Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

In a GLP-compliant guideline 90-day study with reproductive/developmental toxicity screening, no adverse effects on fertility and reproduction were observed at the highest tested dose of 15 mg/kg bw/day. The dose level of 15 mg/kg bw/day was considered to be the NOAEL for reproductive and developmental toxicity. The lowest tested dose of 1 mg/kg bw/day was considered to be a LOAEL for parental toxicity, based on the presence of granulomatous inflammation of the mesenteric lymph nodes with central necrosis at all dose levels with dose-related increase in severity.

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 April 2016 - 15 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2015
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2015
Deviations:
yes
Remarks:
Estrous cycle regularity was not determined during a pretest period (i.e. before start of treatment), due to the young age of the females at commencement of treatment. No blood for thyroid hormone T4 analysis was collected from PND 4 litters.
Qualifier:
according to
Guideline:
other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD 408, Repeated Dose 90-day Oral Toxicity Study in Rodents
Version / remarks:
September 1998.
Deviations:
yes
Remarks:
3 females were treated for 84 days instead of 90
Qualifier:
according to
Guideline:
other: EC No 440/2008 B.7: "Repeated Dose (28 days) Toxicity (oral)"
Version / remarks:
May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
d.d. 3 November 2015
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble and stable in hydrocarbons. Stability in corn oil for at least 5 hours at room temperature and 8 days in the refrigerator is confirmed over the concentration range 1 to 200 mg/mL. Stability in corn oil for at least 5 hours at room temperature is confirmed for 0.2 mg/mL.

OTHER SPECIFICS: no correction factor for purity was applied.
Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
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 and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: ca. 8 weeks
- Weight at study initiation: males 148-178 g, females 150-194 g
- Fasting period before study: no
- Housing: during pre-mating in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm). During 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). During lactation pups were housed 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.
Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum, except during overnight prior to scheduled sacrifice
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY:
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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0-22.9
- Humidity (%): 51-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 April 2016 To: 15 September 2016
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
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/density of the test item (0.83 g/mL) and vehicle (0.92 g/mL). No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed at Charles River Den Bosch and on information provided by the sponsor.
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
- M/F ratio per cage: 1:1, avoiding sibling mating
- Length of cohabitation: a maximum of 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 post-coitum
- After successful mating each pregnant female was caged individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of dose preparations were collected at the test facility on three occasions during the treatment phase, i.e. in Weeks 1, 6 and 13. They were analyzed to assess accuracy of preparation (all groups; Weeks 1, 6 and 13), homogeneity (lowest and highest concentration; Weeks 1, 6 and 13) and stability in vehicle over 5 hours at room temperature under normal laboratory light conditions (lowest concentration; Week 1).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% 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%.
Duration of treatment / exposure:
Males: 90 days (8 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy).
Females that delivered: 91- 104 days, i.e. during 8 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 were not dosed during littering.
Females which failed to deliver healthy offspring were treated for 84 (3 non-pregnant females) or 90 (one female with a total litter loss) days. The slightly shorter treatment duration for a few animals did not adversely affect interpretation of the study results.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.
Frequency of treatment:
Once daily, 7 days per week
Details on study schedule:
- Age at mating of the mated animals in the study: ca. 16 weeks (mated after 8 weeks of treatment)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Concurrent vehicle controls
Dose / conc.:
1 mg/kg bw/day (actual dose received)
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results of a 14-day dose range finding study
Positive control:
Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily. 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.

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment 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 AND COMPOUND INTAKE:
- Time schedule for examinations: 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.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at pre-test, week 13 and during lactation
- Dose groups that were examined: at pre-test: all animals, at week 13: controls and high dose males, during lactation: controls and high-dose females

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight with a maximum of 24 hours
- How many animals: all
- Parameters examined: white blood cells (WBC), differential leucycyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells (RBC), reticulocytes, red blod cell distribution width (RDW), haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelets; prothrombin time (PT), activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of scheduled necropsy
- Animals fasted: Yes, overnight with a maximum of 24 hours
- How many animals: all
- Parameters examined: alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total protein, albumin, total billirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium , potassium, chloride, calcium, inorganic phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of treatment
- Dose groups that were examined: all groups, 5 animals/sex/group
- Battery of functions tested: sensory activity (hearing ability, pupillary reflex, static righting reflex), grip strength (fore- and hind-limb grip strength), locomotor activity

IMMUNOLOGY: No

BLOOD SAMPLE FOR THYROID HORMONE ANALYSIS
F0 generation, males and females: at the end of study blood was collected from all animals at planned necropsy. In males, total thyroxine (T4) was measured, the remaining serum volume was stored for possible future measurement of thyroid-stimulating hormone (TSH).
.
Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to start mating 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.
On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.
Estrous cycle regularity was not determined during a pretest period (i.e. before start of treatment), due to the young age of the females at commencement of treatment. This deviation from the OECD422 guideline does not adversely affect the study; most females showed a regular estrous cycle during the 14-day period preceding the mating phase.
Sperm parameters (parental animals):
Parameters examined in F0 male parental generations: testis weight, epididymis weight.
Testes from all control and high-dose males and all males that failed to sire were examined by a pathologist to examine staging of spermatogenesis.
Litter observations:
F1 generation, PND13-15 pups: blood was collected from two pups per litter (if possible, one male, one female) at planned necropsy. Serum from each sample was divided into 2 aliquots: for measurement of T4 and the remaining volume of serum for possible future measurement of TSH
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after a minimum of 90 days of treatment
- Maternal animals:
Females that delivered: PND 14, 15 or 17
Females that failed to deliver: Post-coitum days 25-27 (females with evidence of mating)
Female with total liter loss: within 24 hours of litter loss.

