Fatty acids, tall-oil, reaction products with formaldehyde and (Z)-N-9-octadecenyl-1,3-propanediamine

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 22 November 2012 to 18 January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented GLP study according to OECD Guideline 422 and EPA OPPTS 870.3650.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650

Deviations:

no

Principles of method if other than guideline:

In addition, the procedures described in the protocol were conform to:
- OECD Guideline 421
- EPA OPPTS 870.3550
- EU method B.7
- OECD Guideline 407
- EPA OPPTS 870.3050

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, tall-oil, reaction products with formaldehyde and (Z)-N-9-octadecenyl-1,3-propanediamine

EC Number:

272-789-1

EC Name:

Fatty acids, tall-oil, reaction products with formaldehyde and (Z)-N-9-octadecenyl-1,3-propanediamine

Cas Number:

68911-83-1

Molecular formula:

C40H76N2O

IUPAC Name:

Fatty acids, tall-oil, reaction products with formaldehyde and (Z)-N-9-octadecenyl-1,3-propanediamine

Details on test material:

- Name of test material (as cited in study report): Reaction product of oleic acid, N1-(9Z)-9-octacecen-1-yl-1,3-propanediamine and paraformaldehyde
- Substance type: Dark brown liquid
- Physical state: Liquid
- Analytical purity: >99%
- Lot/batch No.: 8351213508
- Expiration date of the lot/batch: 5 May 2014
- Stability under test conditions: Stable under storage conditions; stability at higher temperatures: yes, maximum temperature: 50°C, maximum duration: 6 weeks
- Storage condition of test material: at room temperature in the dark

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

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: approximately 11 weeks
- Weight at study initiation: pre-mating day 1: range for males: 326 - 332 grams; range for females: 212 - 215 grams
- Fasting period before study: no
- Housing:
Acommodation:
- Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm)
- 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 weere individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Lactation: Pups were kept 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.
- General: Sterilized sawdust as bedding material and a paper as cage-enrichment/nesting material were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment or bedding material.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24 °C
- Humidity (%): 40 to 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Parental animals:
Dose volume: 5 mL/kg body weight. Actual dose volumes will be calculated according to the latest body weight.

Pups:
Pups were not dosed directly, but could have potentially be exposed to the test substance in utero, via maternal milk or from exposure to maternal urine/feces.

Details on mating procedure:

Following a minimum of 14 days of exposure for the males and females, one female will be cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated day 0 post-coitum. Once mating has occurred, the males and females were separated.
On 20 December 2012 (day 15 of the mating period), group 4 female no. 80 was separated from male no. 40 and cohabited with a proven male from the same group (no. 36).
Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: HPLC
Instrument: Alliance Separation Module 2695 (Waters, Milford, MA, USA)
Detector: Dual lambda Absorbance Detector 2487 (Waters)
Column: Xterra ms C18, 150 mm x 4.6 mm i.d., dp = 5 µm (Waters)
Column temperature: 35°C ± 1 °C
Injection volume: 10 µL
Mobile phase: A - Acetonitrile
B - Ammonia buffer pH 10
C - Tetrahydrofuran
Flow: 1 mL/min
UV detection: 221 nm
Preparation of solutions:
Stock and spiking solutions: stock and spiking solutions of the test substance were prepared in tetrahydrofuran at concentrations of 3376 and 3904 mg/L
Calibration solutions: Calibration solutions in the concentration range of 160 - 2400 mg/L were prepared from two stock solutions. The end solution of the calibration solutions was tetrahydrofuran.
Procedural recovery samples: Approximately 500 mg blank vehicle was spiked with the test substance at a target concentration of 21.7, 64.9 or 214 mg/g. The accuracy samples were treated similarly as the test samples.
Sample injections:
Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analyzed by single injection.
Specifications: Preparation of formulations was considered acceptable if the mean accuracy was in the range of 90-110% of the target concentration and if the coefficient of variation was <=10%. Formulations were considered stable if the relative difference between the stored and freshly taken samples was <= 10%.
Results:
Calibration curves: A calibration curve was constructed using five concentrations. For each concentration, two responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration weighting factor. The coefficient of correlation (r) was > 0.99.
Samples:
Procedural recovery samples:
The mean recoveries of the procedural recovery samples fell within the criterion of 90-110%. It demonstrated that the analytical method was adequate for the determination of the test substance in the test samples.
Test samples:
Accuracy of preparation:
The concentrations analysed in the formulations of group 2, group 3 and group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
Homogeneity:
The formulations of group 2 and group 4 were homogeneous (i.e. coefficient of variation <= 10%).
Stability:
Analysis of group 2 and group 4 formulations after storage yielded a relative difference of <=10%. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions.

