1,1,3,3-tetramethylguanidine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

- Basis for dose level selection
In a test study, TMG was administered by gavage at dose levels of 0, 30, 100 and 300 mg/kg bw/d to groups of each 4 male and 4 female animals.
At 300 mg/kg bw/d, severe clinical findings in one male and 2 female animals occurred, i.e. piloerection and labored respiration. Two female animals had to be sacrificed in a moribund condition. For all males reduced food consumption and loss of body weight was observed. In addition, salivation shortly after administration was observed in all male and female animals. In both sexes, decreased red blood cell counts and hematocrit values were observed together with increased reticulocyte counts. At necropsy, foci in the forestomach and discoloration of glandular stomach were found in all male and 3 female animals, respectively. At 100 mg/kg bw/d, salivation was also observed in all male and 2 female animals in addition to labored respiration in one female. Clinical pathology parameters did not reveal relevant changes, but at necropsy foci in the forestomach and discoloration of the liver were found in one female animal. No findings of concern were observed at 30 mg/kg bw/d. Therefore, the following dose levels were selected for the present study: 10, 30, 100 mg/kg bw/d.

Test material

Test material information

Constituent 1

Reference substance name:

1,1,3,3-tetramethylguanidine

EC Number:

201-302-7

EC Name:

1,1,3,3-tetramethylguanidine

Cas Number:

80-70-6

Molecular formula:

C5H13N3

IUPAC Name:

N,N,N',N'-tetramethylguanidine

Test material form:

liquid

Details on test material:

Puritiy: 99.4 area-% (GC, DB-WAX) and
99.5 area-% (GC, DB 1)
Batch: 2313307

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar Rat; Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Test system and strain: Wistar Rat; Crl:WI(Han)
All animals were free from clinical signs of disease at the start of the study. The females were nulliparous and non-pregnant at the beginning of the study. This was necessary to rule out the possibility of sibling mating

Age (at supply): About 10-11 weeks (male animals), About 8-9 weeks (female animals)

Supplier: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany

Identification:
- Pre-treatment period for cycle determination and randomization: tail-markings
- F0 generation parental animals: ear tattoo (animal number)
- Pups: skin tattoo on PND1
As the pups grow older, the growth of fur makes it impossible to identify them unambiguously by their tattoos.
Thus, the pups will be marked with picric acid between PND 10 and 13.

HOUSING AND DIET
- During pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm); supplied by TECHNIPLAST, Hohenpeißenberg, Germany
- During pre-mating, mating, gestation, lactation, males after mating and females afterweaning: Polycarbonate cages type III
- For motor activity (MA) measurements the animals were housed individually in polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany, with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material
- The food used was ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland. Food and drinking water (from water bottles) were available ad libitum.

Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
Dust-free wooden bedding was used in this study. Wooden gnawing blocks (Lignocel Block Large) der Firma J. Rettenmaier & Söhne GmbH + Co KG, was added for environmental enrichment.
The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.

ENVIRONMENTAL CONDITIONS
The animals were housed in a fully air-conditioned room.
Central air-conditioning guaranteed a range of 20-24°C for temperature and of 30-70% for relative humidity and 15 air changes per hour.
The day/night cycle was 12 hours (12 hours light from 06.00-18.00 h, 12 hours dark from 18.00-06.00 h).
There were no or only minimal deviations from these limits.

The animal room was completely disinfected prior to the study using a disinfector ("AUTEX", fully automatic, formalin-ammonia-based terminal disinfector). The floor and the walls were cleaned once a week with water containing an appropriate disinfectant.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

volume applied: 10 mL/kg bw/d

Details on mating procedure:

MATING OF F0 GENERATION PARENTAL ANIMALS
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group. The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 06.30 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1".

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical investigations of the test substance preparations were carried out as a separate study at the test facility Competence Center Analytics of BASF SE, 67056 Ludwigshafen, Germany, under the responsibility of a Study Director of this test facility. This study was carried out in compliance with the Principles of Good Laboratory Practice.
The stability of 1,1,3,3-Tetramethylguanidine in deionized water over a period of 7 days was proven.
At the beginning (during pre-mating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. These samples were used as a concentration control at the same time. At the time points mentioned above, additionally one sample from the mid concentration was taken for concentration control analysis.
The samples collected at the beginning of the administration period and during the lactation period were analyzed.