GROSS NECROPSY
After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organ weights were determined: 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, uterus including cervix.
The following slides were examined by a pathologist:
- All preserved organs and tissues from controls and high-dose animals
- Testes from all control and high-dose males and all males that failed to sire to examine staging of spermatogenesis
- Bone marrow (sternal), mesenteric lymph nodes, duodenum, jejunim, ileum and spleen of all animals of low- and mid-dose groups, based on (possible) treatment-related changes in these organs in high-dose group
- The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups (3 non-pregnant females and their sires, and one female with a total litter loss and her sire)
- Female mammary gland from the female with a total litter loss
Postmortem examinations (offspring):
SACRIFICE
Culling:
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. 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.
Terminal sacrifice of the remaining pups was performed on PND 13-16.

GROSS NECROPSY
All pups were sexed by both external and internal examination. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.

HISTOPATHOLOGY / ORGAN WEIGTHS
At terminal sacrifice (PND 13-16), the thyroids from 1 male and 1 female pup per litter were preserved in 10% buffered formalin. If possible, these were the same pups as selected for blood sampling.
Organ weights were not determined. No further histopathological evaluation was conducted.
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.
Reproductive indices:
The following indices were calculated:
Mating index (%) = 100 x (number of females mated)/number of females paired
Fertility index (%) = 100 x (number of pregnant females)/(number of females bearing live pups)
Gestation index (%) = 100 x (number of females bearing live pups)/(number of pregnant females0
Duration of gestation: number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
The following indices were calculated:
Post-implantation survival index (%) = 100 x (total number of offspring born)/(total number of uterine implantation sites)
Live birth index (%) = 100 x (number of live offspring on Day 1 after littering)/(total number of offspring born)
Percentage live males at first litter check (%) = 100 x (number of live male pups at first litter check) / (number of live pups at first litter check)
Percentage live females at first litter check (%) = 100 x (number of live female pups at first litter check) / (number of live pups at first litter check)
Viability index (%) = 100 x (number of live offspring on Day 4 before culling) / (number of live offspring on Day 1 after littering)
Lactation index (%) = 100 x (number of live offspring on Day 13 after littering) / (number of live offspring on Day 4 after culling)
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
Any clinical signs noted during the treatment period 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
One female at 5 mg/kg bw/day was sacrificed at PND 12 due to total litter loss.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 15 mg/kg bw/day body weight and body weight gain was slightly reduced in males, starting from approximately Week 6 of the study. A level of statistical significance was achieved on most occasions.
No treatment-related changes in body weight or body weight gain were noted in males treated at 1 or 5 mg/kg bw/day or in females up to 15 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment-related changes in food consumption before or after allowance for body weight were observed in males or females up to 15 mg/kg bw/day.
Any statistically significant differences in (relative) food consumption in females at 15 mg/kg bw/day during the post-coitum period were not considered related to treatment, as these changes were minor and were not consistently recorded with continuing treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmoscopic findings were noted that were considered to be related to treatment.
The nature and incidence of ophthalmoscopic findings noted at pretest examination and at the end of the treatment period were similar between the groups, and occurred within the normal range for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes in haematology parameters distinguished treated animals from control animals:
• Higher total white blood cell count (WBC) in males and females at 15 mg/kg bw/day
• Higher absolute and relative neutrophil counts in males and females at 5 and 15 mg/kg bw/day (not statistically significant for absolute neutrophil count of females at 5 mg/kg bw/day).
• Higher absolute and relative monocyte counts in males at 5 and 15 mg/kg bw/day
• Higher red cell distribution width (RDW) in males at 15 mg/kg bw/day
• Lower haemoglobin in males at 5 and 15 mg/kg bw/day (not statistically significant for males at 5 mg/kg bw/day), and in females at 15 mg/kg bw/day
• Lower mean corpuscular volume (MCV) in males at 5 and 15 mg/kg bw/day and in females at 15 mg/kg bw/day.
• Lower mean corpuscular haemoglobin (MCH) in males and females at 5 and 15 mg/kg bw/day.
• Lower mean corpuscular haemoglobin concentration (MCHC) in females at 15 mg/kg bw/day
• Higher platelet counts in males at 15 mg/kg bw/day.
The lower relative lymphocyte and eosinophil counts in males and females at 5 and/or 15 mg/kg bw/day was attributed to the higher white blood cell counts, since absolute lymphocyte counts were similar to control levels.
Any other statistically significant variations noted in haematology parameters were considered unrelated to treatment as these occurred in the absence of a dose-related trend and/or remained within the range considered normal for rats of this age and strain.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes in clinical biochemistry parameters distinguished treated from control animals:
• Lower albumin in males and females at 15 mg/kg bw/day
• Lower glucose in males at 15 mg/kg bw/day
• Lower cholesterol in males at 15 mg/kg bw/day (an apparent trend towards lower cholesterol was noted across female dose groups).
• The serum T4 level of F0 males was statistically significantly decreased at 15 mg/kg bw/day.
The statistically significant lower alkaline phosphatase activity of males at 15 mg/kg bw/day was considered not toxicologically relevant since the opposite effect would be expected in case of target organ toxicity.
Other statistically significant variations noted in clinical biochemistry parameters were considered unrelated to treatment or not toxicologically relevant as these changes occurred in the absence of a dose-related trend and/or remained within the range considered normal for rats of this age and strain.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Also, grip strength and motor activity were considered to have been unaffected by treatment.
The apparent increase in mean motor activity (ambulations and total movements) of females at 5 and 15 mg/kg bw/day was not attributed to treatment. Instead, this was ascribed to a procedural error in which measurements of the control group (and females at 1 mg/kg bw/day) commenced later than intended, due to which the habituation phase typically lasting 10-15 minutes was most likely exceeded. This was considered to have resulted in a lower motor activity over the first 10 minutes of the measurement interval of the female control group (and females at 1 mg/kg bw/day).