Duration of treatment / exposure:

Males were exposed for 29-32 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 42-57 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 44 (group 1), 52 (group 2), 62, 64 (group 3) and 82 (group 4) were not dosed during littering.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000
Basis:
nominal conc.

No. of animals per sex per dose:

5
Number of pups: 394

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the outcome of a 10-day dose range finding study (project 500748)
- Randomization F0: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

Examinations

Parental animals: Observations and examinations:

MORTALITY/VIABILITY:
At least twice daily. Animals showing pain, distress or discomfort, which is considered not transient in nature or is likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints.

DETAILED CLINICAL OBSERVATIONS: Yes
- Daily from start of treatment onwards up to the day prior to necropsy, detailed clinical obervations were made for all animals immediately after dosing. Once prior to start of treatment and at weekly intervals during the treatement period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

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

FOOD CONSUMPTION: Yes
- Weekly, except for males and females which are 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 on days 1 and 4 of lactation.

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

OPHTHALMOSCOPIC EXAMINATION: No

CLINICAL LABORATORY INVESTIGATIONS:
Blood samples were collected on the day of necropsy from the selected 5 animals/sex/grouop under anaesthesia using isoflurane between 7.00 and 10.30 a.m.
The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin (0.5 mL) for clinical biochemistry parameters. An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.

HAEMATOLOGY: Yes
Parameters checked:
White blood cells, differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells, reticulocytes, red blood cells distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
Parameters checked.
Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total globulin, albumin/globulin ratio, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate (inorg. phos)

URINALYSIS: No

FUNCTIONAL OBSERVATIONS:
The following functional observations were performed on each individual animal of the selected 5 animals/sex/group:
- hearing ability*
- pupillary reflex*
- static righting reflex*
- grip strength*
* score 0 = normal/present, score 1 = abnormal/absent
- locomotor activity (recording period: 1-hour under normal laboratory light conditons, using a computerized monitoring system). Total movement and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.
During motor activity monitoring, all animals were caged individually (pups were housed in their home cages and kept warm using bottles filled with warm water).

The selected males were tested during week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (from lactation day 4 onwards). These tests were performed after observation for clinical signs (incl. arena observation, if applicable) at no specific time point after dosing, but within a similar time period after dosing for the respective animals.

General reproduction data:
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded.
Palpation was used to aid in confirmation of pregnancy. Pregnant females were examined to detect signs of difficult or prolonged parturation, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation of feeding) were examined.

Pups:
Each litter was examined to determine the following, if practically possible:
Mortality/viability:
The numbers of live and dead pups on day 1 of lactation and daily thereafter were determined.
Clinical signs:
At least once daily, detailed clinical observations were made for all animals.
Body weights:
Live pups were weighed on days 1 and 4 of lactation.
Sex:
Sex was determined for all pups on days 1 and 4 of lactation

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight, semincal vesicles including coagulating glands
spermatogenesis staging profiles

Litter observations:

Each litter was examined to determine the following, if practically possible:
Mortality/viability:
The numbers of live and dead pups on day 1 of lactation and daily thereafter were determined.
Clinical signs:
At least once daily, detailed clinical observations were made for al animals.
Body weights:
Live pups were weighed on days 1 and 4 of lactation.
Sex:
Sex was determined for all pups on days 1 and 4 of lactation.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
All males and the selected 5 females/group were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food.
Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetized using isoflurane and subsequently exsanguinated.
Necropsy was conducted on the following days:
Females which deliver: lactation days 5-7
Females which fail to deliver: post-coitum days 26-27 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating)
Females with total litter loss: within 24 hours of litter loss
Males: following completion of mating period (a minimum of 28 days of dose administration)
Euthanized in extremis: when pain, distress or discomfort was considered not transient in nature or is likely to become more severe

