Registration Dossier

Administrative data

Description of key information

In a GLP-compliant guideline 90-day oral study with rats, combined with reproductive/developmental toxicity screening, the lowest tested level of 1 mg/kg bw/day was considered to be a LOAEL, 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.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
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 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:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
September 1998
Deviations:
yes
Remarks:
3 females were treated for 84 days instead of 90
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
August 1998
Deviations:
no
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 421, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2015
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 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
Details on route of administration:
The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
corn oil
Details on oral 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.
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
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
Observations and examinations performed and frequency:
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

OTHER:

ESTROUS CYCLE DETERMINATION: 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.

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).
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.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Males: following completion of the mating period (a minimum of 90 days dosing)
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.
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.
Pups: culling: PND 4, terminal sacrifice: PND 13-16. For spontaneous deaths, necropsy was performed as soon as possible after death and always within 24 hours.

HISTOPATHOLOGY: Yes
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

Other examinations:
Pups were examined for:
Mortality and viability: on PND1 and daily thereafter
Clinical signs: at least once daily
Body weights: PND 1, 4, 7, 13
Sex: PND 1, 4. Sex ratio (% male/ % female) was calculated per group.
Anogenital distance: PND 1, all live pups
Areola/nipple retention: PND 13, all males.
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.
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
Description (incidence and severity):
A dose-dependent statistically significant increase in relative spleen weight was observed in 5 and 15 mg/kg bw/day males (not statistically significant in absolute weights).
All other relative organ weight differences in males at 15 mg/kg bw/day that were statistically significant when compared to the control group were considered to be the result of a test item-related effect on final body weight.
The statistically significant changes in absolute and/or relative spleen weight of females (lower at 1 mg/kg bw/day, higher at 15 mg/kg bw/day) were not considered to be related to treatment because they occurred in the absence of a dose-related trend and microscopic correlates.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related macroscopic findings consisted of:
• Mesenteric lymph node: enlarged in 1/10 females at 1 mg/kg bw/day, 5/10 males and 8/10 females at 5 mg/kg bw/day, and 10/10 males and 9/10 females at 15 mg/kg bw/day.
• Ileum: thickened in 3/10 males at 5 mg/kg bw/day and 6/10 males at 15 mg/kg bw/day.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These necropsy findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
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
Description (incidence and severity):
Length and regularity of the estrous cycle, mating and fertility indices, precoital time, number of implantation sites, gestation index and duration of gestation, partutition/maternal care, post-implantation survival index, live birth index, viability lactation indices were not affected by the treatment.
No clinical signs occurred among pups that were considered to be related to treatment. No toxicologically relevant changes in body weights of pups were noted. Sex ratio, anogenital distance and areola/nipple retention were not affected by the treatment. Serum T4 levels in male and female PND 13-15 pups were not affected by treatment. No macroscopic findings were noted among pups that were considered to be related to treatment.
See the cross-reference for details on reproductive and developmental effects .
Key result
Dose descriptor:
LOAEL
Effect level:
1 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects on reproduction at the highest dose level
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
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 dose level
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 mg/kg bw/day (actual dose received)
System:
immune system
Organ:
mesenteric lymph node
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

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%).

Conclusions:
In a GLP-compliant guideline 90-day oral study with rats, combined with reproductive/developmental toxicity screening, the lowest tested level of 1 mg/kg bw/day was considered to be a LOAEL, 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 adverse changes in in-life parameters (mortality, clinical appearance, functional observations, ophthalmoscopy, body weights and food consumption) were noted up to 15 mg/kg bw/day. A slightly lower body weight and body weight gain was recorded for males at 15 mg/kg bw/day. Since this change was slight and occurred in the absence of any adverse changes in in-life parameters, it was not considered adverse.

Adverse changes were confined to morphologic changes in the lamina propria of the small intestines (at 5 and 15 mg/kg bw/day) and its draining mesenteric lymph nodes (all dose levels), consisting of accumulation of foamy macrophages. In the mesenteric lymph nodes, extensive accumulation of macrophages resulted in a granulomatous inflammation with central necrosis at all dose levels which extended outside the lymph node (extranodal) in several animals at 5 and 15 mg/kg bw/day. The combined occurrence of central necrosis with granulomatous inflammation was considered adverse at all doses. For the accumulation of macrophages in the small intestines, a severity of moderate to marked was considered to be adverse, as recorded at 15 mg/kg bw/day in the jejunum (up to moderate) and ileum (up marked).

Correlating macroscopic lesions consisted of enlarged mesenteric lymph nodes at 1 mg/kg bw/day and higher and thickened ileum at 5 and 15 mg/kg bw/day. Hematological changes that supported these morphological lesions consisted of higher white blood cell counts at 15 mg/kg bw/day and higher relative neutrophil counts at 5 and 15 mg/kg bw/day.