Motor activity of females at 5 and 15 mg/kg bw/day over the remainder of the measurement period appeared similar to those of controls (and females at 1 mg/kg bw/day). Also, motor activity habituation profile of the control group compared to 1 mg/kg bw/day females was similar, as was the case for the 5 and 15 mg/kg bw/day females compared. In addition, mean motor activity (ambulations and total movements) of all female groups over the treatment period remained in the range considered normal for rats of this age and strain. In the opposite sex, motor activity of was similar across all treatment groups, and all male groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Overall, it was considered that there were no treatment-related effects on motor activity in both sexes. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment.
The statistically significantly lower mean hind-limb grip strength in males at 1 and 15 mg/kg bw/day occurred in the absence of a dose-related trend and was therefore considered not to be related to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the mesenteric lymph nodes of males and females at all doses, in the small intestines of males and females at 5 and 15 mg/kg bw/day, in the sternal bone marrow of males at 5 and 15 mg/kg bw/day and females at 15 mg/kg bw/day, and in the spleen of males at 5 and 15 mg/kg bw/day.
In the mesenteric lymph nodes, an increased incidence and severity of foamy macrophages (up to marked degree) and granulomatous inflammation (with central necrosis) (up to massive degree) was present at all doses. Extranodal extension of the (necrotizing) granulomatous processes was present in a few males at 5 mg/kg bw/day and in half of the females at 5 and 15 mg/kg bw/day (up to moderate degree).
In the small intestines, foamy macrophages were present in the lamina propria of the duodenum (up to slight degree) and jejunum (up to moderate degree) in males and females at 15 mg/kg bw/day. In the ileum, foamy macrophages in the lamina propria were present in males and females at 5 mg/kg bw/day (up to slight degree) and 15 mg/kg bw/day (up to marked degree).
In the sternal bone marrow, myeloid hyperplasia was present in males at 5 mg/kg bw/day (minimal degree) and 15 mg/kg bw/day (slight degree) and in females at 15 mg/kg bw/day (up to slight degree).
In the spleen, an increased incidence and severity of extramedullary hematopoiesis was present in males treated at 5 and 15 mg/kg bw/day (up to slight degree). In females, no difference was noted between control and treated animals.
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.
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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment.
Most females had regular cycles of 4 days. Extended di-estrus occurred in a single female at 1 mg/kg bw/day and irregular cycles occurred in two females at 1 mg/kg bw/day, one female at 5 mg/kg bw/day and one female at 15 mg/kg bw/day. These females had normal litters except for one female at 1 mg/kg with irregular cycle, which was not pregnant. Given their incidental nature and absence of a dose-related incidence, these findings did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Spermatogenic staging profiles were normal for all males examined.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index was not affected by treatment. All females showed evidence of mating.
Fertility index was not affected by treatment. One female at 1 mg/kg bw/day, one female at 5 mg/kg bw/day, and one female at 15 mg/kg bw/day were not pregnant (as confirmed by Salewski staining). No abnormalities were seen in the reproductive organs which could account for their non-pregnancy. In the absence of a dose-related incidence, this non-pregnancy was considered not to be related to treatment.
Precoital time and number of implantation sites were not affected by treatment.
For two females both treated at 5 mg/kg bw/day, the number of pups born was slightly higher than the number of implantations. This was considered to be due to normal resorption of these areas as these enumerations were performed on Day 17 of lactation.
Gestation index and duration of gestation were not affected by treatment. 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. No deficiencies in maternal care were observed.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was not affected by treatment. The post-implantation survival indices across the groups ranged from 90 to 97%.
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects on reproduction at the highest tested dose
Key result
Dose descriptor:
LOAEL
Remarks:
parental toxicity
Effect level:
1 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
mesenteric lymph node
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment. Swelling and scabbing of the hind legs was observed in all seven pups of a single litter of 5 mg/kg bw/day group at PND 12. In the absence of similar findings in other pups of females treated at this dose, these clinical signs were considered to be unrelated to treatment. Other clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices across the groups ranged from 96 to 100%.
Three females had a few dead pups at first litter check (one control female, one female at 1 mg/kg bw/day and one female at 5 mg/kg bw/day which had 4, 2 and 4 dead pups, respectively). This
incidental pup mortality was unrelated to treatment with the test item.
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices in the across the groups were 99 or 100%.
Post-natal loss up to PND 4 was limited to one pup at 1 mg/kg bw/day which died spontaneously on PND 3, and one pup at 5 mg/kg bw/day which went missing on PND 3. The missing pup was most likely cannibalised. This incidental post-natal loss was unrelated to treatment with the test item.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes in body weights of pups were noted.
Mean male and female pup body weights appeared slightly lower than controls on PND 13. No level of statistical significance was achieved, means remained well within the range considered normal for pups of this age and strain, differences were small (less than 10% from control means) and no concurrent treatment-related developmental changes were observed. Therefore, these variations were considered not toxicologically relevant.
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 not 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):
No macroscopic findings were noted among pups that were considered to be related to treatment.
All pups of one litter at 5 mg/kg bw/day which were killed in extremis on Day 12 showed swollen hindlegs. In the absence of similar findings in other pups of females treated at this dose, this finding was not considered to be related to treatment.
The nature and incidence of other incidental macroscopic findings noted among pups remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Lactation index was considered unaffected by treatment, ranging from 90 to 100% across the dose groups.
The lower lactation index of 90% and increased breeding loss at 5 mg/kg was due to one female at 5 mg/kg bw/day, which had all pups (2 males and 5 females) sacrificed in extremis on PND 12. Since this only concerned a single litter at 5 mg/kg bw/day, this finding was considered not to be related to treatment.
In the control group and at 1 and 15 mg/kg bw/day, no pups were lost between PND 5 and PND 13. For these groups, the number of live offspring on PND 13 was the same as the number of live offspring on PND 4 (after culling), resulting in a lactation index of 100%.
Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment.
The statistically significantly higher median absolute anogenital distance noted in male pups at 1 mg/kg bw/day occurred in the absence of a dose-related response, and was therefore not considered related to treatment.
Treatment up to 15 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects on development at the highest tested dose
Key result
Reproductive effects observed:
no