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Description of all macroscopic abnormalities were recorded.
The number of former implantation sites and corpora lutea were recorded for all paired females. These numbers were not reported for non-pregnant and non-mated females.
Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution)
Adrenal glands, aorta, brain-cerebellum, mid-brain, cortex, caecum, cervix, clitoral gland, colon, coagulation gland, duodenum, epididymides, eyes (with optic nerve (if detectable) and Harderian gland), female mammary gland area, femur including joint, heart, ileum, jejunum, kidneys, ovaries, (pancreas), peyer's patches (jejunum, ileum) if detectable, pituitary gland, preputial gland, prostate gland, rectum, salvary glands - mandibular, sublingual), sciatic nerve, seminal vesicles, skeletal muscle, (skin), spinal cord - cervical, midthoracic, lumbar, spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid if detectable, (lacrimal gland, exorbital), (larynx), liver, lung infused with formalin, lymph nodes - mandibular, mesenteric, (nasopharynx), (esophagus), (tongue), trachea, urinary bladder, uterus, vagnia, all gross lesions.
Tissues/organs mentioned in paraentheses need only to be examined by the pathologist if changes in macroscopic appearance are indicative of (potential) toxicity.
All remaining females, females which fail to deliver and females with total litter loss: cervix, clitoral gland, coagulation gland, epididymides, mammary gland area, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, vagina, all gross lesions

Necropsy pups:
Pups surviving to planned termination were killed by decapitation between days 5-7 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy data were examined for the presence of milk, if possible.

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of groups 1 and 4
- The additional slides of the testes of the selected 5 males of groups 1 and 4 and all males suspected to be infertile or which died before mating to examine staging of spermatogenesis.
- The preserved organs and tissues of the animals of all dose groups which died spontaneously or were killed in extremis
- All gross lesions of all animals (all dose groups).
- The reproductive organs (including cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina) of alle males that failed to sire and all females that failed to deliver healthy pups
All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.
Of the selected 5 males of the control and high dose group and all males suspected to be infertile or which died before mating, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin.

Organ weights:
Terminal body weight was recorded from all males and selected 5 females/sex/group.
The following organ weights were recorded from the following animals on the scheduled day of necropsy:
Selected 5 animals/sex/group: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus (including cervix), prostate, seminal vesicles incudling coagulating gland, thyroid including parathyroid.
All remaining males:
epidididymides
testes

Postmortem examinations (offspring):

Necropsy pups:
Pups surviving to planned termination were killed by decapitation between days 5-7 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy data were examined for the presence of milk, if possible.

Statistics:

The following statistical methods were used to analyse data:
- If the variables were assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate were applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one-rank test) were 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. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 were accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores). Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations were rounded off before printing. Therefore, two groups might have displayed the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

Mating index (%): (Number of females mated/number of females paired) x 100
Fertility index (%): (Number of pregnant females/number of females paired) x 100
Concenption index (%): (Number of pregnant females/number of females mated) x 100
Gestation index (%): (Number of females bearing live pups/number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Percentage live males at first litter check: (Number of live male pups at first litter check/number of live pups at first litter check) x 100
Percentage live females at first litter check: (Number of live female pups at first litter check/number of live pups at first litter check) x 100
Percentage of postnatal loss: (Number of dead pups before planned necropsy/number of live pups at first litter check) x 100
Viability index: (Number of live pups before planned necropsy/number of pups born alive) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
At 1000 mg/kg, hunched posture, piloerection, rales and/or lean appearance were recorded for the females euthanized in extremis (nos. 75 and 79) in the days preceding their death. Piloerection was also noted incidentally for four of the surviving females at 1000 mg/kg between Day 20 post-coitum and lactation Day 4, and hunched posture was transiently observed for a single surviving female at this dose level. Salivation was noted for all animals at 1000 mg/kg (from Day 9 of the premating period onwards) and for a few males at 300 mg/kg. This was considered to be a physiological response rather than a sign
of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to the taste or possible irritancy of the test substance. For one male at 300 mg/kg a swelling (nodule) was noted on the chest from Day 2 of treatment onwards which increased in diameter from 5 mm to 31 mm (correlating to a carcinoma; see also section 7.2.8). It was not considered test substance related. Based on the observations performed, there was no sign for that this nodule impaired the normal functioning of this male. Other incidental findings noted included alopecia and scales. These findings 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. At the incidence observed, these were considered signs of no toxicological significance.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Reduced body weights and body weight gains were recorded for males at 300 and 1000 mg/kg during the entire treatment period (not always statistically significant). Mean values for body weight and body weight gain were up to 8% and 67%, respectively, lower as compared to the concurrent control. For females at 1000 mg/kg, a trend towards slightly lower body weights and body weight gains from Day 14 of the post coitum period was noted. Since differences from controls were only slight and not statistically significant, this finding was not considered as toxicologically relevant.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
There were no toxicologically relevant effects on food consumption. An apparent dose related trend towards lower food intake was recorded for males during the first days of treatment. As the differences from controls were only slight and changes were transient, they were not considered to be toxicologically relevant.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Not examined

