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Toxicological information

Toxicity to reproduction

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Endpoint:
two-generation reproductive toxicity
Remarks:
two-generation reproduction study combined with 90-day toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2016 to March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
sub-chronic toxicity: oral
Remarks:
combined with a 2-generation reproduction study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2016 to March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
Sept 1998
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
Aug 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD 416, Two-Generation Reproduction Toxicity Study
Version / remarks:
Jan 2001
Deviations:
no
Qualifier:
according to
Guideline:
other: US EPA OPPTS 870.3800, Reproduction and Fertility Effects
Version / remarks:
Aug 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: EC No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.35: "Two-generation Reproduction Toxicity Test". Official Journal of the European Union No. L142
Version / remarks:
May 2008
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for reproduction studies. Charles River Den Bosch has reproductive historical control data in this species from the
same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5-6 weeks old
- Weight at study initiation: 128-161 g for males and 112-139 g for females
- Fasting period before study: no
- Housing:
Pre-mating and Post-weaning: Animals were housed in groups with a maximum of 4 animals/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 4 animals/cage. Since male nos. 81 and 84 were sacrificed in extremis, two males (nos. 85 and 86) of the subsequent cage were selected for blood sampling and were housed separately overnight from 16- 17 October 2016 (Macrolon plastic cage, MIV type, height 18 cm). As a consequence, the other two cage mates (nos. 87 and 88) were housed separately in their home cage with access to food and drinking water to prevent fasting these animals twice. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until one day preceding termination of the dam or until weaning (PND 21) in Macrolon plastic cages (MIII type, height 18 cm).
General Sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
During activity monitoring, animals were housed individually in Macrolon plastic cages (MIII type; height 15 cm) with sterilised sawdust as bedding material.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food for a maximum of 2 hours. The same diets remained in the food hopper for a maximum of 14 days
- Water (e.g. ad libitum): Free access to tap-water. During motor activity measurements, animals did not have access to water for a maximum of 2 hours.
- Acclimation period: at least 5 days prior to treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: July 2016 To: Nov 2016 for F0 and Oct 2016 to March 2017 for F1
Route of administration:
oral: feed
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 substance.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared freshly for use at room temperature for a maximum of 8 days. Following confirmation of stability over 14 days at room temperature under project 512795, diets were prepared freshly for use at room temperature for a maximum of 14 days.
- Mixing appropriate amounts with standard powder rodent diet: The test item was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Water (approximately 15% in total) was added to aid pelleting. The pellets were dried for approximately 24 hours at 35°C before storage. The control animals received similarly prepared pellets but without the test substance.
- Storage temperature of food: Diets were kept in the freezer (≤-15°C) until use, if not used on the day of preparation. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 14 days.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
see attached background material
Duration of treatment / exposure:
Animals received test diet up to the day prior to necropsy.
F0-generation:
Males were exposed for 91 or 92 days, i.e. 10 weeks prior to mating, during mating, and up to the day prior to scheduled sacrifice. F0 females of Groups 1-4 that delivered were exposed for 115-121 days (most females) or 127-128 days (one or two females of Groups 1-3), i.e. during 10 weeks prior to mating, during mating, during post-coitum, and during at least 21 days of lactation (up to the day prior to scheduled necropsy). F0 females which failed to deliver healthy offspring were treated for 99 days (non-pregnant females of Groups 1-3) or 94 days (all 20 non-pregnant females of Group 4).
F1-generation (F0-pups):
The F1-generation could potentially have been exposed to the test item in utero, via maternal milk, from exposure to maternal urine/faeces or via spilled diet from the food hopper, and directly when pups begin eating solid food.After weaning, pups were treated for 77 days prior to mating and continuing until euthanasia; F1 animals received test diet for a total of 113 or 114 days for males and 120-126 days for females.
Frequency of treatment:
continuously, ad libitum
Dose / conc.:
1 500 ppm
Remarks:
F0 group 2, days 1-35
Dose / conc.:
5 000 ppm
Remarks:
F0 group 3, day 1-35
Dose / conc.:
15 000 ppm
Remarks:
F0 group 4, day 1-35
Dose / conc.:
1 000 ppm
Remarks:
Group 2: F0 day 36 - end and F1 parents
Dose / conc.:
3 000 ppm
Remarks:
Group 3: F0 day 36 - end and F1 parents
Dose / conc.:
10 000 ppm
Remarks:
Group 4 F0 day 36 - end
No. of animals per sex per dose:
24
Control animals:
yes, plain diet
Details on study design:
Dose selection rationale:
Initial dose levels as used until Day 35 were based on results of the 14-d dose range finding study (Test Facility Study No. 512946). In this DRF study, 3 females were given 15000 ppm in the diet for 14 days (test item intake 1292 mg/kg bw/d). No mortality, no clinical signs, normal food consumption, no macroscopic abnormalities and normal liver and kidney weight were noted. A slightly lower weight gain for 1/3 females was noted.
From Day 36 of treatment onwards, dose levels in all test item treated groups were lowered in agreement with the Sponsor due to the reduced bodyweight gain of animals in Groups 3 and 4.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to first administration, and at least once daily from start of administration onwards. For all F0 parental animals this was also performed outside the home cage in a standard arena once prior to start of administration and at weekly intervals during the treatment phase .

BODY WEIGHT: Yes
- Time schedule for examinations: 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, 4, 7, 14 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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 during lactation on Days 1, 4, 7, 14 and 21.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

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: Yes
- Time schedule for examinations: at pre-test: First 12 F0 males of each group and all females (including spare animals) Week 13: First 12 F0 males of Groups 1 and 4. First 10 F0 non-pregnant Group 4 females. During lactation: Selected 10 F0 females of Groups 1 and 3.

Blood samples collected for haematology and clinical biochemistry using isoflurane as anaesthetic:
Week 7 (premating) - 10 F0 high dose males and 10 F0 control males3 (these selected animals were not fasted overnight). Samples were collected in support of the severely lower body weight gains in the high dose group (15,000/10,000 ppm).
Week 13 (end of treatment): first 12 F0 males per group and first 10 F0 non-pregnant Group 4 females (collected from the retro-orbital sinus as part of the necropsy procedure).
End of lactation (end of treatment): - selected 10 F0 Group 1-3 females.

HAEMATOLOGY: Yes
- Schedule:
Week 7 (premating) - 10 F0 high dose males and 10 F0 control males (these selected animals were not fasted overnight). Samples were collected in support of the severely lower body weight gains in the high dose group (15,000/10,000 ppm).
Week 13
(end of treatment): - first 12 F0 males per group.
- first 10 F0 non-pregnant Group 4 females (collected from the retro-orbital sinus as part of the necropsy procedure).
End of lactation
(end of treatment): - selected 10 F0 Group 1-3 females.

All animals sampled at the end of the treatment period were fasted overnight, but water was available.
- How many animals: 10-12/sex/group
- Parameters according to OECD 408 were examined.

CLINICAL CHEMISTRY: Yes
- Schedule: see Haematology
- How many animals: 10-12/sex/group
- Parameters according to OECD 408 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 12-13 of treatment: First 12 F0 parental males/group and first 10 F0 non-pregnant Group 4 females, and last week of lactation: First 10 F0 selected Groups 1-3 females.
- Battery of functions tested: hearing ability, pupillary reflex and static righting reflex, grip strength, locomotor activity

OTHER: no other general toxicity parameters were included.
Sacrifice and pathology:
F1 females were not deprived of food overnight prior to scheduled necropsy.
All other animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy.