Analytical verification of the test item concentrations

The concentrations analysed in the formulations of 1, 5 and 15 mg/kg bw/day groups (week 1 formulations) were in agreement with the target concentrations (mean accuracies 99.3, 100.9 and 98.7%, respectively; n = 6, 2 and 6, respectively). The formulations of 15 mg/kg bw/day group were homogeneous (i.e. coefficient of variation 1.0%). The coefficient of variation of the measured concentrations for the formulations of 1 mg/kg bw/day group showed a value of 10.1%, which is slightly above the acceptance criteria for homogeneity (i.e. coefficient of variation ≤ 10%).

The concentrations analysed in the formulations of 5 and 15 mg/kg bw/day groups (week 6 formulations) were in agreement with the target concentrations (mean accuracies 101.0 and 99.5%, n = 2 and 6, respectively). The concentrations analysed in the formulations of 1 mg/kg bw/day group (week 6 formulations) were above the acceptance criteria, mean accuracy of 116.5% ( n = 6). The formulations of 1 and 15 mg/kg bw//day groups were homogeneous (coefficient of variation 1.1% and 1.2%, respectively).

The concentrations analysed in the formulations of 1, 5 and 15 mg/kg bw/day groups (week 13 formulations) were in agreement with the target concentrations (mean accuracies 106.6 (n = 6), 98.5 (n = 2) and 97.2% (n = 6), respectively). The formulations of 1 and 15 mg/kg bw/day groups were homogeneous (i.e. coefficient of variation 1.4% and 1.2%, respectively).

Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours (i.e. relative difference ≤ 10%).

Table 1. Reproduction data

Dose level (mg/kg bw/day)

0

1

5

15

Pregnant/total dams

10/10

9/10

9/10

9/10

Duration of gestation (days, mean ± SD)

21.6 ± 0.7

21.3 ± 0.5

21.3 ± 0.7

21.3 ± 0.5

Implantations (mean ± SD)

12.5 ± 2.3

13.2 ± 1.6

11.8 ± 2.9

11.3 ± 1.7

Dams with living pups on Day 1

10

9

9

9

Females with total litter loss

0

0

1

0

Total number of litters

10

9

9

9

Dead pups at first litter check (litters affected/total number)

1/4

1/2

1/4

0/0

Living pups at first litter check/litter (mean ± SD)

11.1 ± 4.1

12.0 ± 2.0

11.0 ± 2.3

10.2 ± 2.2

Mean live offspring (number)

111

108

99

92

Postnatal loss (% of living pups/litters affected)

0.0/0

0.9/1

1.0/1

0.0/0

Culled pups (total)

37

35

27

21

Living pups PND4/litter (mean ± SD)

7.4 ± 1.9

8.0 ± 0.0

7.9 ± 0.3

7.9 ± 0.3

Breeding loss PND 5-13 (% of living pups at PND4/litters affected)

0.0/0

0.0/0

9.9/1

0.0/0

Living pups PND13/litter (mean± SD)

7.4± 1.9

8.0± 0.0

7.1 ± 2.7

7.9 ± 0.3

Conclusions:
In a GLP-compliant guideline 90-day study with reproductive/developmental toxicity screening, no adverse effects on fertility and reproduction were observed at the highest tested dose of 15 mg/kg bw/day. The dose level of 15 mg/kg bw/day was considered to be the NOAEL for reproductive and developmental toxicity. The lowest tested dose of 1 mg/kg bw/day was considered to be a LOAEL for parental toxicity, based on the presence of granulomatous inflammation of the mesenteric lymph nodes with central necrosis at all dose levels with dose-related increase in severity.
Executive summary:

In a GLP-compliant Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422), the pre-mating period was extended to 8-weeks to cover the male reproduction spermatogenesis period, and to cover full 90-day study requirements of OECD 408. Male and female rats (10/sex/dose) received the test substance at dose levels of 1, 5 and 15 mg/kg bw/day in corn oil by oral gavage. Males were exposed for 90 days, i.e. eight weeks prior to mating, during mating, and up to termination. Females were exposed for 91-104 days, i.e. during eight weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation.

