Reaction product of Aminoguanidine bicarbonate and Tall Oil fatty acid

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10/2011 - 06/2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: 1) OECD 421, Reproduction/Developmental Toxicity Screening Test, July 1995.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: 2) OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

442-600-9

EC Name:

-

Molecular formula:

The substance is a UVCB for which it is not possible to provide a molecular formula

IUPAC Name:

(9E)-N-[(N'-hexadecanoyloctadecanehydrazido)methanimidoyl]octadec-9-enamide

Test material form:

semi-solid (amorphous): gel

Remarks:

migrated information: paste

Details on test material:

- Name of test material (as cited in study report): H7134
- Substance type: Beige to yellow paste
- Storage condition of test material: At room temperature in the dark under nitrogen
- pH: Not indicated

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: approximately 11 weeks.
- Weight at study initiation: mean weight range at start of treatment was 281-307 gr (males) or 188-211 gr (females)
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
Lactation: Pups were kept with the dam until termination in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage-enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 – 22.8
- Humidity (%): 43 - 67
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 Oct 2011 to 19 Dec 2011

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

Method of formulation: Formulations (w/w) wereprepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment were made for the specific gravity of the vehicle (1.036).

Storage conditions of formulations: At ambient temperature.

Justification for use and choice of vehicle: Based on trial formulations performed at NOTOX.

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

The test substance was heated in a water bath to a maximum of 70.2°C for a maximum duration 6 hours 44 minutes. Formulations were heated to a maximum of 68.8°C for a maximum of 39 minutes to facilitate mixing. The formulations were cooled to a maximum of 37.6°C before dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (1-November 2011), according to a validated method (NOTOX Project 496485). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
Stability in the vehicle over 6 hours at room temperature was also determined (highest and lowest concentrations). The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Bases on these analyses, it was concluded that the concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test substance was detected in the Group 1 formulation and Group 2 and Group 4 formulations were homogeneous (i.e. coefficient of variation ≤ 10%) and formulations over the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates)).
- Proof of pregnancy: evidence of sperm in the vaginal lavage, and/or or by the appearance of an intravaginal copulatory plug, referred to as Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- Since less than 9 females of Group 3 (60 mg/kg) showed evidence of mating, Each nonmated female was re-paired once with a proven male of the same group for 2 days. Detection of mating was not confirmed for animal no. 65 which did deliver live offspring. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 postcoitum.
- After successful mating each pregnant female was caged individually in Macrolon cages with bedding.

Duration of treatment / exposure:

Males were exposed for 35-37 days, i.e. 3 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Due to toxicity seen at 250 mg/kg after the initial few days of dosing, the pre-mating period was extended by one week (to 3 weeks instead of 2) to allow animals to at least partially recover at a lower dose level (120 mg/kg) before beginning the mating period. Also, because less than 9 females were pregnant at 60 mg/kg by the end of the mating period, two proven males were retained for 2 days past the normal necropsy period to be used for mating with the non-pregnant females.
Females were exposed for 50-63 days, i.e. during 3 weeks prior to mating (starting with 250 mg/kg bw /day in the high dose group, which was changed to 125 mg/kg bw/day from day 9 onwards), during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). One female (120 mg/kg) was not dosed during littering.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Frequency of treatment:

Once daily for 7 d/w.

Duration of test:

Males: 35-37 days
Females: 50-63 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 15, 60 mg/kg bw/day or 250 mg/kg bw days 1-8 and 120 mg/kg bw days 9 - onwards
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on a previous 28-Day study provided by the Sponsor (CTL/KR1429/Regulatory/Report) where dose levels of 15, 150 and 250 mg/kg in corn oil were administered for 28 days. Relevant findings included salivation, reduced body weights (at 150 and 250 mg/kg, males), decreased haemoglobin and haematocrit (250 mg/kg, females), increased alkaline phosphatase levels (250 mg/kg, both sexes and at 150 mg/kg, females), and higher liver weights (150 and 250 mg/kg, females).Overall, the effects seen at 150 and 250 mg/kg, were fairly minor and were not accompanied by relevant histopathology findings, though the NOAEL was set at 15 mg/kg. Based on these results, 15, 60 and 250 were chosen for the reproduction/developmental toxicity screening test.

- Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.

- Identification of pups:
On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.

- Selection of animals for selected measurements:
5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS
- At least twice daily.

DETAILED CLINICAL OBSERVATIONS
- Daily, detailed clinical observations were made in all animals, at least immediately after dosing. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT
- Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

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 postcoitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION
No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION
No

GENERAL REPRODUCTION DATA
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth.