Necropsy was conducted on the following days:
Condition Day of necropsy
Parental Males : After at least 90 days of administration of F0-parental males and as soon as possible after delivery of litters for F1-parental males.
Females which delivered: F0: Lactation Day 22-24 (i.e. one day after necropsy of pups as dams needed to be fasted for blood sampling).
F1: Lactation Day 21-23.
Females which failed to deliver: Post-coitum Day 25-27 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).
Euthanized in extremis: When pain, distress or discomfort is considered not transient in nature or is likely to become more severe.
All non-pregnant F0 females of Group 4: After 94 days of administration (20 October 2016)

GROSS PATHOLOGY: Yes, for the first 12 F0 males of all groups, the first 10 F0 non-pregnant Group 4 females, the selected 10 Group 1-3 F0 generation females, and all animals that were killed in extremis; organs and tissue according to OECD 408. For all other F0 animals and all F1 animals: adrenal glands, brain (cerebellum, mid-brain and cortex), cerivx, clitoral gland, coagulation gland, epididymidis, female mammary gland area, heart, kidneys, liver, ovaries, pituitary gland, preputial gland, seminal vesicles, spleen, testis, thymus, thyroid incl parathyroid, uterus, vagina, mesenteric lymph nodes, all gross lesions.
HISTOPATHOLOGY: Yes, according to OECD 408, i.e. preserved organs and tissues of all group 1 F0 and F1 males and females, the selected 10 F0 females of Group 3, all group 3 F1 males and females, and all group 4 F0 males and females, all gross lesions of all dose groups and preserved organs and tissues from animals killed in extremis.

Organ weights:
The following organ weights and terminal body weight were recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands Prostate
Brain Seminal vesicles with coagulating glands
Epididymides (total and cauda separately) Spleen
Heart Thyroid including parathyroid
Kidneys Testes
Liver Uterus (including cervix)
Ovaries Thymus
Pituitary gland (after fixation)
Other examinations:
See 7.8.1 2-generation reproduction study part.
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.
-Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup difference.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related clinical signs of toxicity were noted at 10,000 ppm in both sexes and, to a lesser extent, at 3000 ppm in females.
Findings at 10,000 ppm in surviving animals primarily included hunched posture, piloerection and a pale appearance in up to most or all animals, and a lean appearance in up to about one third of the animals. Additional findings in 10,000 ppm females consisted of chromodacryorrhoea in about one third of the females, lethargy in four females, and abnormal gait and swelling of the legs in two females. These clinical signs were mostly noted during the last few weeks of the treatment period, except for abnormal gait and swelling of the skin which were noted only on the last few days of the study.
Treatment-related findings in surviving females at 3000 ppm consisted of piloerection and hunched posture in several females during the last few weeks of the treatment period, and a lean appearance, abnormal gait and swelling of the legs in a single female on the last few days of the study.
No additional clinical signs of toxicity were noted during the weekly arena observations.
Any other clinical signs noted incidentally 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, showed no dose-related trend and/or incidences were similar to those encountered in controls. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two males at 10,000 ppm and one female at 3000 ppm were sacrificed in extremis, which was considered to be related to treatment.
The moribundity of both males (sacrificed at Days 58 and 65, respectively) and of the female (sacrificed at Day 102) was considered to be treatment-related. At the day of sacrifice (or one or more days before), these animals showed clinical signs including but not limited to hunched posture, abnormal gait, piloerection, a lean appearance (one male), ptosis (one male) and/or swelling of the skin of the hindlegs (female). In the week prior to sacrifice, both males lost weight. Major gross lesions consisted of enlarged mesenteric lymph nodes and/or spleen, many nodules/foci of the mesenteric lymph nodes and/or spleen and liver, and/or thickened skin of hindleg(s). Main correlating microscopic findings consisted of necrotizing granuloma(s) of the mesenteric lymph node and/or spleen and liver, extranodal inflammation of the mesenteric lymph node and arthritis of the hindleg(s). These pathology findings were considered to be related with the moribundity of the animals. As similar clinical signs of toxicity, gross lesions and microscopic changes were noted in surviving animals at 3,000 and 10,000 ppm, the moribundity of both males and the female was considered to be related to treatment with the test item.
The moribundity of another female at 3000 ppm (sacrificed at Day 43) was not considered related to treatment with the test item. At the day of sacrifice, she showed tremors, piloerection, abdominal swelling, squeaking and hypothermia. Her body weight development was normal. The cause of her moribundity was a yolk sac carcinoma, a malignant tumor with many metastasis in many organs, correlating with the nodules in many abdominal organs and tissues observed at necropsy. Based on the single occurrence and character of the tumor, this condition was considered to be spontaneous.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights were dose-dependently reduced at 3000 and 10,000 ppm in both sexes throughout the treatment period (statistically significant on most occasions). In males, the differences from controls gradually increased to about 17% (3,000 ppm) and 40% (10,000 ppm) at the end of the treatment period. Mean body weights of females were reduced by about 10% (3000 ppm) or 25% (10,000 ppm) at the end of the pre-mating period and about 15% (3000 ppm) or 30% (10,000 ppm) at the end of the post-coitum period. In the course of the lactation period, the differences from controls remained approximately the same (approximately 9%) at 3000 ppm and decreased to about 16% at 10,000 ppm.
Body weight gain was dose-dependently reduced at 3000 and 10,000 ppm in both sexes throughout the pre-mating period. Body weight gain in males remained reduced at 3,000 and 10,000 ppm until the end of the study. Body weight gain of pregnant females was reduced only at 10,000 ppm during the last week of the gestation period. Higher body weight gain was noted during the second week (10,000 ppm) and third week (3000 and 10,000 ppm) of the lactation period. These variations achieved a level of statistical significance on most occasions.
Note: At 10,000 ppm, evaluation of body weight development during the gestation and lactation periods was based on only four females. The remaining 20 females of this dose group were not pregnant.
Body weight and body weight gain at 1000 ppm were not affected by treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before allowance for body weight was reduced (generally statistically significantly) at 10,000 ppm in both sexes during the pre-mating period, and in females during the post-coitum period. No remarkable differences were noted during the lactation period. Food consumption after allowance for body weight (relative food consumption) at this dose was on most occasions higher than controls (generally statistically significant) throughout the treatment period in males (but not always clearly dose-dependently during the pre-mating period). For females at this dose, relative food consumption was higher during the lactation period and more occasionally during the post-coitum period (not always statistically significant), and was generally similar to that of controls during the pre-mating period.
Relative food consumption was also higher (generally statistically significantly) throughout the treatment period in males and females at 3000 ppm (not clearly dose-dependently during the pre-mating period), which was ascribed to the lower body weights at this dose.
Any other statistically significant differences in absolute or relative food consumption were minor and/or occurred in the absence of a clear dose-related trend and were therefore not considered to be related to treatment.
Note: At 10,000 ppm, evaluation of food consumption during the gestation and lactation periods was based on only four females. The remaining 20 females of this dose group were not pregnant.