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Male no. 40, who failed to sire, showed absence of sperm (aspermia) in the epididymides and absence of all sperm stages in the testes without additional degenerative changes (microscopic correlates of reduced in size noted at necropsy for both organs). Therefore, the testes of this rat were considered to be immature. At this single incidence, it was regarded not treatment related.
For the remaining pairs who failed to sire or deliver healthy pups, no abnormalities were seen in the reproductive organs which could account for their infertility.
Further, the spermatogenic staging profiles were normal for all examined males (except for no. 40 who failed to sire).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No toxicologically relevant effects on reproductive parameters were noted. The mating, fertility and conception indices, precoital time, and number of corporal lutea and implantation sites were unaffected by treatment.

One female at 1000 mg/kg mated during the second pairing period, since the first male (no. 40) she was mated with appeared to be infertile.

For female no. 63 (300 mg/kg), the number of pups (9) was slightly higher than the number of implantations (8). This was considered to be caused by normal resorption of these areas as these enumerations were performed on day 5 of lactation.

One female each at 100 and 1000 mg/kg did not mate; in addition, one female each in the control, 100 and 1000 mg/kg group was not pregnant. Two females at 1000 mg/kg were euthanized in extremis during the post-coitum period (one of them did not mate). As such, there were 9, 8, 10 and 7 litters available for examination in the control, 100, 300 and 1000 mg/kg groups, respectively.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The following (statistically significant) changes in organ weights were noted:
− Higher liver weight for females at 300 mg/kg (absolute; 113% of control) and 1000 mg/kg (absolute and relative; 116% of control).
− Higher liver weight for males at 1000 mg/kg (relative to body weight only; 110% of control).
− Lower thymus weight (absolute: 66% of control and relative: 74% of control) for males at 1000 mg/kg.
− Higher adrenal weight for females at 1000 mg/kg (absolute: 122% of control and relative: 125% of control).
− Higher spleen weight for females at 1000 mg/kg (absolute: 127% of control and relative: 129% of control).
Any other statistically significant changes were considered to be of no toxicological significance as they were due to the lower body weight noted for males, occurred in the absence of a treatment related distribution, remained within the range considered normal for rats of this age and strain and/or no microscopic correlate was found. These findings included a higher relative brain weight and lower absolute weight of seminal vesicles for males at 1000 mg/kg, and higher absolute thymus weight for females at 300 mg/kg.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were treatment-related macroscopic findings in female rats treated at 1000 mg/kg consisting of irregular forestomach epithelium in 2/10 animals and thickened wall of the small intestines in 3/10 animals. A yellowish reddish soft nodule (size 31x26 mm) was noted in the skin/subcutis of the chest region of a male treated at 300 mg/kg. The finding consisted of and was microscopically correlated to a carcinoma of the subcutis. Because of the low incidence, early occurrence and absence of proliferative lesions in the skin/subcutis of other treated rats in this study, this was considered to be an incidental finding, unrelated to treatment. All remaining findings noted for both treated and control animals, occurred at an incidence that was within background levels and/or in the absence of any dose-related distribution. Therefore, they were not considered to be toxicologically relevant.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Adverse histopathological alterations were present in the mesenterial lymph nodes of males and females treated at 300 and 1000 mg/kg, and consisted of foamy macrophage foci and granulomatous inflammation (with or without necrosis). These foamy macrophage foci were considered to be related to intestinal uptake of the test article starting in the villi of the small intestines (recorded as foamy macrophage in the villi of the small intestines in rats treated at 300 mg/kg and 1000 mg/kg; macroscopic correlate: thickened wall of the small intestine) and from there transported to the mesenteric lymph node. When taking up more material at higher doses (300 and 1000 mg/kg), the foci in the mesenteric lymph node increased in incidence and/or severity. Additionally, development into granulomas (sometimes with central necrosis, up to marked degree) were recorded in rats treated at 300 and 1000 mg/kg. The presence of foamy macrophage foci up to slight degree in the mesenteric lymph node in the majority of rats treated at 100 mg/kg without development of granulomas was considered non-adverse in nature. Other treatment-related morphologic findings were recorded in the lung (alveolar macrophages) of males and females at 300 and 1000 mg/kg, liver (hepatocellular centrilobular hypertrophy; corresponding increased organ weight) of males and females at 1000 mg/kg, spleen (decrease in hematopoietic foci) of males at 1000 mg/kg, and adrenals (hypertrophy of the zona fasciculate with corresponding increased adrenal weights) of females at 1000 mg/kg. Also local effects on the forestomach (diffuse hyperplasia of the squamous epithelium; correlating macroscopic finding: irregular epithelium) were noted for males and females at 1000 mg/kg. Additional microscopic findings in one female (no. 79) at 1000 mg/kg killed in extremis on Day 8 of the mating period consisted of moderate lymphoid atrophy in the thymus and moderate bone marrow atrophy.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive NOAEL

