehicle controls. The grip strength of the hind limbs was similar in all dose groups and comparable to that in controls in males as well as in females.

In the motor activity test, comprising measurement of movements and ambulations in 12 successive intervals of 5 minutes each, all male groups showed a similar habituation profile with very high activity in the first interval that gradually decreased over the duration of the test period.
However, in one male treated at 3 mg/kg/day (no. 14) an aberrant habituation pattern was observed. Relatively high movements and ambulations were recorded over the 3rd to 7th time interval for this male, resulting in high total values for these parameters of more than twice the average of the other four males of this group. Exclusion of male no 14 from the group resulted in similar group mean values for movements and ambulation compared to the other groups. The aberrant habituation pattern in this male was considered a fortuitous finding and not related to treatment.

In females, the habituation pattern in the controls and low and mid dose were similar to that observed in males, but a different habituation pattern was observed in high dose females, receiving 30 mg/kg bw/day. After a normal, high activity in these high dose females in the first interval, the activity in the second interval was immediately decreased to a (very) low level, for both total movements and ambulations, and remained low until the end of the test.
One high dose female (no. 71), however, showed an increase in activity in the 3rd interval which remained relatively high over the complete test period. As a result this female showed the highest activity of all females in study. Exclusion of female no 71 from the group resulted in high dose group mean values for total movements and ambulations that were clearly lower than the other groups and a standard deviation of the group mean that was comparable to those of the other group means. On the other hand, as the effects on motor activity in the high dose females were suspected to be treatment-related, the high variation in total number of movements and total number of ambulations among the individual females of the high dose group might be the result and do not legitimate exclusion of any female from the high dose group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with LOWINOX® 22IB46 were noted in the liver and thyroid gland of the 30 mg/kg/day group males.
Liver: Hepatocellular hypertrophy was recorded in the liver of all males at 30 mg/kg/day at minimal degree.
Thyroid gland: An increased incidence and severity of follicular cell hypertrophy, up to a slight degree, was present in males treated at 30 mg/kg/day.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone analyses:
The lower serum levels of T4 observed in the low and mid dose F0-males, achieving levels of statistical significance when compared to controls, were considered to have arisen as a result of slightly high control values. Therefore, and in the absence of a treatment-related distribution these changes were considered not to be toxicologically relevant. Moreover, all values were within normal limits.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment.
An irregular cycle was noted for one high dose female treated at 30 mg/kg/day (no. 79) prior to start of mating, but this female gave birth to a normal litter. Given their incidental nature and absence of an apparent correlation to pregnancy status, this finding did not indicate a relation with treatment.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were 2/10 couples (males no. 22 and 27 and females no. 62 and 67) of the 10 mg/kg/day group with no offspring. No abnormalities were seen in the reproductive organs, which could account for their lack of offspring.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and spermatogenic staging profiles were normal for all males examined.

Reproduction Data
No toxicologically relevant effects on reproductive parameters were noted.
There were 2/10 couples (male 22/female 62 and male 27/females 67) at 10 mg/kg/day without offspring. Based on (low) body weight gain during the post coitum phase and the lack of implantations sites, female 67 has not been pregnant. Based on body weight gain during the post coitum phase, female 62 was supposed to be pregnant. This female was scheduled for necropsy on day 26 post coitum because it failed to deliver. At necropsy, this female showed a body weight loss of approximately 15% compared to on day 20 post coitum and the presence of (one) implantation site. However, no evidence of resorption(s) was found.
It was considered possible that littering (of one pup) was missed and that this female could have had a total litter loss. Furthermore, no abnormalities were seen in the reproductive organs which could account for their lack of healthy offspring. In the absence of a dose related incidence, this lack of healthy offspring in two mid dose females was considered not to be related to treatment.

Mating index
Mating index was considered not to be affected by treatment. All females showed evidence of mating. In two females (control no. 41 and low dose no. 52) evidence of mating was obtained indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the daily vaginal lavage and also explains the continuation of di-estrous during the mating in these females.