The mean daily intake of the test item per kg body weight during the different phases of the study is given in the table below.
Test item intake (mg test item/kg bw/day)(1)

Group 2 Group 3 Group 4
1000 ppm(2) 3000 ppm(2) 10,000 ppm(2)
Males
Pre-mating 96 326 1025
Mating 60 201 953
Mean of means(3) 88 299 1010

Females
Pre-mating 104 360 996
Post-coitum 76 252 836
Lactation 73 253 890
Mean of means(3) 90 309 930

1 Values are the overall group means in the periods indicated.
2 These dietary concentrations were used from Day 36 of the study (lower concentrations were used during the lactation period, see section 5.5). Before Day 36, higher concentrations were used: 1,500 (Group 2), 5,000 ppm (Group 3) and 15,000 ppm (Group 4).
3 Mean of means of all periods, weighed for number of measurement intervals per period:
Males: ((11 x mean premating) + (3 x mean mating)) / 14
Females: ((11 x mean premating) + (6 x mean post-coitum) + (4 x mean lactation)) / 21
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
At 10,000 ppm, mean food conversion efficiency (g body weight gained per g food consumed) of males was reduced with statistical significance during most of the pre-mating period. For females at this dose, mean food conversion efficiency was reduced during the initial 4 weeks of the premating period. Females at 10,000 ppm also showed lower mean food conversion efficiency on several occasions during the post-coitum period.
At 3000 ppm, mean food conversion efficiency was reduced for males over Days 1-36 of the premating period and during a single occasion during the mating period, and for females only over Days 1-8 and 15-22 of the premating period.
At 1000 ppm, mean food conversion efficiency was only reduced during the first week of treatment for males.
Overall, mean over mean food conversion efficiency during the premating phase (males and females) and post-coitum phase showed an apparent downward trend over the dose groups. During lactation however, there was no apparent dose-related trend in mean over mean food conversion efficiency. It should be noted that the concentration in the test diets was lowered during lactation.
The statistically significantly lower mean food conversion efficiency of males at 1000, 3000 and 10,000 ppm over Days 8-15 of the mating period was not considered to be related to treatment since this difference occurred in the absence of a dose related trend and was not consistently noted with continuing treatment.
The higher mean food conversion efficiency of females at 10,000 ppm over Days 14-21 of lactation was attributed as secondary to the lower mean litter size.
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):
Note: The 10,000 ppm females selected for haematology were non-pregnant (selected females of the other groups were lactating) and they were treated 3-4 weeks shorter compared to females of the other groups. When assessing the possible relation with treatment of differences in haematology values between 10,000 ppm females and concurrent controls, it was taken into consideration that physiological status may influence haematology parameters (Ref 1, Ref 2).
Ref. 1 Urasoko Y., He X.J., Masao T., Kinoshita Y., Edamoto H., Hatayama K., Asano Y., Tamura K, Mochizuki M.. Changes in blood parameters and the expression of coagulation-related genes in lactating Sprague-Dawley rats. Journal of the American Association of Laboratory Animal Science 51, 144-149 (2012).
Ref. 2 Menke A., Wolberbeek A., Snel C., Bruijntjes J., Groot D. de, Oostrum L. van, Waalkens I., Kuper C.F.. Potentially increased sensitivity of pregnant and lactating female rats to immunotoxic agents. Toxicologic Pathology 40, 255-260 (2012).

The following (mostly statistically significant) changes in haematology parameters distinguished treated animals from control animals:
• Higher total white blood cell counts (WBC) at 3000 and 10,000 ppm in both sexes. Values in 10,000 ppm females were higher (not statistically significantly) relative to concurrent (lactating) and historical (nulliparous) controls.
• Higher percentage of neutrophils and lower percentage of lymphocytes at 3,000 and 10,000 ppm in males and from 1000 ppm onwards in females. Values in 10,000 ppm females differed from concurrent (lactating) controls (neutrophils not statistically significantly) and historical (nulliparous) controls.
• Lower percentage of eosinophils at 1000 and 3000 ppm in females.
• Higher percentage of reticulocytes in females at 3000 and 10,000 ppm. Values in 10,000 ppm females were higher relative to concurrent (lactating) and historical (nulliparous) controls.
• Lower haemoglobin concentration and haematocrit at 10,000 ppm in both sexes. Values for haemoglobin in 10,000 ppm females were lower relative to concurrent (lactating) and historical (nulliparous) controls, values for haematocrit were lower only relative to concurrent controls.
• Lower mean corpuscular volume (MCV) in males at 3000 and 10,000 ppm, and in females at 10,000 ppm (relative to concurrent (lactating) and historical (nulliparous) controls).
• Lower mean corpuscular haemoglobin (MCH) in males from 1000 ppm onwards, and in females at 10,000 ppm (relative to concurrent (lactating) and historical (nulliparous) controls).
• Higher platelets at 10,000 ppm in both sexes. Values in 10,000 ppm females were higher relative to concurrent (lactating) and historical (nulliparous) controls.
• Lower activated partial thromboplastin time (APTT) at 10,000 ppm in males.

The lower percentage of monocytes (statistically significant) noted in 10,000 ppm females (compared to concurrent controls) was considered to be due to the difference in physiological status. Monocyte values in 10,000 ppm females were in the normal range for nulliparous control females.
Other statistically significant variations noted in haematology parameters at the end of the treatment period were considered unrelated to treatment as they occurred in the absence of a dose-related trend.
Haematology conducted in Week 7 of the pre-mating period in males of the control and 10,000 ppm groups showed similar changes in haematology parameters as noted at the end of the treatment period, with the following exceptions:
• Higher percentage of monocytes in Week 7 (no change at the end of treatment).
• Higher number of red blood cells (RBC) in Week 7 (no change at the end of treatment).
• Higher haemoglobin and haematocrit in Week 7 (lower at the end of treatment).
• Higher prothrombin time (PT) in Week 7 (no change at the end of treatment).
• No change in APTT in Week 7 (lower at the end of treatment).

Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Note: The 10,000 ppm females selected for clinical biochemistry were non-pregnant (selected females of the other groups were lactating) and they were treated 3-4 weeks shorter compared to females of the other groups. When assessing the possible relation with treatment of differences in clinical biochemistry values between 10,000 ppm females and concurrent controls, it was taken into consideration that physiological status may influence clinical biochemistry parameters (Ref. 1).

Ref. 1 Urasoko Y., He X.J., Masao T., Kinoshita Y., Edamoto H., Hatayama K., Asano Y., Tamura K, Mochizuki M.. Changes in blood parameters and the expression of coagulation-related genes in lactating Sprague-Dawley rats. Journal of the American Association of Laboratory Animal Science 51, 144-149 (2012).