Stability analysis
The stability of the test substance in deionized water was demonstrated over a period of 7 days at room temperature (study No. 17L00150). As the mixtures were stored no longer than this time period, the stability was guaranteed.
Homogeneity control analyses
Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that 1,1,3,3-Tetramethylguanidine was distributed homogeneously in deionized water.
Concentration control analyses
The concentrations of 1,1,3,3-Tetramethylguanidine in deionized water were found to be in the range of 90-110% of the nominal concentration.
The results demonstrated the correctness of the concentrations of 1,1,3,3-Tetramethylguanidine in deionized water.

Duration of treatment / exposure:

dosing of males: 28 days
dosing of females: up to 55 days
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 for both sexes lasted up to one day prior to sacrifice.

Frequency of treatment:

daily at the same time in the morning (exception: no administration to animals being in labor).

Details on study schedule:

F0 generation parental animals and their progeny
On the day of arrival, the animals were subjected to an acclimatization period during which they received ground diet and drinking water ad libitum. 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. Prior to the first detailed clinical observation, the animals were distributed according to weight among the individual test groups, separated by sex. The weight variation of the animals used did not exceed 20 percent of the mean weight of each sex. The list of randomization instructions was compiled by a computer. After the acclimatization period, the test substance was administered orally via gavage to the F0 generation parental animals, daily at the same time in the morning (exception: no administration to animals being in labor). The treatment lasted up to one day prior to sacrifice. The animals of the control group were treated in the same way with the vehicle only. The calculation of the administered volume was generally based on the most recent individual body weights. Two weeks after the beginning of treatment, the surviving males and females from the same test group were mated overnight in a ratio of 1:1.

Standardization of litters (F1 generation pups)
On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If individual litters did not have 4 pups/sex, the litters were processed in such a way that the most evenly distributed 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups). Surplus pups or 2 preferably female pups per litter were sacrificed. Standardization of litters was not performed in litters with smaller then or equal to 8 pups.

Pups after standardization
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood were sampled for possible determination of thyroid hormone concentrations.
All pups were examined externally and eviscerated; their organs were assessed macroscopically.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

TEST SUBSTANCE PREPARATIONS AND PREPARATION FREQUENCY
1,1,3,3-Tetramethylguanidine was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, deionized water was filled up to the desired volume and subsequently released with a magnetic stirrer. The test substance preparations were produced weekly, at least.

FOOD ANALYSES
The supplier assayed the food used in the study for chemical and microbiological contaminants:

DRINKING WATER ANALYSES
The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring Department of BASF SE as well as for the presence of microorganisms by a contract laboratory.

BEDDING AND ENRICHMENT ANALYSES
The bedding and the enrichment are regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).

EXPERIMENTAL PROCEDURE
see field "details on study schedule"

Examinations

Parental animals: Observations and examinations:

Parental animals
Mortality
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

Clinical observations
A cageside examination was conducted 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. Abnormalities and changes were documented for each animal. The parturition and lactation behavior of the dams was generally evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver or umbilical cord not cut) were documented on an individual dam basis. On weekdays (except Saturdays, Sundays and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered to be the 24-hour period from about 15:00 h of one day until about 15:00 h of the following day.

Detailed clinical observations
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were 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

Food consumption
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
- Food consumption was not determined after 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 7, 14 and 20.
- Food consumption of the females which gave birth to a litter was determined for PNDs 4, 7, 10 and 13.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

Water consumption
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

Body weight data
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). The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
- During the premating phase, body weight was determined once a week, i.e. on study days 0, 7 and 13.
- During the mating period, the 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.
- Females without litter and after weaning (PND 13) were weighed once a week.

Functional observational battery
A functional observational battery was performed in 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 at the end of the administration period starting at about 10.00 h. The 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 findings were ranked according to the degree of severity, if applicable. 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, Nose discharge, Lacrimation, Eyes/pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/stereotypes, Gait abnormalities, Activity/arousal level, 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
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. 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. The program required a file name for the measured data to be stored. This name consisted of the reference number and a serial number.

Male reproduction data
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. For the males, mating and fertility indices were calculated for F1 litters.