Fertility and Conception index
Fertility and conception index were considered not to be affected by treatment.
One mid dose female treated at 10 mg/kg/day (no. 67) was not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was considered not to be related to treatment.

Precoital time
Precoital time was considered not to be affected by treatment.
The median as well as the mean precoital time in high dose females treated at 30 mg/kg/day was higher than observed in the other groups. This was considered to be caused by the fact that by accident no high dose females were found positive of mating on the first day of the mating period. Since all high dose females were found positive within 4 days, similar as in the other groups, no toxicological significance was attached to this finding.

Number of implantation sites
Number of implantation sites was considered not to be affected by treatment.
For one mid dose female (no.57), the number of pups was higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during (the 16 days of) lactation. No toxicological relevance was attached to this finding in the current study.

Developmental Data
Gestation index and duration
Gestation index and duration of gestation were considered not to be affected by treatment.

Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females and no deficiencies in maternal care were observed.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.

Post-implantation survival index
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

other: behavioural changes observed in the functional observations tests

Key result

Critical effects observed:

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Several pups among litters from high dose females (10 pups distributed over three litters) showed less or no milk early in the lactation period. Four of these pups were found dead shortly thereafter, whereas the other six pups recovered and survived until scheduled necropsy. No further clinical signs occurred among pups that were considered to be related to treatment.

Clinical symptoms were incidentally observed among single pups. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age.
Moreover, distribution over the dose groups did not show a relation to treatment. These findings were therefore considered not to be toxicologically relevant.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Live birth index
A minimal tendency to a dose related increase in the number of litters with one or more dead pups at first litter check was observed. The number of litters with dead pups at first litter check were 1, 2, (total of six dead pups), 3 (total of three dead pups) and 5 (total of seven dead pups), for the control, 3, 10 and 30 mg/kg/day treated animals, respectively. In five out of seven of the dead pups in litters from high dose females no milk was found in the stomach, whereas no findings were observed in the other dead pups at macroscopic examination. These results are excluding the possible total litter loss (of possibly one pup) in one female treated at 10 mg/kg/day.

Viability index
A number pups went missing between PND 1 and PND 4 (before culling). The number of pups that went missing was statistically significantly higher among litters from high dose females compared to that in the litters of other groups. The total number of missing pups between Days 1 to 4 were 0, 1, 0 and 9 (distributed over five litters), for the control, 3, 10 and 30 mg/kg/day treated animals, respectively. The missing pups were most likely cannibalized.

Lactation index
The number of live offspring on PND 13 compared to the number of live offspring on PND 4 (after culling) was considered not to be affected by treatment. No pups were found dead/missing between lactation Days 5 and 13.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Lower body weights of male and female pups from high dose dams treated at 30 mg/kg/day were observed during lactation, achieving levels of statistical significance for female pups on PND 7 and PND13 when compared to controls. The effect was slightly more prominent in female pups than in male pups and the difference in mean body weight between high dose pups and controls increased during the progress of the lactation phase.

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:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 13-15 pups were considered not to be affected by treatment.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Among litters from high dose females, five (out of seven) pups that were found dead at first litter check showed no milk in the stomach, whereas no findings were observed in the dead pups at first litter check from the other dose groups at macroscopic examination. No macroscopic findings were noted among pups surviving until scheduled necropsy that were considered to be related to treatment.

Macroscopic findings were incidentally observed among pups at necropsy. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant. Moreover, the distribution of the findings over the groups did not indicated a relation to treatment.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment.

Anogenital distance
The median for the absolute anogenital distance in female pups from high dose dams treated at 30 mg/kg/day was statistically significantly reduced when compared to controls. The anogenital distance normalized for body weight in female pups was similar to that in the other groups.
The anogenital distance in male pups (absolute and normalized for body weight) was considered not to be affected by treatment.
The statistically significantly higher median anogenital distance of male pups from dams treated at 3 mg/kg/day was considered an incidental finding and in the absence of a dose related-trend no toxicological significance was attached to this change.