The following (mostly statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
• Higher activity of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) at 3000 and 10,000 ppm in males.
• Lower (not statistically significant) alkaline phosphatase activity (ALP) at 10,000 ppm in both sexes. Values in 10,000 ppm females were in the normal range for nulliparous control females.
• Lower total protein at 10,000 ppm in males and females (not statistically significant for females; the mean was lower relative to concurrent (lactating) and historical (nulliparous) controls).
• Lower albumin at 3000 ppm in males and at 3000 and 10,000 ppm in males and females (not statistically significant for females; the mean was lower relative to concurrent (lactating) and historical (nulliparous) controls).
• Higher urea at 3000 ppm in females.
• Lower glucose at 10,000 ppm in males.
• Lower total cholesterol at 10,000 ppm in both sexes. Although values in 10,000 ppm females were normal compared to historical (nulliparous) controls, it cannot be ruled out that the lower cholesterol in these females was related to treatment taken in the context of the treatment-related reduction of cholesterol in males.
• Higher potassium at 10,000 ppm in males.
The statistically significantly lower values for ALAT and urea, and the higher values for sodium and chloride noted in 10,000 ppm females (compared to concurrent controls) were considered to be due to the difference in physiological status. Values for these parameters in 10,000 ppm females were in the normal range for nulliparous control females.
Clinical chemistry conducted in Week 7 of the pre-mating period in males of the control and 10,000 ppm groups showed similar changes in clinical chemistry parameters as noted at the end of the treatment period, with the following exceptions:
• Higher urea and creatinine in Week 7 (no change at the end of treatment).
• Statistically significantly lower ALP (not statistically significant at the end of treatment).
• No statistically significant difference in potassium in Week 7 (higher at the end of treatment).
• No statistically significant difference in albumin in Week 7 (lower at the end of treatment).
• Lower inorganic phosphate in Week 7 (no change at the end of treatment).
Other statistically significant variations noted in clinical biochemistry parameters were considered unrelated to treatment or not toxicologically relevant due to the slight magnitude and/or direction of the differences.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
In males, statistically significantly lower fore- and hindlimb grip strength values were noted at all dose levels. The differences from controls showed a dose-related trend.
In females, the statistically significantly lower fore- and hindlimb grip strength noted at 10,000 ppm was ascribed to the difference in physiological status and time of testing of 10,000 ppm females and concurrent controls (10,000 ppm females: non-pregnant, tested in Week 12-13 of the treatment period; controls: lactating, tested towards the end of the lactation period after approximately 17 weeks of treatment). Compared to historical control results for female rats of this strain and age, grip strength values in females at 10,000 ppm were within normal limits.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Note: 20/24 females of the 10,000 ppm group were not pregnant and treated for three to about five weeks shorter than females of the other groups (most of which had offspring). These differences in physiological status and age at sacrifice were taken into account in the evaluation of the organ weight results.
The following test item-related changes in organ weights were observed (relative differences from controls are shown in the table below):
• Higher spleen weight (relative to body weight) at 3000 and 10,000 ppm in both sexes.
• Higher liver, kidney and adrenal gland weights (relative to body weight) at 3000 and 10,000 ppm in males.
• Lower prostate gland weight (absolute and relative to body weights) at 10,000 ppm in males.

Males Females
Dose level (ppm): 1000 3000 10000 1000 3000 10000

SPLEEN
Absolute 1 0 -12** 6 10 -8
Relative to body weight 5 19** 46** 5 18** 29**

LIVER Not applicable
Absolute -1 -9** -31**
Relative to body weight 4 10** 16**

KIDNEYS Not applicable
Absolute -1 -11** -36**
Relative to body weight 3 6** 8**

ADRENAL GLANDS Not applicable
Absolute 4 -2 -25**
Relative to body weight 8 23** 31**

PROSTATE GLAND Not applicable
Absolute -6 -14** -53**
Relative to body weight -1 2 -22**
*: P<0.05, **: P<0.01

Many statistically significant organ weight differences (mostly lower absolute organ weights) were considered to be due to the test item-related decrease in body weight, particularly in rats treated at 10,000 ppm which had on average 40% (males) or about 20% lower terminal body weights than concurrent controls (these females had about 23% lower terminal body weights compared to nulliparous historical control females of this strain and age).
Other organ weight changes were caused by lack of pregnancy (such as the higher relative thymus weights in females at 10,000 ppm) and shorter treatment periods, as was the case for most Group 4 females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At scheduled necropsy, test item-related macroscopic findings were observed in F0-males at 10,000 ppm and in F0-females at all dose levels, as listed below. The number of animals examined at scheduled sacrifice was 24/sex/group, except in the 10,000 group of males and the 3000 ppm group of females which included 22 survivors.
• Emaciation in 14 males and 10 females at 10,000 ppm.
• Prostate gland, seminal vesicle, and preputial gland: reduced in size at 10,000 ppm in 10, 5 and 3 males, respectively. There was no microscopic correlate.
• Lungs: grown together with pleura in one female at 10,000 ppm. The microscopic correlate was inflammation of the pleura with adhesions (including diaphragm).
• Pituitary gland: discoloration in 17, reduced in size in six, and gelatinous contents in one female at 10,000 ppm. There were no microscopic correlates.
• Liver: many gray-white foci in two females at 10,000 ppm. The microscopic correlate was necrotizing granulomas.
• Spleen: enlarged in three females at 10,000 ppm. The microscopic correlate was increased hematopoiesis (except for female no. 179, with no correlate).
• Mesenteric lymph node: enlarged in four females at 1000 ppm, 11 females at 3000 ppm and in 12 males and 13 females at 10,000 ppm; yellowish discoloration in three females at 10,000 ppm. Microscopic correlates were necrotizing granulomas, extranodal inflammation and/or foamy macrophages.
• Skin/subcutis of the hindleg(s): thickened/thickening in one female at 3000 ppm and in two females at 10,000 ppm, correlating with arthritis and edema.
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.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Due to the large number of tables for microscopy, details are presented under "Any other information on results incl. tables.
Histopathological findings: neoplastic:
no effects observed
Details on results:
See 7.8.1 two-generation reproduction study part for parameters such as sperm parameters and estrous cycle, and effects on F1 parents.
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Effect level:
79 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
72 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 ppm
System:
cardiovascular
Organ:
mesenteric lymph node
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
72 mg/kg bw/day (actual dose received)
System:
cardiovascular
Organ:
mesenteric lymph node
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Chemical analysis of diet preparations

Accuracy

During the Week 2 analysis, the concentrations analyzed in the diets of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%).

During the Week 8 analysis, the concentrations analyzed in the diets of Group 2, Group 3 and Group 4 the mean accuracy was above the target concentration (i.e. 171%, 142% and 141% of the target respectively).

During the Week 15 analysis of Group 2 (400, 667 and 1000 ppm). Group 3 (1200, 1500, 2000 and 3000 ppm) and Group 4 (10000 ppm), the concentrations analyzed in the diets were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%). For the diet of Group 2 (500 ppm), the mean accuracy was below the target concentration (i.e. 73% of the target). For the diet of Group 4 (4000 ppm), the mean accuracy was above the target concentration (i.e. 161% of the target). For the diet of Group 4 (5000 and 6667 ppm), the mean accuracy was above the target concentration (i.e. 76% and 70% of the target respectively).

During the Week 22 analysis, the concentrations analyzed in the diets of Group 2 were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%). For the diet of Group 3, the mean accuracy was above the target concentration (i.e. 130% of the target).

During the Week 31 analysis, the mean accuracy of the Group 2 samples was below the target concentration (i.e. 72% of the target). The mean accuracy of the Group 4 samples was above the target concentration (i.e. 144% of the target).

During the Week 34 analysis, the mean accuracies of the Group 2 and Group 3 samples were above the target concentration (i.e. 147% and 185% of the target respectively).

In the Group 1 diets, no test item was detected.

In summary, these data indicated that accuracy of preparation was within criteria on most occasions. Variability in quality control sample values related to the method employed may have accounted for secondary deviations in accuracy values. Overall, it was considered that these results indicated that accuracy of diet preparation was sufficient for the purpose of this study.

Homogeneity

The diets of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%) during the Week 2, Week 8 and Week 31 analysis. During the Week 15 analysis, Group 4 was homogeneous and the homogeneity of the Group 2 samples was slightly above the criterion (i.e. 11%). During the Week 22 analysis, Group 2 was homogeneous and the homogeneity of the Group 4 samples was above the criterion (i.e. 21%). During the Week 34 analysis, Group 4 was homogeneous and the homogeneity of the Group 2 samples was above the criterion (i.e. 20%).