Female reproduction and delivery data
The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females. For the females, mating, fertility and gestation indices were calculated for F1 litters.
The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters.
The implantations were counted and the postimplantation loss (in %) was calculated. To determine the number of implantation sites, the apparently non-pregnant uteri were stained for about 5 minutes in 1% ammonium sulfide solution according to the method of SALEWSKI (Salewski, E.; 1964). Then the uteri were rinsed carefully in physiologic salt solution (0.9% NaCl). Thereafter the implantation sites were recorded for calculation of the postimplantation loss.

CLINICAL PATHOLOGY
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units. The parameters listed below were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.

Hematology
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, differential blood count, Reticulocytes
Further: prothrombin time

Clinical chemistry
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the following clinicochemical parameters: ALT, AST, ALP, GGT, Na, K, Cl, inorganic Phosphate, Ca, Urea, Creatinine, Glucose, total Bilirubin, total protein, Albumin, Globulins, Triglycerides, Cholesterol, Bile acids.

Thyroid Hormones
Blood samples were taken from all surplus pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling. All generated serum samples were frozen at -80°C until measurement. Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH). The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisa was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

Oestrous cyclicity (parental animals):

For all females of the pool estrous cycle normality was evaluated before the beginning of the administration. 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):

The morphology and distribution of the different successive stages during spermatogenesis was examined according to Russell et al., 1990.

Litter observations:

Pup number and status at delivery: All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

Pup viability/mortality: In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PNDs 1-4, 5-7 and 8-13 were determined. Pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day of birth (PND 0), and on lactation days 4, 7 and 13. Furthermore, viability and survival indices were calculated.

Sex ratio: On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at PND 0 and PND 13.

Pup clinical observations: The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

Pup body weight data: The pups were weighed on the day after birth (PND 1) as well as on PNDs 4, 7 and 13. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization of the litters. “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

Nipple/areola anlagen: All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13 of the lactation phase. The number of nipple/areola anlagen was counted.

Anogenital distance (AGD): AGD (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements was done blind to treatment in a randomized order, using a measuring ocular, on all live male, female and uncertain pups on day 1 after birth. Subsequently, the anogenital index was caluclated.

Postmortem examinations (parental animals):

Necropsy
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

Organ weights
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (terminal body weight), Epididymides, Ovaries, Prostate, Seminal vesicles with coagulating glands, Testes, Thyroid glands (fixed), Uterus (with cervix)
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus

Organ/tissue fixation
The following organs or tissues of all parental animals were fixed in in 4% neutral-buffered formaldehyde 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 gland, 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 (uteri of all apparently nonpregnant animals or empty uterus horns were stained according to Salewski E, 1964), Vagina

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table attached ("Histopathology").
Following histotechnical processing, all histological slides, tables of individual macroscopic findings as well as organ weight tables were sent to InSight Pathology BV, Chopinlaan 6, NL-5343EM Oss, The Netherlands. Light microscopical examination of the hematoxylineosin stained slides and assessment of findings was performed under the responsibility of the Principal Investigator Eric van Esch at the test site InSight Pathology BV under the test site phase number ISP 17231.

Special attention was given to the stages of spermatogenesis in the testes and histopathology of interstitial testicular cell structure.
The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).
A correlation between gross lesions and histopathological findings was attempted.

Peer review
After completion of the histopathological assessment by the PI pathologist Eric van Esch an external peer review was performed by Dr. Maria Cecilia Rey Moreno (BASF SE, Ludwigshafen) including the stomachs of all female animals, and all organs of one male and one female control animal (Nos. 2 and 104) and one high dose male and female animal (Nos. 32 and 135).

Genital organs were assessed in two additional animals/sex (males No. 23, 24, and 36; females No. 123, 124, and 136). Results presented in the pathology phase report reflect the consensus opinion of the principal investigator and the peer review pathologist and are documented in the finding tables of the phase report (pathology).

Postmortem examinations (offspring):

Pup necropsy observations
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and the organs were assessed macroscopically. On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for further processing. All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic
evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

Statistics:

Parameter: Food consumption, body weight (bw) and bw change (parental animals and pups; for pup weights, litter means were used), gestation days, anogenital distance, anogenital index
Statistical test: Simultaneous comparison of all dose groups with control group (CoG) using DUNNETT test (2sided)

Parameter: Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups
Statistical test: Pair-wise comparison of each dose group with CoG using FISHER'S EXACT test (onesided)

Parameter: Mating days until day 0 p.c., %postimplantation loss, pups stillborn, %perinatal loss, nipple development, Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index
Statistical test: Pair-wise comparison of the dose group with CoG using WILCOXON test (onesided) with BONFERRONI-HOLM adjustment