Areola/nipple retention
Treatment up to 30 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

Key result

Critical effects observed:

Key result

Reproductive effects observed:

Lowest effective dose / conc.:

30 mg/kg bw/day (nominal)

Treatment related:

REPRODUCTION DATA SUMMARY

GROUP 1

CONTROL

GROUP 2

3 MG/KG

GROUP 3

10 MG/KG

GROUP 4

30 MG/KG

Females paired

Females mated

Pregnant females

Females with implantations only

Females with living pups on Day 1

Mating index (%)

(Females mated/Females paired ) * 100

Fertility index (%)

(Pregnant females/Females paired) * 100

Conception index (%)

(Pregnant females/Females mated) * 100

Gestation index (%)

(Females with living pups on Day 1/Pregnant females) * 100

100

PRECOITAL TIME

F0-GENERATION – POST COITUM

DAY OF THE

PAIRING PERIOD

GROUP 1

CONTROL

GROUP 2

3 MG/KG

GROUP 3

10 MG/KG

GROUP 4

30 MG/KG

NUMBER OF FEMALES MATED

MEDIAN PRECOITAL TIME

MEAN PRECOITAL TIME

2.3

2.4

1.9

2.9

IMPLANTATION SITES SUMMARY

FEMALES

		GROUP 1 CONTROL	GROUP 2 3 MG/KG	GROUP 3 10 MG/KG	GROUP 4 30 MG/KG
NECROPSY
Implantations	MEAN ST. DEV N	11.4 3.0 10	13.3 1.9 10	11.9 4.1 9	14.2 2.9 10

DEVELOPMENTAL DATA

F0-GENERATION – LACTATION

GROUP 1

CONTROL

GROUP 2

3 MG/KG

GROUP 3

10 MG/KG

GROUP 4

30 MG/KG

LITTERS

TOTAL

DURATION OF GESTATION

MEAN (+)

ST. DEV

21.4

0.7

21.7

0.7

21.5

1.1

21.7

0.5

DEAD PUPS AT FIRST LITTER CHECK

LITTERS AFFECTED (#)

TOTAL

MEAN (+)

ST. DEV

0.1

0.3

0.6

1.6

0.4

0.5

0.7

0.9

LIVING PUPS AT FIRST LITTER CHECK

% OF MALES/FEMALES (#)

TOTAL

MEAN (+)

ST. DEV

49/51

108

10.8

2.7

53/47

119

11.9

1.8

45/55

11.4

0.9

50/50

111

11.1

2.7

POSTNATAL LOSS

% OF LIVING PUPS

LITTERS AFFECTED (#)

TOTAL (#)

MEAN (+)

ST. DEV

0.0

0.8

0.1

0.3

0.0

8.1

9 ##

0.9

1.3

CULLED PUPS

TOTAL

LIVING PUPS DAY 4 P. P.

TOTAL

MEAN (+)

ST. DEV

7.6

1.3

8.0

0.0

8.0

0.0

7.8

0.6

BREEDING LOSS DAYS 5-13 P.P.

% OF LIVING PUPS AT DAY 4 P. P.

LITTERS AFFECTED (#)

TOTAL (#)

MEAN (+)

ST. DEV

0.0

LIVING PUPS DAY 13 P. P.