In summary, these data indicated that homogeneity of preparation was within criteria on most occasions. Variability in quality control sample values related to the method employed may have accounted for secondary deviations in homogeneity values.

Overall, it was considered that these results indicated that homogeneity of diet preparation was sufficient for the purpose of this study.

Stability

Analysis of Group 2 and Group 4 diets after storage yielded a relative difference of ≥ 10%. These results have been caused by notable changes of the sensitivity of the MS detector in time, therefore are not seen as reliable.

Since in Test Facility Study No. 512795 the trial diets were stable for at least 8 days when stored at room temperature under normal laboratory light conditions and the relative difference after storage of 25 days in this study was slightly above ≥ 10% it was considered that diets were stable at room temperature under normal laboratory light conditions.

Histopathology P0

Note: 20/24 females of the 10,000 ppm group were not pregnant and treated for three to about five weeks shorter than females of the other groups (most of which had offspring).

Test item-related microscopic findings were noted in the small intestines, mesenteric lymph node, spleen, liver, adrenal glands, kidneys, lungs, thymus (males only), sternal bone marrow, ovaries and skin/subcutis of the hindleg. These changes are summarized and described in the following table.

Summary Test Item-Related Microscopic Findings F0– Small Intestines

 

Males

Females

Dose level (ppm):

0

1000

3000

10000

0

1000

3000

10000

 

 

 

 

 

 

 

 

 

DUODENUMa

12

12

12

14

10

10

13

10

   Foamy macrophages villi

 

 

 

 

 

 

 

 

      Minimal

-

-

-

11

-

-

-

6

      Slight

-

-

-

3

-

-

-

1

JEJUNUMa

12

12

12

14

12

10

13

11

   Foamy macrophages villi

 

 

 

 

 

 

 

 

      Minimal

-

-

3

-

-

-

2

1

      Slight

-

-

-

4

-

-

3

4

      Moderate

-

-

-

9

-

-

-

4

      Marked

-

-

-

1

-

-

-

2

ILEUMa

12

12

12

14

10

10

13

10

   Foamy macrophages villi

 

 

 

 

 

 

 

 

      Minimal

-

-

5

10

-

-

1

4

      Slight

-

-

-

3

-

-

1

-

      Moderate

-

-

-

-

-

-

-

1

a = Number of tissues examined from each group.

In the small intestines, the following changes were observed:

·    Foamy macrophages of the duodenum at 10,000 ppm in both sexes, up to slight degree.

·    Foamy macrophages of the jejunum from 3000 ppm onward in both sexes, up to marked degree.

·    Foamy macrophages of the ileum from 3000 ppm onward in both sexes, up to moderate degree.

Summary Test Item-Related Microscopic Findings F0– Mesenteric lymph node

 

Males

Females

Dose level (ppm):

0

1000

3000

10000

0

1000

3000

10000

 

 

 

 

 

 

 

 

 

MESENTERIC LYMPH NODEa

12

12

12

14

10

11

15

16

   Necrotizing granuloma(s)

 

 

 

 

 

 

 

 

      Minimal

-

3

2

-

-

-

1

1

      Slight

-

2

5

3

-

3

5

3

      Moderate

-

-

1

3

-

-

6

9

      Marked

-

-

-

2

-

-

-

1

   Extranodal inflammation

 

 

 

 

 

 

 

 

      Minimal

-

-

-

3

-

-

5

2

      Slight

-

-

1

-

-

1

2

3

      Moderate

-

-

-

2

-

-

-

4

   Foamy macrophages

 

 

 

 

 

 

 

 

      Minimal

-

3

-

1

-

4

1

1

      Slight

-

9

4

6

-

6

6

6

      Moderate

-

-

7

4

-

1

8

8

      Marked

-

-

1

3

-

-

-

1

   Macrophage foci

 

 

 

 

 

 

 

 

      Minimal

1

2

1

-

2

2

-

-

      Slight

-

1

-

-

-

7

1

-

      Moderate

-

1

-

-

-

1

-

-

   Lymphangectasia

 

 

 

 

 

 

 

 

      Slight

-

-

-

1

-

-

1

-

a = Number of tissues examined from each group.

In the mesenteric lymph node, the following changes were observed:

·    Necrotizing granuloma(s) (i.e. granuloma with central necrosis) from 1,000 ppm onward in both sexes, up to marked degree.

·    Extranodal inflammation (peritonitis) from 3000 ppm onward in both sexes from, up to moderate degree.

·    Foamy macrophages (foci and in sinusoids) from 1000 ppm onward in both sexes, up to marked degree.

·    Macrophage foci (not foamy) at increased incidence and severity (up to moderate) at 1000 ppm in both sexes.

·    Lymphangectasia at slight degree in one 3000 ppm female and one 10,000 ppm male.

Summary Test Item-Related Microscopic Findings F0– Kidneys

 

Males

Females

Dose level (ppm):

0

1000

3000

10000

0

1000

3000

10000

 

 

 

 

 

 

 

 

 

KIDNEYSa

12

12

12

14

10

10

12

11

   Vacuolation glomerular podocytes, foamy

 

 

 

 

 

 

 

 

      Minimal

-

-

-

1

-

-

-

-

      Slight

-

-

-

3

-

-

-

2

      Moderate

-

-

-

10

-

-

-

9

   Intranuclear inclusion bodies, tubular

 

 

 

 

 

 

 

 

      Minimal

-

-

-

7

-

-

-

2

      Slight

-

-

-

1

-

-

-

5

      Moderate

-

-

-

-

-

-

-

2

a = Number of tissues examined from each group.

In the kidneys, the following changes were observed:

·    Foamy vacuolation of glomerular podocytes at 10,000 ppm in both sexes, up to moderate degree.

·    Intranuclear inclusion bodies, tubular at 10,000 ppm in both sexes, up to moderate degree.

Summary Test Item-Related Microscopic Findings F0– Bone Marrow (sternum)

 

Males

Females

Dose level (ppm):

0

1000

3000

10000

0

1000

3000

10000

 

 

 

 

 

 

 

 

 

BONE MARROWa

12

12

12

14

10

10

12

10

   Increased myelopoiesis

 

 

 

 

 

 

 

 

      Minimal

-

6

11

6

2

2

9

3

      Slight

-

-

-

3

-

-

-

4

a = Number of tissues examined from each group.

In the bone marrow (sternal), the following change was observed:

·   Increased incidence of increased myelopoiesis in males starting at 1,000 ppm and in females starting at 3000 ppm, up to slight degree.

Summary Test Item-Related Microscopic Findings F0– Ovaries

 

Females

Dose level (ppm):

0

1000

3000

10000

 

 

 

 

 

OVARIESa

24

11

13

24

   Necrotizing granuloma(s)

 

 

 

 

      Slight

-

-

-

3

   Foamy cell aggregates

 

 

 

 

      Minimal

-

-

5

5

      Slight

-

-

4

4

a = Number of tissues examined from each group.

In the ovaries, the following changes were observed:

·    Necrotizing granuloma(s) (i.e. granuloma with central necrosis) at 10,000 ppm, at slight degree.

·    Foamy cell aggregates starting at 3000 ppm, up to a slight degree.