Non-adverse histopathological lesions at 5 and 15 mg/kg bw/day that were considered to have occurred secondary to the granulomatous inflammation process in the mesenteric lymph nodes consisted of very low severity grades of myeloid hyperplasia in the sternal bone marrow. The very mildly increased incidence and severity of extramedullary hematopoiesis in the spleen of males barely exceeded the background level and was therefore not considered to be adverse. A higher spleen weight recorded at 15 mg/kg bw/day and several small hematological changes at 5 and 15 mg/kg bw/day (including higher red cell distribution width, and lower haemoglobin mean corpuscular volume, and mean corpuscular haemoglobin (concentration)) were attributed to the observed extramedullary splenic hematopoiesis. These findings were considered not adverse.

Thyroid hormone T4 was lower in males treated at 15 mg/kg bw/day, but means remained within the range considered normal for rats of this age and strain. Also, this finding was not accompanied by changes in weight or morphology of the thyroid. As such, the lower T4 levels were considered not to represent an adverse change.

Other clinical biochemistry changes in males and/or females at 15 mg/kg bw/day that were considered to be related to treatment consisted of lower albumin, glucose and cholesterol. For none of these changes an apparent correlation with histopathological lesions could be made, and changes were essentially slight in degree. As such, these changes were not considered adverse.

Based on these results, the lowest tested dose level of 1 mg/kg bw/day was considered to be a LOAEL for parental toxicity, based on presence of granulomatous inflammation of the mesenteric lymph nodes with central necrosis at all dose levels with dose-related increase in severity.

In summary the findings consist of

- accumulation of foamy macrophages in the mesenteric lymph nodes, resulting in a granulomatous inflammation with central necrosis at all dose levels which extended outside the lymph node (extranodal) in several animals at 5 and 15 mg/kg bw/day.

- infiltration of foamy macrophages in lamina propria intestines at 5 and 15 mg/kg bw/d

- Increased neutrophil counts at 5 and 15 mg/kg bw/d in males and females

- a lower bw in males at 15 mg/kg bw/d.

 

This result pattern does very well compare to effects seen for cationic surfactants in general.

The severity of the foamy macrophages (see attached graph) in the mesenteric lymph nodes at 1 mg/kg bw is at the high end of what sometimes is seen in control groups. However, in three animals the observed minimal to slight central necrosis is considered adverse. But further extrapolation with a factor 3 to dose levels of 0.33 mg/kg bw/d, would indicate a NOAEL.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
1 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP-compliant guideline study.
System:
gastrointestinal tract
Organ:
mesenteric lymph node

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

The most significant treatment-related changes are effects on the small intestine and mesenteric lymph nodes. A relatively strong inflammatory reaction is also observed at high dose levels. These effects in the gastro-intestinal tract have consistently been observed with cationic surfactants.

A mode of action has not been established but it is possible to suspect the known corrosivity to be at least partially involved. The observed effects are local and they are by some interpreted as phospholipidosis, something commonly observed following treatment with cationic amphiphilic material, including marketed pharmaceuticals, and generally considered to be non-adverse. When taking into consideration the relatively strong corrosive effects of this substance and the route of administration, it cannot be excluded that the overall toxicity reflects a point-of-first-contact effect.

Phospholipidosis is a plausible mechanism. 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. Noteworthy in this respect is that recent research shows that the log distribution coefficient for cationic surfactants between water and phospholipid are possibly several orders of magnitude higher than between water and oil. (log Pow diamine methylated is 4.06 based on ratio solubility octanol and water) The complex of cationic surfactant and phospholipids are difficult to digest by the macrophages, and they accumulate with the lysosomes. Recent (unpublished) studies have shown various cationic surfactants to be lysosomotropic, and scored positive for phospholipidosis inin vitrostudies with HepG2 cells.

Additional information

oral:

First a 14-day range finding study was performed involving two dose groups of 100 and 30 mg/kg bw/d in corn oil, 5 ml/kg bw; in groups of 3 females.

 

Parameter

30 mg/kg

100 mg/kg

Mortality

No mortality.

All animals sacrificed in extremis on Day 12.

Clinical appearance

No findings.

Hunched posture, piloerection, lean appearance, ptosis and/or salivation (all animals, primarily in the second week).

Body weight

Slight weight loss(-2 or -5% for all animals over Days 1-8, continuing for two animals to   -5% and -6% over Days 1-14, with slight weight gain for one animal over Days 8-14).

Weight loss (-11 or -15% for all animals over Days 1-8).

Food consumption

Slightly reduced (approximately two thirds of the intake expected for rats of this age and strain).

Notably reduced (approximately one third of the intake expected for rats of this age and strain).

Liver and kidney weight

Considered unaffected by treatment

Not determined; all animals sacrificed in extremis.

Macroscopic examination

No abnormalities noted.