% OF MALES/FEMALES (#)

TOTAL

MEAN (+)

ST. DEV

50/50

7.6

1.3

53/48

8.0

0.0

48/52

8.0

0.0

53/47

7.8

0.6

Viability index = (Number of alive pups on day 4 p.p. / Number of pups born alive) * 100

Weaning index = (Number of alive pups on day 13 p.p. / Number of alive pups in day 4 p.p.) * 100

+/++ Steel-test significant at 5% (+) or 1% (++) level

#/## Fisher’s Exact test significant at 5% (#) or 1% (##) level

DEVELOPMENTAL DATA

GROUP 1

CONTROL

GROUP 2

3 MG/KG

GROUP 3

10 MG/KG

GROUP 4

30 MG/KG

Total number of offspring born

Total number of uterine implantation sites

Number of live offspring on Day 1 after littering

Number of live offspring on Day 4 (before culling)

Number of live offspring on Day 4 (after culling)

Number of live offspring on Day 13 after littering

109

114

108

125

133

119

118

107

118

142

111

102

Post-implantations survival Index (%)

(Total number of offspring born/Total number of uterine implantation sites) * 100

Live birth Index (%)

(Number of live offspring on Day 1 after littering/Total number of offspring born) * 100

Viability Index (%)

(Number of live offspring on Day 4 (before culling)/Number of live offspring on Day 1 after littering) * 100

Lactation Index (%)

(Number of live offspring on Day 13 after littering/Number of live offspring on Day 4 (after culling)) * 100

100

BODY WEIGHTS OF PUPS (GRAM)

F0-GENERATION – LACTATION

DAY

SEX

GROUP 1

CONTROL

GROUP 2

3 MG/KG

GROUP 3

10 MG/KG

GROUP 4

30 MG/KG

MEAN

ST. DEV

6.7

0.9

6.7

0.8

6.6

0.8

6.3

0.7

MEAN

ST. DEV

6.5

0.9

6.3

0.8

6.3

0.8

6.0

0.8

M+F

MEAN

ST. DEV

6.6

0.9

6.5

0.8

6.4

0.8

6.1

0.7

MEAN

ST. DEV

9.9

1.8

9.6

1.2

9.4

1.1

8.8

1.3

MEAN

ST. DEV

9.7

1.9

9.0

1.2

9.1

1.3

8.4

1.1

M+F

MEAN

ST. DEV

9.8

1.8

9.3

1.2

9.2

1.2

8.7

1.2

MEAN

ST. DEV

16.2

2.8

15.7

1.9

15.7

1.7

14.3

1.8

MEAN

ST. DEV

16.1

2.8

15.0

1.8

15.1

1.9

13.5*

1.5

M+F

MEAN

ST. DEV

16.2

2.7

15.3

1.9

15.4

1.8

13.9

1.8

MEAN

ST. DEV

30.6

4.3

29.5

2.6

29.7

2.6

27.8

3.8

MEAN

ST. DEV

30.3

3.9

28.4

2.6

28.7

2.8

26.7*

3.6

M+F

MEAN

ST. DEV

30.5

4.1

29.0

2.6

29.2

2.6

27.3

3.9

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

ANOGENITAL DISTANCE AND NIPPLE RETENTION PER GROUP

FEMALES

F0-GENERATION – LACTATION

GROUP 1

CONTROL

GROUP 2

3 MG/KG

GROUP 3

10 MG/KG

GROUP 4

30 MG/KG

Anogenital dist M mm

MEAN

MEDIAN (+)

2.46

2.43

2.51

2.53++

2.45

2.46

2.48

2.45

Anogenital dist F mm

MEAN

MEDIAN (+)

0.91

0.93

0.88

0.90

0.94

0.83

0.84++

Number of nipples

MEAN

MEDIAN (+)