HAEMATOLOGY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 20 hours, or 21 hours for 1 animal). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: White blood cells, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets
- Clotting Potential was determined (Prothrombin time and Activated Partial thromboplastin time)

CLINICAL CHEMISTRY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Animals fasted: Yes (with a maximum of 20 hours, or 21 hours for 1 animal). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked in table were: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids

URINALYSIS
No

NEUROBEHAVIOURAL EXAMINATION
No

Ovaries and uterine content:

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

Fetal examinations:

SACRIFICE
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

HISTOPATHOLOGY / ORGAN WEIGHTS
No.

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.

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. No statistical analysis was performed on histopathology findings.

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 indices:
- 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
- Percentage of postnatal loss Days 0-4 of lactation: (Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check) x 100
- Viability index: (Number of live pups on Day 4 of lactation / Number of pups born alive) x 100

Historical control data:

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. NOTOX B.V. 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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY
No mortality occurred during the study period.

CLINICAL SIGNS
After a few days of treatment at 250 mg/kg, hunched posture and salivation were noted for all animals; piloerection was also seen for all females. Lean appearance and hypothermia were also noted, though less commonly, during this time. These symptoms persisted for a few days after the dose level was lowered to 120 mg/kg (from Day 9 onwards), but then the piloerection, hunched posture, lean appearance and hypothermia were no longer seen (piloerection was noted for one male and two females much later during the treatment period as well). In contrast, salivation was noted throughout the treatment period for animals at 120 mg/kg.
At 60 mg/kg, salivation was also noted for all animals during most of the treatment period. In all cases, salivation was considered to be a physiological response rather than a sign of systemic toxicity since it was noted right after dosing and was not accompanied by other relevant signs of systemic toxicity. This may be related to the taste or possible irritancy of the test substance.

Incidental findings noted included scales on the flank or abdomen, alopecia, scabs on the cheek, rales, swelling of the abdomen and exophtholamos of the right eye. These findings occurred within the range of background findings to be expected for rats of this age and strain and/or were isolated occurrences. At the incidence observed, these were not considered toxicologically relevant.

BODY WEIGHTS
High dose animals had significant body weight loss after the first 8 days of dosing at 250 mg/kg. After switching to the lower dose level of 120 mg/kg (from Day 9 onwards), animals showed a substantial recovery, though body weight and body weight gains did not catch up to the pre-dose levels seen at Day 1. However, when body weight gains were re-calculated from Day 8 to reflect when the dose level was lowered, animals at 120 mg/kg gained significantly more weight compared to controls supporting that these animals recovered from the toxicity seen at 250 mg/kg.
Body weights and body weight gains were significantly lower for high dose animals through the entire pre-mating and mating periods. Absolute body weights were also lower for females during the post-coitum and lactation periods, though body weight gains remained similar to control levels during this time.
As the condition of the animals clearly improved after switching to 120 mg/kg on Day 9, the body weight gains were substantial, and the adverse clinical signs subsided, the lower body weights were consequently not considered to be adverse.

There were no other toxicologically relevant effects on body weights or body weight gains.
Body weight gain was significantly lower for females at 60 mg/kg on Day 4 of the post-coitum period, but as this was transient and the difference from controls was only slight, it was not considered to be treatment related.

FOOD CONSUMPTION
Absolute and relative food consumption was lower Days 1-8 of the pre-mating period for high dose females. This is also reflective of the treatment related toxicity seen with the initial exposure of 250 mg/kg. These values returned to levels comparable to controls after treatment was switched to 120 mg/kg. At 120 mg/kg, absolute food consumption values were lower for females from Days 4-20 of the post-coitum period. This likely reflects less food consumption due to their comparatively lower body weights because relative food consumption was only very slightly lower for most of the post coitum period (though it was significantly lower from controls on Days 17-20 post-coitum). This was not considered to be toxicologically relevant.

HAEMATOLOGY
There were no toxicologically relevant effects on haematology parameters up to 120 mg/kg. Females at 120 mg/kg had significantly lower lymphocytes and white blood cells and higher neutrophils (white blood cells were also significantly lower for females at 60 mg/kg). However, no supporting evidence of toxicity was seen in other relevant parameters. These changes are likely a secondary response to stress, and do not reflect adverse toxicological effects. There were no other effects on haematology parameters.

CLINICAL BIOCHEMISTRY
At 120 mg/kg, the following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
-Higher aspartate aminotransferase (ASAT)
-Higher potassium (also at 15 mg/kg)
-Higher chloride
-Lower calcium
-Lower inorganic phosphate

These changes at 120 mg/kg were in most cases only slightly different from control values. Furthermore, these were not accompanied by any relevant changes in organ weights or macroscopic findings, and remained within the normal range of values available (with the exception of potassium at 120 mg/kg, only). Furthermore, elevations in endpoints like creatinine and protein were not seen and the increase in ASAT was not accompanied by corresponding increases in alanine aminotransferase (ALAT), alkaline phosphatase (ALP) and bilirubin that could be indicative of renal or hepatic toxicity, respectively. Taken together, the evidence collectively supports that these changes were not adverse, and were thus not considered to be toxicologically relevant.