No reproduction toxicity was observed up to the highest dose level tested (15 mg/kg).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Screening study for fertility; high quality OECD 422/408 guideline in compliance to GLP.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a GLP-compliant Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422), the pre-mating period was extended to 8-weeks to cover the male reproduction spermatogenesis period, and to cover full 90-day study requirements of OECD 408. Male and female rats (10/sex/dose) received the test substance at dose levels of 1, 5 and 15 mg/kg bw/day in corn oil by oral gavage. Males were exposed for 90 days, i.e. eight weeks prior to mating, during mating, and up to termination. Females were exposed for 91-104 days, i.e. during eight weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation.

No reproduction toxicity was observed up to the highest dose level tested (15 mg/kg).

Parental results:

Parental NOAEL is below 1 mg/kg (based on presence of granulomatous inflammation of the mesenteric lymph nodes with central necrosis at all dose levels.

Reproductive results:

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Reproduction NOAEL is at least 15 mg/kg.

 

Considerations on the mode of action show that the most significant treatment-related changes in the studies performed on Diamine methylated involve local irritating/corrosive effects. In physiological circumstances, Diamine methylated has a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar tails easily dissolve in the membranes, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule is not expected to easily pass membrane structures, and consequently, reproduction toxicity is not a likely concern.

 

Fertility by inhalation:

Exposure of humans to Diamine methylated via inhalation is not likely taking into account the low vapour pressure as well as low possibility of exposure to aerosols or droplets of an inhalable size. Furthermore, as the substance is classified as corrosive, local effects will be dose-limiting and preclude sufficient uptake for systemic effects to develop.

 

Fertility by dermal:

Diamine methylated is corrosive. Effects will be characterized by local corrosive effects that are related to duration, quantity and concentration, rather than by systemic toxicity due to dermal uptake. Dermal absorption is expected to be much lower compared to oral absorption. Related to its corrosive properties, exposures are likely to be limited.

Effects on developmental toxicity

Description of key information

In a GLP-compliant guideline 90-day study with reproductive/developmental toxicity screening, no adverse effects on reproduction and development were observed at the highest tested dose of 15 mg/kg bw/day. The NOAEL for reproductive and developmental toxicity was set at 15 mg/kg bw/day. The lowest tested dose of 1 mg/kg bw/day was considered to be the LOAEL for parental toxicity, based on the presence of granulomatous inflammation of the mesenteric lymph nodes with central necrosis at all dose levels with dose-related increase in severity. A prenatal developmental toxicity in rats (OECD 414) showed no developmental toxicity up to the highest tested dose level of 15 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 May 2019 - 23 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study was initiated after final decision of ECHA.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Purity/Composition correction factor: No correction factor required.
Test item handling: No specific handling conditions required.
Stability for at least 5 hours at room temperature is confirmed over the concentration range 0.2 to 200 mg/mL and stability for 8 days in the refrigerator is confirmed over the concentration range 1 to 200 mg/mL, Charles River Study No. 512048 and ABL Project 16059).
Species:
rat
Strain:
other: Wistar Han
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks
- Weight at study initiation: 177-257 g
- Fasting period before study: no
- Housing: Individually in Macrolon plastic cages containing appropriate bedding
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Municipal tap water, ad libitum
- Acclimation period: at least 5 days

No known contaminants were expected to be present in the feed that would interfere with the objectives of the study. Periodic analysis of the water was performed, and it was considered that there were no known contaminants in the water that would interfere with the objectives of the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 45-62
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 05 May 2019 To: 23 May 2019
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 5 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: the vehicle was chosen based on trial formulations performed at Charles River Den Bosch and on information provided by the sponsor.
- Concentration in vehicle: 0.3, 1.3 and 3.8 mg/mL for the low, mid and high dose, respectively.
- Amount of vehicle (if gavage): 4 mL/kg bw
Stability for at least 5 hours at room temperature was previously confirmed over the concentration range 0.2 to 200 mg/mL and stability for 8 days in the refrigerator is confirmed over the concentration range 1 to 200 mg/mL, Charles River Study No. 512048 and ABL Project 16059.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure (Ardena Bioanalytical Laboratory Study No. ABL16059).
Dose formulation samples were collected for analysis during the first week of treatment for concentration analysis (all groups) and analysis of homogeneity (low and high dose groups only). In the second week of treatment, dose formulation samples were collected for concentration analysis and analysis of homogeneity (low dose group only).
All samples were shipped on dry ice to the analytical laboratory on the date of preparation.
Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was below or at 10%.
Details on mating procedure:
The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
Duration of treatment / exposure:
Females were dosed on Day 6 to 20 post-coitum (15 days in total)
Frequency of treatment:
Once daily
Duration of test:
Females were sacrificed Day 21 post-coitum
Dose / conc.:
1 mg/kg bw/day (actual dose received)
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