Summary Test Item-Related Microscopic Findings F0– Spleen and Liver

 

Males

Females

Dose level (ppm):

0

1000

3000

10000

0

1000

3000

10000

 

 

 

 

 

 

 

 

 

SPLEENa

12

12

12

14

10

11

12

11

   Necrotizing granuloma(s)

 

 

 

 

 

 

 

 

      Marked

-

-

-

2

-

-

-

-

   Increase in granulocytes

 

 

 

 

 

 

 

 

      Present

-

-

9

13

-

4

8

9

LIVERa

12

12

12

14

10

12

12

12

   Necrotizing granuloma(s)

 

 

 

 

 

 

 

 

      Minimal

-

-

-

1

-

-

-

1

      Slight

-

-

-

1

-

-

-

1

      Moderate

-

-

-

1

-

-

-

3

   Serosal inflammation

 

 

 

 

 

 

 

 

      Minimal

-

-

-

-

-

-

-

1

   Increased mitosis

 

 

 

 

 

 

 

 

      Slight

-

-

-

1

-

-

-

-

      Moderate

-

-

-

1

-

-

-

-

   Sinusoidal changes

 

 

 

 

 

 

 

 

      Present

-

11

12

14

-

3

9

12

a = Number of tissues examined from each group

In the spleen, the following changes were observed:

·    Necrotizing granuloma(s) (i.e. granuloma with central necrosis) at 10,000 ppm in males, at marked degree.

·    Increase in granulocytes in the vascular sinus in females from 1000 ppm onward and in males from 3000 ppm onward.

In the liver, the following changes were observed:

·    Necrotizing granuloma(s) (i.e. granuloma with central necrosis) at 10,000 ppm of both sexes, up to moderate degree.

·    Increased mitosis in two 10,000 ppm males, up to moderate degree.

·    Serosal inflammation in one 15000/10000 ppm female (minimal).

·    Sinusoidal changes from 1000 ppm onward in both sexes. The main observation was a multifocal, scattered sinusoidal dilation, with sometimes eosinophilic contents. Occasionally, there was some Kupfer cell hypertrophy and/or increase in number of granulocytes in the bloodstream.

Summary Test Item-Related Microscopic Findings F0– Thymus

 

Males

Females

Dose level (ppm):

0

1000

3000

10000

0

1000

3000

10000

 

 

 

 

 

 

 

 

 

THYMUSa

12

12

12

14

11

4

2

10

   Lymphoid Atrophy

 

 

 

 

 

 

 

 

      Minimal

1

-

1

9

1

-

-

1

a = Number of tissues examined from each group

In the thymus, the following change was observed:

·   Lymphoid atrophy at increased incidence at 10,000 ppm in males, at minimal degree.

Summary Test Item-Related Microscopic Findings F0– Adrenal glands

 

Males

Females

Dose level (ppm):

0

1000

3000

10000

0

1000

3000

10000

 

 

 

 

 

 

 

 

 

ADRENAL GLANDSa

12

12

12

14

10

10

12

10

   Infiltrate inflamm.cells,

 

 

 

 

 

 

 

 

      Minimal

-

-

-

3

-

2

-

3

      Slight

-

-

-

2

-

-

-

-

   Vacuol.z. fasc. multifocal

 

 

 

 

 

 

 

 

      Minimal

-

1

9

9

-

4

9

5

      Slight

-

-

3

1

-

-

-

-

   Degeneration. z. reticul.

 

 

 

 

 

 

 

 

      Slight

-

-

-

-

-

-

-

1

   Serosal inflammation

 

 

 

 

 

 

 

 

      Minimal

-

-

-

1

-

-

-

-

      Slight

-

-

-

1

-

-

-

-

a = Number of tissues examined from each group

 

In the adrenal glands, the following changes were observed:

·    An increased incidence and severity of inflammatory cell infiltrate, mainly lymphocytic, at 10,000 ppm in males, up to slight degree.

·    Multifocal scattered vacuolation of the zona fasciculata from 1,000 ppm onward in both sexes, up to slight degree.

·    Degeneration of the zona reticularis in one 10,000 ppm female, at slight degree.

·    Serosal inflammation in two 10,000 ppm males, up to slight degree.

Summary Test Item-Related Microscopic Findings F0– Lung

 

Males

Females

Dose level (ppm):

0

1000

3000

10000

0

1000

3000

10000

 

 

 

 

 

 

 

 

 

LUNGa

12

12

12

14

10

10

12

11

   Alv.macroph. aggregation

 

 

 

 

 

 

 

 

      Minimal

2

4

5

4

-

1

4

5

      Slight

-

-

-

2

-

-

-

2

   Inflammation pleura

 

 

 

 

 

 

 

 

      Moderate

-

-

-

-

-

-

-

1

a = Number of tissues examined from each group

In the lung, the following changes were observed:

·    Alveolar macrophage aggregation at increased incidence and severity at 10,000 ppm in both sexes, up to slight degree.

·    Inflammation of the pleura in one 10,000 ppm female, at moderate degree.

Summary Test Item-Related Microscopic Findings F0– Skin/Subcutis Hindleg

 

Males

Females

Dose level (ppm):

0

1000

3000

10000

0

1000

3000

10000

 

 

 

 

 

 

 

 

 

SKIN/SUBCUTIS HINDLEGa

0

0

0

1

0

0

2

2

   Arthritis

 

 

 

 

 

 

 

 

      Slight

-

-

-

1

-

-

-

-

      Moderate

-

-

-

-

-

-

1

1

      Marked

-

-

-

-

-

-

1

1

   Edema subcutis

 

 

 

 

 

 

 

 

      Moderate

-

-

-

1

-

-

1

1

      Marked

-

-

-

-

-

-

1

1

a = Number of tissues examined from each group

In the skin/subcutis of the hindleg, the following changes were observed:

·    Arthritis in females starting at 3000 ppm and in one male at 10,000 ppm, up to marked degree.

·    Edema of the subcutis in females starting at 3000 ppm and in one male at 10,000 ppm, up to marked degree.

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.

Conclusions:
The parental NOAEL of this 90-day study combined with a 2-generation reproduction study is <1000 ppm (corresponds to 90 and 79 mg/kg bw/d in F0 and F1 females, resp.) for females and 1000 ppm for males (corresponds to 88 and 72 mg/kg bw/d in F0 and F1 males, resp.) based on necrotizing granulomas with extranodal inflammation in the mesenteric lymph nodes, with a dose-response relationship. Test-item related mortality, body weight reduction, effects on haematology and clinical biochemistry parameterswere noted at higher dose levels, as well as microscopic findings at higher dose levels in the small intestines, mesenteric lymph node, spleen, liver, adrenal glands, kidneys, lungs, thymus (males only), sternal bone marrow, ovaries and skin/subcutis of the hindleg.
Executive summary:

A combination of a two-generation reproduction and 90 -day toxicity study was performed according to guidelines and GLP.

The test substance was administered during two generations by dietary administration to SPF-bred Wistar Han rats. In the F0-generation one control group and three treated groups were tested, each consisting of 24 males and 24 females (Groups 1-4, respectively). The dose levels (dietary concentrations) were 1000 (low dose), 3000 (mid dose) and 10,000 ppm (high dose). Up to and including Day 35, dose levels were 1500 (low dose), 5000 (mid dose) and 15,000 ppm (high dose). These initial dose levels were reduced on account of reduced body weight gain at the mid (5000 ppm) and high (15,000 ppm) dose. The F1-generation consisted of one control group and two treated groups (1000 and 3000 ppm) with 24 animals/sex/group. In the F1-generation, no 10,000 ppm group was included since the F0-generation produced only four litters at this dose level.