0.00

CORRECTED ANOGENITAL DISTANCE SUMMARY

FEMALES

GROUP 1

CONTROL

GROUP 2

3 MG/KG

GROUP 3

10 MG/KG

GROUP 4

30 MG/KG

AT LACTATION

Norm anog dist M mm

MEAN

ST. DEV

1.31

0.08

1.33

0.04

1.31

0.06

1.35

0.06

Norm anog dist F mm

MEAN

ST. DEV

0.49

0.05

0.48

0.04

0.51

0.03

0.46

0.06

+/++ Steel test significant at 5% (+) or 1% (++) level

CLINICAL BIOCHEMISTRY SUMMARY

MALES

GROUP 1

CONTROL

GROUP 2

3 MG/KG

GROUP 3

10 MG/KG

GROUP 4

30 MG/KG

PND 13-15

Total T4

μg/dL

MEAN

ST. DEV

6.87

1.25

7.66

1.21

6.98

1.33

7.00

1.29

FEMALES

GROUP 1

CONTROL

GROUP 2

3 MG/KG

GROUP 3

10 MG/KG

GROUP 4

30 MG/KG

PND 13-15

Total T4

μg/dL

MEAN

ST. DEV

6.41

1.31

6.77

1.08

6.42

0.64

6.09

0.83

+/++ Steel-test significant at 5% (+) or 1% (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Conclusions:

Lowinox® 22IB46 was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 3, 10 and 30 mg/kg. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 31 days). The females that delivered were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 49-56 days). Females that failed to deliver healthy offspring were exposed for 40-42 days.

Based on the results of formulation analysis it was considered that the formulations were prepared accurately and homogenously, and were stable for at least 5 hours at room temperature down to concentrations as low as 0.6 mg/mL (equivalent to a dose level of 3 mg/kg).

Parental results:
Uncoordinated movements were observed for a major part of the study period in both high dose males and females treated at 30 mg/kg/day. In some of these females, temporary behavioural changes, including lethargy, restlessness or fearfulness, were occasionally observed. The functional observation test performed towards the end of the treatment period showed decreased fore limb grip strength in both high dose males and females and an accelerated decrease in movements and ambulations in the motor activity test in high dose females only in contrast to a more gradual decrease in activity in a normal habituation pattern. There might be a relation between the clinical signs and behavioural changes which might be interpreted as signs of neurotoxicity caused by treatment with LOWINOX® 22IB46 at 30 mg/kg/day. In the absence of changes in gait/motility and effects on motor activity in the mid dose animals, the minimal decreases in fore limb grip strength observed at this dose level were considered to be non-adverse and of no toxicological significance.
Small increases in liver weights were observed in high dose males. Histopathology of the liver revealed minimal hepatocellular hypertrophy, but in the absence of any degenerative findings this was considered to be a non-adverse finding.
Furthermore, histopathology also revealed follicular cell hypertrophy of the thyroid gland in males at 30 mg/kg/day. The minor increase in incidence and/or severity (up to slight degree) and the fact that it is usually an adaptive response to induction of hepatic enzymes, this finding was regarded to be an adaptive change and considered to be non-adverse.

No treatment-related and/or toxicologically significant changes were noted in any of the other parental parameters investigated in this study (i.e. body weight, food consumption, clinical laboratory investigations and macroscopic examination).

Reproductive results:
No reproduction toxicity was observed up to the highest dose level tested (30 mg/kg/day).

Developmental results:
A minimal tendency to a dose related increase in the number of litters with one or more dead pups at first litter check and a higher number of pups that went missing in litters from the high dose females. The total of numbers pups loss up to PND 4 (before culling) were 1, 7 (among three litters), 3 (among three litters) and 16 (among seven litters). No pups loss was observed between PND 4 and PND 13-15 in any litter of all dose groups. Lower body weights in male and female pups of high dose dams treated at 30 mg/kg/day were observed during the lactation phase. The median for the absolute anogenital distance in female pups from high dose dams treated at 30 mg/kg/day was reduced. Since the normalized anogenital distance in female pups was similar to that in the other groups, the change in anogenital distance was considered to be related to the lower body weights observed in the female pups.
Therefore, no toxicological significance was attached to the changes in anogenital distance itself, but this finding must be evaluated in combination with the lower body weights and might indicate a retarded development of the high dose pups in comparison with control pups.
It was striking that less or no milk in the stomach was observed among pups from high dose females only early in the lactation phase, whereas this phenomenon was not observed in pups from females in the other dose groups. The reduced amount of milk in the stomach in nine (out of sixteen) the pups that were found dead (at first litter check) or went missing in the high dose group, might have been related to its death. It could not be established from the results obtained in this study if the reduced amount of milk was the result of effects in the females or of effects in the pups itself.
No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, lactation indices, duration of gestation, parturition, sex ratio, maternal care, clinical signs, areola/nipple retention (PND 13 males), T4 thyroid hormone levels (PND 4 and 13-15) and macroscopy.

