Amines, N-(C16-18 and C18-unsatd. alkyl)-N,N',N'-trimethyltrimethylenedi-

Repeated dose toxicity: oral

Administrative data

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

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 for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2015

Deviations:

no

Qualifier:

according to guideline

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

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

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

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 or test system 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).

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

13 April 2016 - 15 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OEC 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

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

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

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 test animals or test system 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 have interfered 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.

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%.

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).

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

Duration of test:

At least 90 days

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

Maternal 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).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: no, the females were allowed to litter naturally and were sacrificed on PND 13-15.

Fetal examinations:

Each litter was examined to determine the following, if practically possible:
Mortality/viability: PND 1 and daily thereafter
Clinical signs: at least once daily
Body weights: PND 1, 4,7, 13 (live pups)
Sex: all pups on PND 1 and 4; sex ratio (m/f) calculated per group
Anogenital distance: PND1, normalized to the cube root of body weight
Areola/nipple retention: PND13, all males
Thyroid hormone analysis: PND 13-15, 2 pups per litter for the measurement of thyroxine and possible measurement of TSH
Gross necropsy: PND4 (culling) and PND13-16

- Head examinations: No

Statistics:

The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• 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.

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 females)
Duration of gestation: number of days between confirmation of mating and the beginning of parturition
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)

Historical control data:

Not applicable

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 and including 15 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

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 for 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.
See the cross-reference for details on reproductive and developmental effects .

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

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%.
Pre-implantation losses were not examined.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

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.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Duration of gestation was not affected by treatment.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Gestation index and duration of gestation were not affected by treatment.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

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.

Other effects:

no effects observed

Description (incidence and severity):

Precoital time was 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.

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

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.

Basis for effect level:

other: no adverse effects on fertility and reproduction at the highest tested dose

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: mesenteric lymph nodes

Description (incidence and severity):

Granulomatous inflammation of the mesenteric lymph nodes with central necrosis at all dose levels with dose-related increase in severity.

Fetal body weight changes:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): 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.

Reduction in number of live offspring:

no effects observed

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.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was not affected by treatment

Changes in litter size and weights:

no effects observed

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.

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

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 which died spontaneously on PND 3, and one pup at 5 mg/kg 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.

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

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 bw/day 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%.
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 (one litter at 5 mg/kg bw/day) 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.
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.
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. 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.

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects on development at the highest tested dose

Key result

Abnormalities:

no effects observed

Key result

Developmental 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 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.

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 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

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.

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

May 2008

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

August 1998

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

Test guideline 6

Qualifier:

according to guideline

Guideline:

other: OECD 421, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

d.d. 3 November 2015

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine

EC Number:

695-101-5

Cas Number:

1275611-65-8

Molecular formula:

Not applicable

IUPAC Name:

N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Diamine methylated
- Substance type:Amber liquid
- Physical state:Liquid
- Storage conditions of test material:At room temperature
- Content (certified): 100% (UVCB–Substance)
- Batch/Lot number:211401A Morris
- Expiration date of the lot/batch:24 March 2018

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.

Test animals

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 or test system 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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Concurrent vehicle controls

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

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

Examinations

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.

Results and discussion

Results of examinations

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 .

Effect levels

1

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

2

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

3

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

Target system / organ toxicity

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

Any other information on results incl. tables

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%).

Applicant's summary and conclusion

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.