The dose levels were selected based on the results of a previously performed 14-day dose range finder (Charles River Study No. 521047) and an extended OECD 422 study (Charles River Study No. 521048) in rats and in an attempt to produce graded responses to the test item. During the dose range finder, animals were dosed at 30 and 100 mg/kg bw/day by oral gavage. At 100 mg/kg bw/day, all animals were moribund after 11 days of treatment. At 30 mg/kg bw/day, effects were limited to body weight loss (-2 or -5% for all animals over Days 1-8, continuing for two animals to -5% and -6% over Days 1-14) and slightly reduced food intake. Based on these results, 1, 5 and 15 mg/kg/day were selected as dose levels for the extended OECD 422 study. During this study, animals received the test item for approximately 90 days. No adverse changes were noted in in-life parameters (i.e. mortality, clinical appearance, functional observations, body weights and food consumption) of females up to 15 mg/kg bw/day. As the effects on body weight at 30 mg/kg/day were considered unacceptable for the current study, the same dose levels as used in the extended OECD 422 study were selected.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Animals were observed for general health/mortality and moribundity twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Clinical observations were performed at least once daily, beginning on Day 2 post-coitum and lasting up to the day prior to necropsy. The time of onset, grade and duration of any observed sign was recorded. Cage debris was examined to detect premature birth.

BODY WEIGHT: Yes
- Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION: Yes
- Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Blood was collected on the day of scheduled necropsy (unfasted), concentration of Triiodothyronine (T3) Thyroxine (T4) and Thyroid-Stimulating Hormone (TSH) in serum was determined.
- All animals were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No organs (except for the uterus and thyroid gland) were weighed.
- Thyroid glands were weighed at necropsy for all animals. Paired organs were weighed together. Organ to body weight ratio (using the body weight on Day 21 post coitum) were calculated.
- Thyroid glands of all animals of the control and the high dose group were histopathologically examined. Thyroids of all groups were collected, but it was considered of no added value to examine tissues of the low and mid dose group, since no test item-related changes were observed in the highest dose group.
Ovaries and uterine content:
Each ovary and uterine horn of all animals were dissected and examined as quickly as possible to determine: the number of corpora lutea, the weight of the gravid uterus, the number of implantation sites, the number and distribution of live and dead fetuses, the number and distribution of embryo-fetal deaths and the sex of each fetus based on the ano-genital distance.
Fetal examinations:
Live fetuses were euthanized. All litters were subjected to detailed external, visceral and skeletal examinations.
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).
Each viable fetus was sexed, examined in detail to detect macroscopic visible abnormalities and their weight was determined. The anogenital distance (AGD) was measured for all viable fetuses. The AGD was normalized to the cube root of the fetal body weight.
The numbers of fetuses (litters) available for morphological examination were 230 (22), 263 (22), 243 (22) and 255 (22) in the control, 1, 5 and 15 mg/kg bw/day groups, respectively. External examination was done for all fetuses, visceral examination was done for approximately half of the fetuses of all groups, and skeletal examination was done for the other half of fetuses. Moreover, the visceral examination was added for two fetuses (one from the control group and one from the mid dose group) as during eviscerating prior to skeletal staining prominent visceral malformations were noticed.

Visceral Examinations:
The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The heads were removed from this one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. As visceral malformations were suspected for two pups in the high dose group which were selected for skeletal examination, these fetuses were also subjected to visceral examination. All carcasses, including the carcasses without heads, were eviscerated, skinned, labeled and fixed in 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal Examinations:
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson by a method similar to that described by Dawson and Inouye. Subsequently, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads). All specimens were archived in glycerin with bronopol as preservative. A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.






Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: low dose group vs. control group, mid dose group vs. control group, high dose group vs. control group. Datasets with at least 3 groups (the designated control group and at least 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test. Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.

Indices:
Maternal Variables
Body Weight Gains: Calculated against the body weight on Day 6 post-coitum;
Corrected Body Weight Gains: Body weight on Day 21 post-coitum minus the body weight on Day 6 post-coitum and the weight of gravid uterus;
Relative Food Consumption: Calculated against the body weight for scheduled intervals;
Organ Weight Relative to Body Weight: Calculated against the body weight on Day 21 post-coitum.

Reproduction and Developmental Variables
For each group, the following calculations were performed:
Pre-implantation loss (%): ((number of corpora lutea - number of implantation sites)/number of corpora lutea)*100
Post-implantation loss (%): ((number of implantation sites - number of live fetuses)/number of implantation sites)*100

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:
Viable fetuses affected/litter (%): ((number of viable fetuses affected/litter)/number of viable fetuses/litter)*100


Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were noted during the observation period.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights, body weight gain and weight gain corrected for gravid uterus were considered to have been unaffected by treatment up to 5 mg/kg bw/day. Mean body weight gain of females at 15 mg/kg bw/day was slightly reduced from start of treatment onwards. On Day 21 post coitum, mean body weight gain of these females was 7% lower than concurrent controls (statistical significance was achieved on Day 18 post coitum only). In addition, mean relative weight gain corrected for gravid uterus weight was statistically significantly lower at 15 mg/kg/day when compared with controls (7.7% vs 12.8%). The mean relative weight gain remained just within the historical control range. The correction for gravid uterus weight made clear that two pregnant females at 15 mg/kg bw/day actually lost weight over the treatment phase, i.e. Day 6 to Day 21 post coitum.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was considered unaffected by treatment up to 5 mg/kg bw/day.
At 15 mg/kg bw/day, mean food consumption was decreased throughout the treatment period when compared with concurrent control. Overall, relative food consumption was decreased with 8% when compared with concurrent control (mean of means). The differences in relative food consumption between females at 15 mg/kg bw/day and concurrent control were statistically significant and ranged from 7-11% on separate intervals.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum levels of total T3 and T4 and TSH were considered to be unaffected by treatment up to 15 mg/kg bw/day. All mean values remained within the historical control range. The statistically significantly lower mean TSH level in females at 1 mg/kg/day was considered to be a chance finding, in the absence of a dose-related response.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant higher thyroid gland weights (absolute and relative to body weights) were noted in the 15 mg/kg bw/day group females when compared with the control group females. All relative thyroid weights remained within normal historical control limits. Moreover, no treatment-related effects were noted in any of the thyroid hormones or during microscopic evaluation of the thyroid. The increase noted in thyroid organ weight was therefore considered a chance finding and unrelated to treatment with the test item.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic findings in the thyroid gland of the control and high dose group animals.
Number of abortions:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on litter size of any group. Mean litter sizes were 10.5, 12.0, 11.0 and 11.6 fetuses/litter for the control, 1, 5 and 15 mg/kg bw/day groups, respectively.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were in the range of normal biological variation and considered unaffected by treatment up to 15 mg/kg bw/day. The statistically significant lower percentage of pre-implantation loss for females at 5 mg/kg bw/day was attributed to the relatively high concurrent control mean and in the absence of a dose related response considered unrelated to treatment.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The litter incidence of early and late resorptions and post-implantation loss was considered unaffected by treatment up to 15 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed up to and including the highest dose tested
Remarks on result:
other: 15 mg/kg bw/day was selected as highest dose because the body weight loss observed at 30 mg/kg bw/day in a 14-day dose range finder in non-pregnant rats was considered unacceptable for the current study.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The fetal weights (male, female and combined) in treatment groups were comparable with the controls and remained within the historical control range of the Test Facility. Mean combined (male and female) fetal body weights were 5.3, 5.3, 5.3 and 5.1 gram for the control, 1, 5 and 15 mg/kg bw/day groups, respectively.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 15 mg/kg bw/day.
External malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on external morphology following treatment up to 15 mg/kg bw/day.
Two externally malformed fetuses were observed in this study. One fetus at 5 mg/kg/day had a meningocele, substantiated skeletally, and in addition appeared to have fusion of the mandibles. The other fetus was a control fetus that had microcephaly with according skeletal findings of the skull. Due to the single occurrence and/or occurrence in a control fetus, these malformations were considered spontaneous in origin.
External variations were not seen in any group.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on skeletal morphology following treatment up to 15 mg/kg bw/day.
Aside from the underlying malformations of the two fetuses with an external skull finding (one in the control and one in the 5 mg/kg/day group, respectively), the latter fetus also had fusion of the mandibles. In addition, bent limb bones were observed in one fetus at 1 mg/kg bw/day. Both malformations were observed previously in historical controls and the group distribution did not indicate a treatment relationship. Therefore, they were considered chance findings.
Skeletal variations were observed in 75.0%, 77.3%, 81.0% and 80.8% of fetuses per litter in the control, 1, 5 and 15 mg/kg bw/day groups, respectively. All the ones noted occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data. Therefore, they were considered unrelated to treatment.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on visceral morphology following treatment up to 15 mg/kg bw/day.
Visceral malformations occurred in two fetuses at 1 and 15 mg/kg bw/day each. At the high dose, one fetus had a malpositioned atrium and ventricular septum defect and one fetus had a small eye and right-sided aorta. At the low dose, both affected fetuses had situs inversus. Due to the group distribution, single occurrence and/or occurrence in historical control fetuses, these malformations were considered unrelated to treatment.
Only two visceral variations (dilated ureter and convoluted ureter) were observed in this study and both occurred in one fetus at 15 mg/kg bw/day. The single occurrence did not indicate a treatment relationship. Moreover, both variations were observed previously among historical control fetuses.
Other effects:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on fetal ano-genital distance (both sexes) noted after treatment up to 15 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to and including the highest dose tested
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Based on the results of a prenatal developmental toxicity study performed in rats according to OECD guideline 414 and GLP principles, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Diamine methylated was established to be 15 mg/kg bw/day, as no adverse effects were seen at the highest dose tested (15 mg/kg bw/day).
Executive summary:

The objectives of this study were to determine the potential of Diamine methylated to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats from Day 6 to 20 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.

The dose levels in this study were selected to be 0, 1, 5 and 15 mg/kg/day, based on the results of a 14-day dose range finder (Test Facility Study No. 512047) and an extended OECD 422 study (Test Facility Study No. 512048). 15 mg/kg/day was selected as highest dose because the body weight loss observed at 30 mg/kg (described in a 14-day dose range finder in non-pregnant rats; Test Facility Study No. 512047) was considered unacceptable for the current study. Test substance was administered in corn oil at 4 mL/kg bw to 22 females per dose group.