Males were exposed for 91 or 92 days, i.e. 10 weeks prior to mating, during mating, and up to the day prior to scheduled sacrifice. F0females of Groups 1-4 that delivered were exposed for 115-121 days (most females) or 127-128 days (one or two females of Groups 1-3), i.e. during 10 weeks prior to mating, during mating, duringpost-coitum, and during at least 21 days of lactation (up to the day prior to scheduled necropsy). F0females which failed to deliver healthy offspring were treated for 99 days (non-pregnant females of Groups 1-3) or 94 days (all 20 non-pregnant females of Group 4). Between Days 4 and 6 of lactation, litters were reduced in size to eight pups by random culling of F1-pups. After weaning, one F1-male and one F1-female of each litter of the control group and the 1,000 and 3,000 ppm groups were selected for mating with a pup of another litter of the same dose group to produce an F2-generation. F1-females were allowed to produce and rear a litter until Days 21-23 of lactation. After weaning, pups were treated for 77 days prior to mating and continuing until euthanasia; F1animals received test diet for a total of 113 or 114 days for males and 120-126 days for females.

Evaluated parameters

The following observations and examinations were evaluated: mortality / viability, clinical signs, functional observations, body weight and food consumption, food conversion efficiency, clinical biochemistry, macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio, sexual maturation and postnatal pup development (mortality, clinical signs, body weight, macroscopy and organ weights). Chemical analyses of diets were conducted on several occasions to assess accuracy, homogeneity and stability.

Results/discussion

Accuracy, homogeneity and stability of diet preparations were considered acceptable for the purpose of this study.

Formulation analysis showed that the accuracy, homogeneity and stability of diet preparations were acceptable for the purpose of this study.

Parental results:

In both generations parental toxicity was observed from 1000 ppm onwards.

Three animals of the F0-generation, i.e. one female at 3000 ppm and two males at 10,000 ppm, were prematurely sacrificed for humane reasons (between study Days 58 and 102). Their moribund condition was related to the presence of necrotizing granuloma(s) within the mesenteric lymph node, spleen and/or liver and extranodal inflammation of the mesenteric lymph node and/or arthritis of the hindleg(s). Several of these morphological findings were also noted as adverse findings in surviving animals as discussed below.

One of the key findings in this study was the occurrence of accumulation of foamy macrophages within the villi at 3000 and 10,000 ppm in parental rats of the F0- and F1-generation. This was considered to be due to absorption of the test item in the small intestines. The draining mesenteric lymph node, however, already showed an increased incidence of small foci with non-foamy and foamy macrophages (foci and in sinusoids) at the low dose (1000 ppm). At the higher doses (3000 and 10,000 ppm), those foci developed into small to medium sized foamy macrophage aggregates (foci and in sinusoids) that most likely merged into large necrotizing granulomas (i.e. a centre of necrosis, surrounded by a rim of inflammatory cells like neutrophils and (giant) macrophages) and/or extranodal inflammation into the abdominal cavity (i.e. peritonitis). This was regarded to have resulted in serosal inflammation as noted in a few other organs. Besides in the mesenteric lymph nodes, foamy cell aggregations and/or necrotizing granulomas were also noted in other organs like lung, liver, spleen and ovaries.

Secondary effects of these inflammatory processes were noted in the liver and spleen, in the form of trafficking of especially granulocytes through the vasculature of spleen and/or liver. The increased myelopoiesis observed in the sternal bone marrow (from 1000 ppm in males, from 3000 ppm in females) was believed to be secondary due to increased demand of inflammatory cells (neutrophils and monocytes), as supported by hematological data.

Occasionally, arthritis of a leg was noted at 3000 and 10,000 ppm. Since there were only a few macrophages at the site of the arthritis, there might be another pathway for this, like direct exposure after absorption and transport through blood or lymph stream, or by immune mediated processes.

The cytoplasmic foamy vacuolation of glomerular podocytes of the kidneys, observed at 10,000 ppm in both sexes, suggest that the test-item or related degradation products was/were able to pass the glomerular basement membrane. This cytoplasmic change could potentially have its effect on the filtration process.

Based on cases of moribundity at 3000 and 10,000 ppm, and severity and degenerative character of microscopic findings (granulomas/necrotizing inflammation, serosal inflammation suggestive of peritonitis/pleuritis) or passage through basement membranes (kidney)), the following findings were considered adverse for the F0-generation:

Adverse findings at 1000 ppm:

·      Mesenteric lymph node: necrotizing granulomas with extranodal inflammation in a female.

Adverse findings starting at 3000 ppm:

·      Mesenteric lymph node: necrotizing granulomas at increased incidence and severity and/or extranodal inflammation in both sexes.

·      Hindleg: arthritis in a few females.

Adverse findings at 10,000 ppm:

·      Spleen: necrotizing granulomas in males.

·      Liver: necrotizing granulomas in both sexes.

·      Kidneys: the presence of foamy cells in the podocytes of the glomeruli in both sexes.

·      Ovaries: necrotizing granulomas in females.

·      Adrenal glands: serosal inflammation in males.

·      Lung: inflammation pleura in a female.

·      Hindleg: arthritis with marked edema in a male.

Histopathological changes in parental rats of the F1-generation consisted of a non-adverse ovarian change at 1000 and 3000 ppm (described below under reproductive results). In addition, there were treatment-related macroscopic findings, the main finding being enlargement of the mesenteric lymph nodes (in two males at 1000 ppm and all animals at 3000 ppm). The other findings were for the spleen of a 3000 ppm male (grown together with peritoneum) and the skin/subcutis of a foreleg (nodule) of another 3000 ppm male. The microscopic findings in these organs with gross lesions were generally similar in nature as in the F0-generation. Based on severity and degenerative character (inflammation and/or necrosis), the following findings (all gross lesions) were considered adverse for the F1-generation:

Adverse findings at 1000 ppm:

·      Mesenteric lymph node: necrotizing granulomas with extranodal inflammation in a male.

Adverse findings at 3,000 ppm:

·      Mesenteric lymph node: necrotizing granulomas at increased incidence and severity and/or extranodal inflammation in both sexes.

·      Spleen: serosal inflammation in a male.

·      Foreleg: arthritis and edema in a male.

In-life findings in the F0and F1generation included clinical signs of toxicity, lower body weights, food intake and food conversion efficiency. For theF0generation, clinical signs were noted at 3000 and 10,000 ppm, mostly during the last few weeks of the treatment period, and mainly consisted of hunched posture, piloerection and a pale appearance.Abnormal gait and swelling of the hindlegs were occasionally observed in a few of these animals, one of which was sacrificed for humane reasons. In theF1-generation, only piloerection was observed at increased frequency at 3000 ppm, but only on one or a few days during treatment. 

Parental body weights were reduced in both sexes at 3000 (F0and F1generation)and 10,000 ppm (F0-generation)throughout the treatment period. In males, the differences from controls were about 15% (3000 ppm) and 40% (10,000 ppm) at the end of the treatment period. Body weights of females were reduced by approximately 10% (300 ppm) or 25% (10,000 ppm) at the end of the pre-mating period and approximately 15% (3000 ppm) or 30% (10,000 ppm) at the end of the post-coitum period. During the lactation period, the differences from controls became smaller. The reduced body weight gain was accompanied by a higher relative food consumption at 10,000 ppm and lower food conversion efficiency (g body weight gain per g food consumed) at 3000 and 10,000 ppm.