MACROSCOPIC EXAMINATION
There were no treatment related macroscopic findings noted up to 120 mg/kg. Incidental findings noted for control and/or treated animals included yellowish, soft nodules on the tail of the epididymides, isolated dark red focus on the cranial pole of the right kidney, reduced size of the right seminal vesicle, isolated tan focus on the preputial glands or the clitoral gland, enlarged liver, thickened ureter, watery-clear fluid in the ureter and kidney, soft, watery-clear cyst on the urinary bladder, and alopecia of the abdominal region or foreleg. These remained within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. As such, they were not considered toxicologically relevant.

ORGAN WEIGHTS
Organ weights and organ to body weight ratios among the dose groups were similar to control levels.

MICROSCOPIC EXAMINATION
There were no treatment-related microscopic findings in the reproductive organs. Three females failed to mate, conceive, or deliver healthy offspring: Two females (dosed with 15 mg/kg) and one female dosed with (120 mg/kg). No cause of infertility was found for the these animals. All microscopic findings recorded in animals surviving to the end of the assigned study period were within the normal range of background pathology encountered in Wistar Han rats of this age and strain.

REPRODUCTIVE DATA
No toxicologically relevant effects on reproductive parameters were noted up to and including 120 mg/kg.
At 120 mg/kg, there were significantly fewer corpora lutea than for control females. This was attributable to unusually high values for two control females, with 20 and 24 corpora lutea, respectively. Furthermore, there was no difference in the number of implantation sites and the mean number of living pups per litter at 120 mg/kg was comparable to controls (120 mg/kg: 10.1, Controls: 11.6). As such, this was not considered to be treatment related.
The mating, fertility and conception indices, precoital time, and number of implantation sites were unaffected by treatment.

GESTATION
The gestation index and duration of gestation were unaffected up to and including 120 mg/kg.

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

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

>= 120 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

>= 120 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.

Details on embryotoxic / teratogenic effects:
Early postnatal pup development
The number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopic exam did not reveal treatment-related findings.

Mortality
One pup at 15 mg/kg and 3 pups at 120 mg/kg were found dead or went missing during the first days of lactation. The single missing pup was most likely cannibalized. There were no dead or missing pups in the control and 60 mg/kg groups. No toxicological relevance was attributed to the dead/missing pups since the mortality incidence remained within the range considered normal for pups of this age.

Clinical signs
Incidental clinical symptoms of pups consisted of blue spot on the nose or head, noted for two pups in the control group. These are not unexpected from pups of this age and were seen only in controls, thus they were not treatment related or toxicologically relevant.

Body weights
Body weights of pups were not significantly different from controls up to 120 mg/kg.

Macroscopy
No milk in the stomach was the only incidental finding noted for pups that were found dead. There were no macroscopic findings noted for pups surviving to the scheduled necropsy.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In an oral OECD 421 screening study, no adverse effects were seen in the parental animals, nor in the offspring at the highest dose tested. Based on these data, a maternal and developmental No Observed Adverse Effect Level (NOAEL) of ≥120 mg/kg/day was determined.

Executive summary:

An OECD 421 study titled “Reproductive/developmental toxicity screening test of H7134 in rats by oral gavage” (Zmarowski A, 2012) was performed that had a NOEAL of 120 mg/kg bw/day. Additional measurements particularly affecting clinical biochemistry were completed during this study so that it was similar to an OECD 422 study without the sensory reactivity to stimuli, assessment of grip strength, and other activity assessments. No toxicologically relevant effects were observed up to the highest dose level tested (120 mg/kg). The intial concentrations used were 0. 15, 60, and 250 mg/kg bw/day. However, the Group 4 animals, that were initially exposed to 250 mg/kg, had significantly reduced body weights, body weight gains, absolute and relative food consumption and adverse clinical signs were seen beginning after 4 days of treatment. After treatment was lowered to 120 mg/kg from Day 9 onwards, the condition of the animals improved substantially. While the body weights and body weight gains remained lower for these animals (except for body weight gains during post-coitum and lactation) through the treatment duration, this was not considered adverse since the body weight gains were substantially higher than controls when re-calculated from the time the dose level was lowered. There were several changes noted in clinical biochemistry parameters at 120 mg/kg bw/day, however these were not accompanied by any relevant changes in organ weights or macroscopic findings, and remained within the normal range of values available. Furthermore, increases were not observed in several endpoints that would be indicative of renal or hepatic toxicity. Furthermore, there were no toxicologically relevant effects on any of the remaining parental parameters investigated in this study (clinical observations, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). Taken together, the body weight differences and the clinical biochemistry changes were not considered to be toxicologically relevant. No effects that were indictative of reproductive or developmental toxicity were seen. Therefore, the NOAEL was set at 120 mg/kg bw/day.