 

Chemical analyses of formulations were conducted twice during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (T3, T4, and TSH), gross necropsy findings, number of corpora lutea, organ weights ((gravid) uterus and thyroid gland), uterine contents and histopathologic examination (thyroid gland).

In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, ano-genital distance, external, visceral and skeletal malformations and developmental variations.

 

Results:

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels for Group 3 and 4 formulations. 

Formulations of Group 2 were below the target concentration, however as no adverse effects were noted in any of the study parameters up to 15 mg/kg/day, there was no impact on the study outcome. 

No test item-related effects were observed in the 1 and 5 mg/kg/day groups.

Mean food consumption and body weight gain was reduced throughout the treatment period in females at 15 mg/kg/day. In addition, body weight gain corrected for gravid uterus weight was statistically significantly lower for these females. Based on the magnitude of change, absence of any clinical symptoms and as values remained within the normal range, these effects were considered not adverse. 

No developmental toxicity was observed in the 1, 5 and 15 mg/kg/day groups.

 

In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Diamine methylated was established as being 15 mg/kg/day. 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Prenatal developmental toxiicty study; high quality OECD 414 guideline in compliance to GLP.
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

In a GLP-compliant Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422), the pre-mating period was extended to 8-weeks to cover the male reproduction spermatogenesis period, and to cover full 90-day study requirements of OECD 408. Male and female rats (10/sex/dose) received the test substance at dose levels of 1, 5 and 15 mg/kg bw/day in corn oil by oral gavage. Males were exposed for 90 days, i.e. eight weeks prior to mating, during mating, and up to termination. Females were exposed for 91-104 days, i.e. during eight weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation.

Parental results: Parental NOAEL is below 1 mg/kg (based on presence of granulomatous inflammation of the mesenteric lymph nodes with central necrosis at all dose levels.

Developmental results: No developmental toxicity was observed up to the highest dose level tested (15 mg/kg).

No toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance (PND 1), areola/nipple retention (PND 13 males), serum concentration of thyroid hormone T4 (PND 13-15) and macroscopy.

Diamine methylated was further evaluated in a GLP-compliant prenatal developmental toxicity in rats (OECD 414). The test substance was administeredorally by gavage to time-mated female Wistar Han rats from Day 6 to 20 post-coitum, inclusive. The dose levels in this study were selected to be 0, 1, 5 and 15 mg/kg/day, based on the results of a 14-day dose range finder and the extended OECD 422 study mentioned above. 15 mg/kg/day was selected as highest dose because the body weight loss observed at 30 mg/kg (described in a 14-day dose range finder in non-pregnant rats) was considered unacceptable for the current study. Test substance was administered in corn oil at 4 mL/kg bw to 22 females per dose group.

No test item-related effects were observed in the 1 and 5 mg/kg/day groups. Mean food consumption and body weight gain was reduced throughout the treatment period in females at 15 mg/kg/day. In addition, body weight gain corrected for gravid uterus weight was statistically significantly lower for these females. Based on the magnitude of change, absence of any clinical symptoms and as values remained within the normal range, these effects were considered not adverse. 

No developmental toxicity was observed in any of the 1, 5 and 15 mg/kg/day groups.

An additional consideration is that specific teratogenic effects or specific organ interactions from Diamine methylated is unlikely. In physiological circumstances, Diamine methylated has a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces as cellular membranes.The apolar tails easily dissolve in the membranes, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule will not easily pass membrane structures. Cytotoxicity at the local site of contact through disruption of cell membrane will is considered the most prominent mechanism of action for toxic effects.

Developmental tox by inhalation:

Exposure of humans to Diamine methylated via inhalation is not likely taking into account the low vapour pressure as well as low possibility of exposure to aerosols or droplets of an inhalable size. Furthermore, as the substance is classified as corrosive, local effects will be dose-limiting and preclude sufficient uptake for systemic effects to develop.

 

Developmental tox by dermal:

Diamine methylated is corrosive. Effects will be characterized by local corrosive effects that are related to duration, quantity and concentration, rather than by systemic toxicity due to dermal uptake. Dermal absorption is expected to be much lower compared to oral absorption. Related to its corrosive properties, exposures are likely to be limited.

Mode of Action Analysis / Human Relevance Framework

Based on structure and mechanism of cytotoxicity, reproduction toxicity is not expected. The observed effects are local, reflecting a point-of-first-contact effect.In physiological circumstances, Diamine methylated has a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar hydrophobic tails are pushed out of solution and easily dissolve in the lipid bilayer, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule will not easily pass membrane structures. Cytotoxicity through disruption of cell membrane at exposure site will occur rather than absorption over the cell membrane. Consequently, significant uptake followed by placental transfer is not expected to occur.

Justification for classification or non-classification

Available data do not indicate a concern for reproduction or developmental toxicity.

However, as a firm conclusion from an adequate EOGRTS with this compound is lacking, no definite conclusion might be drawn for classification purposes.

But based on limited exposures by dermal route (substance is severely irritating/ corrosive) or by inhalation (very low vapour pressure), as well as lack of indication for concerns regarding reproductive toxicity from repeated dose studies and a reproduction toxicity screening study there are no immediate concerns.