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
One pup at 100 mg/kg went missing on day 2 of the lactation period. It was most likely cannibalized. No abnormalities were noted for this pup at first litter check. At the single incidence no toxicological relevance was ascribed to this missing pup.

CLINICAL SIGNS (OFFSPRING)
The only clinical sign seen was endorotation of the leg, which was noted for a single control pup. This was a spontaneous abnormality.

BODY WEIGHT (OFFSPRING)
Body weights of pups were unaffected by treatment

GROSS PATHOLOGY (OFFSPRING)
Endorotation of the leg was the only macroscopic finding seen, which was noted for a single control pup. This was a spontaneous findings.

HISTOPATHOLOGY (OFFSPRING)
The change in the mean sex ration in group 4 was only slight, reaching no statistical significance when comparing to the controls. The laboratory's control data (122 screening studies in the period from 2008 until now) includes two studies with a percentage of 63 -64% for male pups. As these two historical studies show the ultimate limit of the historical data range, a possible relationship to treatment could not be excluded for the tendency to a changed sex ratio in the high dose group. It should be noted that in this type of study the sex of the pups is determined externally by the anogenital distance and sex organs are not examined.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

List of deviations:

1. Females 58 and 59 were interchanged at start of mating. Consequently, the clinical signs, body weights and food consumption data and the mating date recorded for animal no. 58 during the mating period actually belongs to female 59, and vice versa. Upon discovery of the interchange, females 58 and 59 were reallocated as 81 and 82, respectively. All subsequent data were collected as animals 81 and 82. Additionally, these animals were not re-tattooed with the new animal number, though they had their original permanent identification.

Evaluation: Since both animals were within the same treatment group, summary data were unaffected by this interchange. The animals still had their original tattoo and the new numbers were clearly documented on the cage cards.

2. Animal no. 55 (300 mg/kg) was not weighed in the tox program as scheduled on 03-Jan-2013.

She was weighed a few days later, on 07-Jan-2013. Evaluation: The dosing volume (1.3 mL) remained the same for this animal, so there was no impact on dosing and the few days difference between body weight collection has no impact.

3. No body weight was collected for female no. 44 on lactation Day 4 and no food consumption data are available from lactation Days 1-4.

Evaluation: Sufficient data is available for a thorough evaluation.

4 On Day 4 no observations were entered online for pup 2 from litter 44 (control), and no observations were entered online on Day 6 for all pups from litter 66 (300 mg/kg).

Evaluation: Sufficient data is available for a thorough evaluation, and no clinical signs were

noted for pups in the treated groups during the study.

5. The organs and tissues from animal no. 52 were fixed in Modified Davidson’s solution instead of formalin.

Evaluation: Only a skin abnormality was required for microscopic examination; this was unaffected by the difference in fixatives.

The study integrity was not adversely affected by the deviations.

Applicant's summary and conclusion

Conclusions:

No reproductive toxicity was observed for treatment up to 1000 mg/kg. A change (slight and reaching not statistical significance compared to controls) was observed in the sex ratio at 300 mg/kg. No other developmental parameters were affected.

As a possible treatment related effect in the sex ratio at 1000 mg/kg could not be excluded based on the limited data available from this screening study, the following NOAELs values were derived:
Reproductive NOAEL: at least 1000 mg/kg
Developmental NOAEL: 300 mg/kg