Parameter: %live male day x, %live female day x
Statistical test: WILCOXON test (2sided)

Parameter: Number of cycles and Cycle Length, Rearing, grip strength of fore limbs and hind limbs, landing foot-splay test, motor activity
Statistical test: Non-parametric one-way analysis using KRUSKALWALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair-wise comparison of the dose groups with CoG was performed using WILCOXON test (2sided)

Blood parameters
Statistical test: Parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting pvalue was equal or less than 0.05, a pairwise comparison of each dose group with CoG was performed using WILCOXON-test (2sided)

Weight parameters
Statistical test: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting pvalue was equal or less than 0.05, a pairwise comparison of each dose group with CoG was performed using WILCOXON-test (2sided)

Reproductive indices:

Male reproduction data
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.
For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:
Male mating index (%) = (number of males with confirmed mating / number of males placed with females) x 100
Male fertility index (%) = (number of males proving their fertility / number of males placed with females) x 100

Female reproduction and delivery data
The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.
For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:
Female mating index (%) = (number of females mated / number of females placed with males) x 100
Female fertility index (%) = (number of females pregnant / number of females mated) x 100
Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant) x 100

The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters according to the following formula:
Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100

The implantations were counted and the postimplantation loss (in %) was calculated according the following formula:
Postimplantation loss (%) = ((number of implantations – number of pups delivered) / number of implantations) x 100

To determine the number of implantation sites, the apparently non-pregnant uteri were stained for about 5 minutes in 1% ammonium sulfide solution according to the method of Salewski, E.; 1964. The implantation sites were recorded for calculation of the postimplantation loss.

Offspring viability indices:

In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PNDs 1-4, 5-7 and 8-13 were determined. Pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day of birth (PND 0), and on lactation days 4, 7 and 13. Furthermore, viability and survival indices were calculated according to the following formulas:
Viability index (%) = (number of live pups on day 4 after birth / number of live pups on the day of birth) x 100
Survival index (%) = (number of live pups on day 13 after birth / number of live pups on day 4 after birth) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Summary clinical observations for males and females
Treatment-related, but not adverse signs of toxicity were observed.
Salivation after treatment was observed in animals of test group 3 (100 mg/kg bw/d), i.e. male animal No. 31 on premating day 11, in male animal No. 34 on premating day 13 and in female animal No. 131 on premating days 10 and 13. In addition, salivation was observed in female animal (No. 111) of test group 1 (10 mg/kg bw/d) on premating days 12-13. From the temporary, short appearance immediately after dosing (or shortly before) it was concluded that both kind of findings were induced by a bad taste of the test substance or local affection of the upper digestive tract.
Soft feces were observed in female animal No. 111 of test group 1 (10 mg/kg bw/d) on premating days 12-13.
Palpable mass through skin was observed in female animal No. 123 of test group 2 (30 mg/kg bw/d) between mating day 2 and gestation day 35.
Both findings were assessed to be incidental and not related to treatment.

Summary clinical observations for females during gestation
Palpable mass through skin was observed in female animal No. 123 of test group 2 (30 mg/kg bw/d) between mating day 2 and gestation day 35.
Salivation after treatment was observed in female animal No. 131 on gestation days 0 and 2, in female animal No. 134 on gestation day 9 and in female animal No. 137 on gestation days 7-8 of test group 3 (100 mg/kg bw/d).

Clinical observations for females during lactation
Pale skin was observed in female animal No. 114 of test group 1 (10 mg/kg bw/d) on lactation day 0. In addition, piloerection was observed in female animal Nos. 137 (between lactation days 0 to 3), No. 138 (between lactation days 0 to 2), and No. 136 (on lactation day 3).
Piloerection was also observed in female animal Nos. 108 (between lactation days 0 to 2) and 114 (on lactation day 0).

Detailed clinical observations
During detailed clinical observations (DCO), additional findings which did not already occur during the daily clinical examinations were not observed.
Palpable mass through skin was observed in female animal No. 123 of test group 2 (30 mg/kg bw/d) on study days 21, 28, 35, 42 and 49.

Mortality:

no mortality observed

Description (incidence):

No animal died prematurely in the present study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant changes in mean body weights and mean body weight change values were observed for male and female animals of test groups 1-3 (10, 30 and 100 mg/kg bw/d) when compared to the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in male and female animals during the entire study.