In conclusion, treatment with LOWINOX® 22IB46 by oral gavage in male and female Wistar Han rats at dose levels of 3, 10 and 30 mg/kg revealed parental and developmental toxicity at 30 mg/kg/day. No reproduction toxicity was observed for treatment up to 30 mg/kg/day.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 10 mg/kg/day (based on clinical signs and behavioural changes observed in the functional observations tests)
Reproduction NOAEL: at least 30 mg/kg (the highest dose level tested in this study)
Developmental NOAEL: 10 mg/kg/day (based on increased pup loss during early development and slight retardation of development of pups in the lactation phase)

Executive summary:

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of LOWINOX® 22IB46 in rats by oral gavage, including preliminary dose range finder.

Guidelines

The study was based on the following guidelines:

-OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2015.

-OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

-OECD 421, Reproduction/Developmental Toxicity Screening Test, July 2015

-OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

-EC No 440/2008 B.7: "Repeated Dose (28 days) Toxicity (oral)", May 2008.

-OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.

-OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

Rationale for dose levels

Based on the results of a 10-day dose range finder, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 3, 10 and 30 mg/kg.

Study outline

The test item, formulated in propylene glycol, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Sampling and analysis of dose formulations was performed on two occasion during the treatment phase to show accuracy, homogeneity and stability of the test item in vehicle, propylene glycol.

Males were exposed for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 49-56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were exposed for 40-42 days.

Evaluated parameters

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment and PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: mating, fertility and conception indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy).

Results/discussion

Based on the results of formulation analysis it was considered that the formulations were prepared accurately and homogenously, and were stable for at least 5 hours at room temperature down to concentrations as low as 0.6 mg/mL (equivalent to a dose level of 3 mg/kg).

Parental results:

Uncoordinated movements were observed for a major part of the study period in both high dose males and females. In some of these females, temporary behavioural changes, including lethargy, restlessness or fearfulness, were occasionally observed. The functional observation test performed towards the end of the treatment period showed decreased fore limb grip strength in both high dose males and females and an accelerated decrease in movements and ambulations in the motor activity test in high dose females only in contrast to a more gradual decrease in activity in a normal habituation pattern. There might be a relation between the clinical signs and behavioural changes which might be interpreted as signs of neurotoxicity caused by treatment with LOWINOX® 22IB46 at 30 mg/kg/day. In the absence of changes in gait/motility and effects on motor activity in the mid dose animals, the minimal decreases in fore limb grip strength observed at this dose level were considered to be non-adverse and of no toxicological significance.

Small increases in liver weights were observed in high dose males. Histopathology of the liver revealed minimal hepatocellular hypertrophy, but in the absence of any degenerative findings this was considered to be a non-adverse finding.

Furthermore, histopathology also revealed follicular cell hypertrophy of the thyroid gland in high dose males. The minor increase in incidence and/or severity (up to slight degree) and the fact that it is usually an adaptive response to induction of hepatic enzymes, this finding was regarded to be an adaptive change and considered to be non-adverse.

No treatment-related and/or toxicologically significant changes were noted in any of the other parental parameters investigated in this study (i.e. body weight, food consumption, clinical laboratory investigations and macroscopic examination).

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (30 mg/kg/day).