Irregular surface of the forestomach, gelatinous contents of the gastro-intestinal tract and reduced size of the liver and thymus (all animals)

Based on the results of this range finding study, dose levels suggested for the main study are: 1, 5 and 15 mg/kg body weight. No clear peak effect of occurrence of clinical signs was observed in the range finding study. Therefore, clinical observations in the main study will be conducted immediately after dosing, and functional observation tests will be conducted after dosing at no specific time point, but within a similar time period after dosing for the respective animals.

In the subsequent 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 adverse changes in in-life parameters (mortality, clinical appearance, functional observations, ophthalmoscopy, body weights and food consumption) were noted up to 15 mg/kg bw/day. A slightly lower body weight and body weight gain was recorded for males at 15 mg/kg bw/day.

 

Adverse changes were confined to morphologic changes in the lamina propria of the small intestines (at 5 and 15 mg/kg bw/day) and its draining mesenteric lymph nodes (all dose levels), consisting of accumulation of foamy macrophages. In the mesenteric lymph nodes, extensive accumulation of macrophages resulted in a granulomatous inflammation with central necrosis at all dose levels which extended outside the lymph node (extranodal) in several animals at 5 and 15 mg/kg bw/day. The combined occurrence of central necrosis with granulomatous inflammation was considered adverse at all doses. For the accumulation of macrophages in the small intestines, a severity of moderate to marked was considered to be adverse, as recorded at 15 mg/kg bw/day in the jejunum (up to moderate) and ileum (up marked).

Correlating macroscopic lesions consisted of enlarged mesenteric lymph nodes at 1 mg/kg bw/day and higher and thickened ileum at 5 and 15 mg/kg bw/day. Hematological changes that supported these morphological lesions consisted of higher white blood cell counts at 15 mg/kg bw/day and higher relative neutrophil counts at 5 and 15 mg/kg bw/day.

Non-adverse histopathological lesions at 5 and 15 mg/kg bw/day that were considered to have occurred secondary to the granulomatous inflammation process in the mesenteric lymph nodes consisted of very low severity grades of myeloid hyperplasia in the sternal bone marrow. The very mildly increased incidence and severity of extramedullary hematopoiesis in the spleen of males barely exceeded the background level and was therefore not considered to be adverse. A higher spleen weight recorded at 15 mg/kg bw/day and several small hematological changes at 5 and 15 mg/kg bw/day (including higher red cell distribution width, and lower haemoglobin mean corpuscular volume, and mean corpuscular haemoglobin (concentration)) were attributed to the observed extramedullary splenic hematopoiesis. These findings were considered not adverse.

 

In summary the findings consist of

- accumulation of foamy macrophages in the mesenteric lymph nodes, resulting in a granulomatous inflammation with central necrosis at all dose levels which extended outside the lymph node (extranodal) in several animals at 5 and 15 mg/kg bw/day.

- infiltration of foamy macrophages in lamina propria intestines at 5 and 15 mg/kg bw/d

- Increased neutrophil counts at 5 and 15 mg/kg bw/d in males and females

- a lower bw in males at 15 mg/kg bw/d.

 

This result pattern does very well compare to effects seen for cationic surfactants in general.

The severity of the foamy macrophages in the mesenteric lymph nodes at 1 mg/kg bw is at the high end of what sometimes is seen in control groups. However, in three animals the observed minimal to slight central necrosis is considered adverse. But further extrapolation with a factor 3 to dose levels of 0.33 mg/kg bw/d, would indicate a NOAEL.

 

Based on these results, the lowest tested dose level of 1 mg/kg bw/day was considered to be a LOAEL for parental toxicity, based on presence of granulomatous inflammation of the mesenteric lymph nodes with central necrosis at all dose levels with dose-related increase in severity.

 

Inhalation:

Diamine methylatedhas a low vp pressure (2.3E-04 Pa at 20°C, experimental) andits use is limited to industrial settings that do not involve the forming of aerosols, particles or droplets of an inhalable size. So exposure to humans via the inhalation route will be unlikely to occur.

Furthermore, as the substance is classified as corrosive, local effects will be dose-limiting and preclude sufficient uptake for systemic effects to develop.

 

Dermal:

Diamine methylatedis corrosive to the skin and is not expected to easily pass the skin. The skin is therefore not a preferred route when studying repeated dose systemic toxicity.

Justification for classification or non-classification

The range finding study with Diamine methylated showed severe toxicity at 100 mg/kg bw/day, which resulted to all animalsto besacrificedin extremison Day 12. As this clearly would indicate severe toxicity below 100 mg/kg bw/day STOT-RE Cat.2 criteria are definitely fulfilled.

The effects following 90-days exposure duration of granulomatous inflammation with central necrosis extending outside the lymph node (extranodal) in several animals at 5 and 15 mg/kg is considered adverse. With these effects observed below 10 mg/kg bw results to classification STOT-RE Cat. 1 with H372: Causes damage to organs (mesenteric lymph nodes) through prolonged or repeated exposure.