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

no effects observed

Description (incidence and severity):

No treatment-related, adverse findings were observed.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Examination included in DCO.

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related, but not adverse changes among clinical chemistry parameters were observed.
In females of test groups 2 and 3 (30 and 100 mg/kg bw/d) triglyceride values were higher compared to controls, although not statistically significant in test group 3. However, this was the only altered parameter among these individuals and therefore, it was regarded as treatment-related but not adverse (ECETOC Technical Report No. 85, 2002).
In females of test group 1 (10 mg/kg bw/d) total protein and albumin values were significantly increased. These changes were not dose-dependent and therefore they were regarded as incidental and not treatment-related.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observational battery
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, without a dose-response relationship or occurred in single animals only, these observations were considered as incidental.
The following examinations were performed during FOB and are assessed individually: Home cage observations/Open field observations/Sensorimotor tests/reflexes/Quantitative parameters: No test substance-related effects were observed.

Motor activity measurement
Regarding the overall motor activity, no test substance-related deviations were noted for male and female animals.
Comparing the single intervals with the control groups, one significantly increased value was measured for male animals of test group 1 (10 mg/kg bw/d) at interval 10. The change was regarded to be incidental and not related to treatment as neither other single intervals nor the overall motor activity was affected. No significant deviations to control values were observed for female animals in test groups 1 to 3 (10, 30 and 100 mg/kg bw/d) when compared to the control group.

Immunological findings:

no effects observed

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):

Glandular stomach: minimal mucosal erosions (few) were observed in a single female of the group treated with 30 mh/kg bw/d and minimal to slight erosions were observed in 3 out of 10 females of the group treated with 100 mg/kg bw/d 1,1,3,3-Tetramethylguanidine. In the latter animals, the lesion was found focal in 2 females (132, 137), while female 139 showed few erosions.

Reproductive organs: thorough microscopic examination of the male and female reproductive organs did not reveal any relevant histopathologic change, also not in the couples that did had no offspring: the findings in the reproductive organs from these couples such as inflammatory cell infiltrate in the prostate of male 24 and 36, and the foci of squamous metaplasia in the uterus of female 124 were considered spontaneous findings that did not influenced the fertility. The morphology and distribution of the different successive stages during spermatogenesis (Russell et al., 1990) was normal for all males examined.

Other microscopic findings are considered spontaneous or incidential and consistent with the usual pattern of findings in animals of this strain and age.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data revealed regular cycles in the rearing F1 females of all test groups including the control. The mean estrous cycle duration in the different test groups (0-3) was 4 days.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The morphology and distribution of the different successive stages during spermatogenesis (Russell et al., 1990) was normal for all males examined.

Reproductive performance:

no effects observed

Description (incidence and severity):

The male mating index calculated after the mating period to produce F1 litter was 100% in all test groups.

Male fertility index: Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter.
Male animal No. 11 of test group 1 (10 mg/kg bw/d), which was mated with female animal No. 111, male animal Nos. 23 and 24 of test group 2 (30 mg/kg bw/d), which were mated with female animal Nos. 123 and 124, and male animal Nos. 34 and 36 of test group 3 (100 mg/kg bw/d), which were mated with female animal Nos. 134 and 136, did not generate F1 pups.
Note: Female animal Nos. 111 and 134 had implants but did not deliver pups. Therefore, male animal No. 34 was assessed to be fertile. Thus, the male fertility index was 100% in test groups 0 and 1, 80% in test group 2 and 90% in test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data

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 4.6 days for test group 0, 3.2 days for test group 1, 2.8 days for test group 2 and 3.4 days for test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Female fertility index: Most sperm positive rats delivered pups with the exception of female animal No. 111 of test group 1 (10 mg/kg bw/d), which was mated with male animal No. 11, female animal Nos. 123 and 124 of test group 2 (30 mg/kg bw/d), which were mated with male animal Nos. 23 and 24, and female animal Nos. 134 and 136 of test group 3 (100 mg/kg bw/d), which were mated with male animal Nos. 34 and 36, did not generate F1 pups. These female animals had sperm in vaginal smear but did not deliver pups. Female animal Nos. 111 and 134 showed implants and female animal Nos. 123, 124 and 136 did not show
any implants.
Thus, the female fertility index was 100% in test groups 0 and 1, 80% in test group 2 and 90% in test group 3. These values reflected the normal range of
biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

The mean duration of gestation was 22.4 days in control group 0, 22.7 days in test group 1, 22.8 days in test group 2 and 22.6 days in test group 3.
The gestation index was 100% in test groups 0 and 2 (0 and 30 mg/kg bw/d), 90% in test group 1 (10 mg/kg bw/d) and 88.9% in test group 3 (100 mg/kg bw/d).