Developmental results:

A minimal tendency to a dose related increase in the number of litters with one or more dead pups at first litter check and a higher number of pups that went missing in litters from the high dose females. The total of numbers pups loss up to PND 4 (before culling) were 1, 7 (among three litters), 3 (among three litters) and 16 (among seven litters). No pup loss was observed between PND 4 and PND 13-15 in any litter of all dose groups. Lower body weights in male and female pups of high dose dams treated at 30 mg/kg/day were observed during the lactation phase. The median for the absolute anogenital distance in female pups from high dose dams was reduced. Since the normalized anogenital distance in female pups was similar to that in the other groups, the change in anogenital distance was considered to be related to the lower body weights observed in the female pups. Therefore, no toxicological significance was attached to the changes in anogenital distance itself, but this finding must be evaluated in combination with the lower body weights and might indicate a retarded development of the high dose pups in comparison with control pups.

It was striking that less or no milk in the stomach was observed among pups from high dose females only early in the lactation phase, whereas this phenomenon was not observed in pups from females in the other dose groups. The reduced amount of milk in the stomach in nine (out of sixteen) the pups that were found dead (at first litter check) or went missing in the high dose group, might have been related to its death. It could not be established from the results obtained in this study if the reduced amount of milk was the result of effects in the females or of effects in the pups itself.

No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, lactation indices, duration of gestation, parturition, sex ratio, maternal care, clinical signs, areola/nipple retention (PND 13 males) and T4 thyroid hormone levels (PND 4 and 13-15).

Conclusion

Treatment with LOWINOX® 22IB46 by oral gavage in male and female Wistar Han rats at dose levels of 3, 10 and 30 mg/kg revealed parental and developmental toxicity at 30 mg/kg/day. No reproduction toxicity was observed for treatment up to 30 mg/kg/day.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: 10 mg/kg/day (based on clinical signs and behavioural changes observed in the functional observations tests)

Reproduction NOAEL: at least 30 mg/kg (the highest dose level tested in this study)

Developmental NOAEL: 10 mg/kg/day (based on increased pup loss during early development and slight retardation of development of pups in the lactation phase).

Effect on fertility: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 30 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: K1

Effect on fertility: via inhalation route

Endpoint conclusion:: no study available

Effect on fertility: via dermal route

Endpoint conclusion:: no study available

Additional information

Reproduction/developmental toxicity screening test

Evaluated parameters

Results/discussion

Parental results:

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (30 mg/kg/day).

Conclusion

Treatment with LOWINOX® 22IB46 by oral gavage in male and female Wistar Han rats at dose levels of 3, 10 and 30 mg/kg revealed parental toxicity at 30 mg/kg/day. No reproduction toxicity was observed for treatment up to 30 mg/kg/day.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: 10 mg/kg/day (based on clinical signs and behavioural changes observed in the functional observations tests)

Reproduction NOAEL: at least 30 mg/kg (the highest dose level tested in this study)

Effects on developmental toxicity

Description of key information

Reproduction/developmental toxicity screening test

Parental NOAEL: 10 mg/kg/day (based on clinical signs and behavioural changes observed in the functional observations tests).

Developmental NOAEL: 10 mg/kg/day (based on increased pup loss during early development and slight retardation of development of pups in the lactation phase).

Effect on developmental toxicity: via oral route

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 10 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: K1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:: no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:: no study available

Additional information

Reproduction/developmental toxicity screening test

Evaluated parameters

Results/discussion

Parental results:

Developmental results:

Conclusion

Treatment with LOWINOX® 22IB46 by oral gavage in male and female Wistar Han rats at dose levels of 3, 10 and 30 mg/kg revealed parental and developmental toxicity at 30 mg/kg/day.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: 10 mg/kg/day (based on clinical signs and behavioural changes observed in the functional observations tests)

Developmental NOAEL: 10 mg/kg/day (based on increased pup loss during early development and slight retardation of development of pups in the lactation phase)

Justification for classification or non-classification

Additional information

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