The rate live birth indices were 100% in test groups 1 and 3 (10 and 100 mg/kg bw/d), 98.4% in test group 0 (control group) and 98.1% in test group 2 (30 mg/kg bw/d). These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

The postimplantation loss was 14.3% in control group 0, 18.7% in test group 1 (10 mg/kg bw/d), 5.5% in test group 2 (30 mg/kg bw/d) and 17.6% in test group 3 (100 mg/kg bw/d). The value for test group 1 was slightly above the historical control data, but the value for test group 3 was still within the range. As no dose-response relationship occurred and all respective values were within or just slightly above the range the changes were assessed to be incidental and not related to treatment

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no relevant systemic effects observed

2

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: based on findings in glandular stomach of mid and high dose females

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Four female pups (Nos. 103-10, 103-11, 104-14, 107-14) and one male pup (No. 104-01) of the control group (0 mg/kg bw/d), one female pup (No. 116-13) of test group 1 (10 mg/kg bw/d) and 3 female pups (Nos. 126-13, 126-14, 126-15) of test group 2 (30 mg/kg bw/d) died prematurely.
Female pup No. 101-08 (control group) showed loss of tail end on study days 4-10 and pigtail on study days 10-13.
All other F1 pups in all test groups (0-3) did not show adverse clinical signs up to scheduled sacrifice on PND 4 and PND 13.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The rate live birth indices were 100% in test groups 1 and 3 (10 and 100 mg/kg bw/d), 98.4% in test group 0 (control group) and 98.1% in test group 2 (30 mg/kg bw/d).
The viability index indicating pup mortality between PND 0 and 4 was 97.9% in test group 0, 100% in test group 1, 99.0% in test group 2 and 100% in test group 3.
The survival index indicating pup mortality between PND 4 and 13 was 100% in test groups 0, 2 and 3 and 98.6% in test group 1.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group.
One male runt was found in one litter of test group 0 (0 mg/kg bw/d), two female runts were found in in each one litter of test group 1 (10 mg/kg bw/d) and 2 male and 2 female runts were found in each one litter of test group 2 (30 mg/kg bw/d). A relation to dosing was not observed, test substance-related effects did not occur.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed in all test groups.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed up to high dose

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of 1,1,3,3-Tetramethylguanidine to Wistar rats revealed signs of local toxicity at dose levels of 30 mg/kg bw/d and above in female animals indicated by erosions in the glandular stomach.
However, the no observed adverse effect level (NOAEL) for general systemic toxicity was set to 100 mg/kg bw/d for male and female Wistar rats.
The NOAEL for reproductive performance and fertility was also set to 100 mg/kg bw/d for male and female Wistar rats.
The NOAEL for developmental toxicity was 100 mg/kg bw/d.

Executive summary:

In a GLP-compliant OECD422 study (28 days repeated dose + reproduction/developmental toxicity screening) TMG was given to rats orally ba gavage (2018). TMG was given daily as a solution to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0, 10, 30, and 100 mg/kg bw/d. Deionized water served as vehicle. 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 test substance preparation analyses confirmed: Stability of the test-substance preparations for a period of 7 days at room temperature, homogeneous distribution of the test substance in the vehicle, correctness of the prepared concentrations. Effects: The following test substance-related, relevant findings were noted: Local effects on the glandular stomach in individual female animals related to the corrosive potential of the test item. For all other organs/endpoints/parameters exmanied: No treatment-related, adverse effects were observed.

CONCLUSION: Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of 1,1,3,3-Tetramethylguanidine to Wistar rats revealed signs of local toxicity at dose levels of 30 mg/kg bw/d and above in female animals indicated by erosions in the glandular stomach. However, the no observed adverse effect level (NOAEL) for general systemic toxicity was set to 100 mg/kg bw/d for male and female Wistar rats. The NOAEL for reproductive performance and fertility was also set to 100 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 100 mg/kg bw/d.