Fore- and hindlimb grip strength were dose-dependently reduced inF0-males starting at 1000 ppm. Values in these animals remained in the normal range for male rats of this strain and age. Moreover, there were no corroborative clinical signs or changes in other measures in the neuromuscular domain (including gait, air righting reflex and motor activity). Therefore, these changes in grip strength were considered not to reflect impaired neuromuscular function. A relationship to the observed arthritis/edema in several animals at 3000 and 10000 ppm was not considered likely since these morphological changes were also noted in females for which mean grip strength appeared unaffected by treatment.

Several macroscopic findings at 10,000 ppm were considered to be related to the significant reduction in body weight gain at this dose level. These findings included emaciation in both sexes, pituitary changes in females (discoloration and/or reduced in size and/or gelatinous contents, without histologic correlate), reduced size and weight of the prostate gland, and reduced size of the seminal vesicles and preputial gland (without histologic correlate).

Haematology, conducted in F0parental rats at the end of the treatment period, showed treatment-related changes at 10,000 ppm and, to a lesser extent, at the lower dose levels. The 10,000 ppm animals showed changes in white blood cell parameters (higher total white blood cell count, higher percentage of neutrophils and lower percentage of lymphocytes in both sexes; lower percentage of eosinophils in females), red blood cell parameters (lower haemoglobin, haematocrit, mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) in both sexes, higher percentage of reticulocytes in females) and coagulation parameters (higher platelet count and activated partial prothrombin time in both sexes). At 3000 ppm, total white blood cells, neutrophils, lymphocytes (both sexes), MCV and MCH (males) and reticulocytes (females) were affected. Changes at 1000 ppm were limited to higher neutrophils and lower lymphocytes in females, and lower MCH in males.

The increases in neutrophils and total white blood cells were considered to be related to morphologic changes involving inflammatory cells as described above. The changes in MCV, MCH and reticulocytes at 1000 and 3000 ppm occurred in the absence of changes in main red blood cell parameters (haemoglobin, number of red blood cells) or supportive morphological changes and were therefore considered non-adverse.    

Clinical biochemistry values in F0parental rats at the end of the treatment period were affected at 3000 and 10,000 ppm, particularly in males. F0-males had higher plasma activities of alanine and aspartate aminotransferase and lower albumin from 3,000 ppm onwards, and higher potassium and lower total protein, glucose, total cholesterol and alkaline phosphatase activity at 10,000 ppm. Females at 10,000 ppm had lower values for total protein, albumin and total cholesterol.  

Treatment-related organ weight changes in F0parental rats consisted of higher relative weights of the spleen in both sexes and of the liver, kidneys and adrenal glands in males from 3000 ppm onward, and lower prostate weights at 10,000 ppm (the latter considered being secondary to the lower body weights). Test item-related higher spleen weights (absolute and relative to body weights) were noted in the 1000 ppm F1females (relative to body weights) and 3000 ppm F1males and females (absolute and relative to body weights) and higher liver weights (relative to body weights) were noted in 3000 ppm F1males. In F0-rats there were histologic correlates for the increases in liver and spleen weightsin the form ofnecrotizing granulomas (in F1-rats liver and spleen were not examined microscopically, except for one spleen with a gross lesion).

Based on these results, the parental NOAEL was <1000 ppm based on adverse morphologic changes (necrotizing granulomas with extranodal inflammation) in the mesenteric lymph nodes of F0 females starting at 1000 ppm (corresponds to 90 and 79 mg/kg bw/d in F0 and F1 females, resp.) .

The most critical effects at lower dose levels involve the foamy macrophages, which were also seen in the OECD 422 study at the lowest dose of 300 mg/kg bw. The attached graph compares the combined findings from the OECD 422 study with the results from the F0 and F1 in this study and shows that the results are very comparable. Extrapolation indicates a NOAEL of around 20 mg/kg bw/day (conservative; extrapolation of the three lines results to 28 - 52 mg/kg bw as NOEL), as threshold for the effects irrespective duration of the study (45 day in OECD 422 or 90+-days), but with increasing duration the severity of effects at higher dose levels tend to increase.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
Jan 2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Version / remarks:
Aug 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.35 (Two-Generation Reproduction Toxicity Test)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EU method B.26: “Sub-chronic Oral Toxicity Test: Repeated dose 90-day toxicity study in rodents". Official Journal of the European Union No. L142
Version / remarks:
May 2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD 408, "Repeated Dose 90-day Oral Toxicity Study in Rodents"
Version / remarks:
September 1998
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: US EPA OPPTS 870.3100 "90-day Oral Toxicity in Rodents"
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): di-C16-C18 (evennumbered) alkyl tripropylenetetramine
- Purity: 100% (UVCB substance)
- Purity test date: 08 July 2016
- Batch No.: 1330539
- Expiration date of the batch: 04 August 2018
- Appearance: Viscous liquid
- Storage condition of test material: At room temperature container flushed with nitrogen
- pH: 8.5-10.5 at concentration of 1%
- Specific gravity/density: 0.864 at 20°C

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 reproduction studies. Charles River Den Bosch has reproductive historical control data in this species from the
same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
5-6 weeks old
- Weight at study initiation:
128-161 g for males and 112-139 g for females
- Fasting period before study:
no
- Housing:

Pre-mating and Post-weaning: Animals were housed in groups with a maximum of 4 animals/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 4 animals/cage. Since male nos. 81 and 84 were sacrificed in extremis, two males (nos. 85 and 86) of the subsequent cage were selected for blood sampling and were housed separately overnight from 16- 17 October 2016 (Macrolon plastic cage, MIV type, height 18 cm). As a consequence, the other two cage mates (nos. 87 and 88) were housed separately in their home cage with access to food and drinking water to prevent fasting these animals twice. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until one day preceding termination of the dam or until weaning (PND 21) in Macrolon plastic cages (MIII type, height 18 cm).
General Sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
During activity monitoring, animals were housed individually in Macrolon plastic cages (MIII type; height 15 cm) with sterilised sawdust as bedding material.
- Diet (e.g. ad libitum):
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food for a maximum of 2 hours. The same diets remained in the food hopper for a maximum of 14 days
- Water (e.g. ad libitum):
Free access to tap-water. During motor activity measurements, animals did not have access to water for a maximum of 2 hours.
- Acclimation period:
at least 5 days prior to treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
18-24
- Humidity (%):
40-70
- Air changes (per hr):
at least 10
- Photoperiod (hrs dark / hrs light):
12/12

IN-LIFE DATES: From: July 2016 To: Nov 2016 for F0 and Oct 2016 to March 2017 for F1

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency):
Diets were prepared freshly for use at room temperature for a maximum of 8 days. Following confirmation of stability over 14 days at room temperature under project 512795, diets were prepared freshly for use at room temperature for a maximum of 14 days.
- Mixing appropriate amounts with standard powder rodent diet:
The test item was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Water (approximately 15% in total) was added to aid pelleting. The pellets were dried for approximately 24 hours at 35°C before storage. The control animals received similarly prepared pellets but without the test substance.
- Storage temperature of food:
Diets were kept in the freezer (≤-15°C) until use, if not used on the day of preparation. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 14 days.
Details on mating procedure:
Following a minimum of 70 days of exposure for the males and females, one female was 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 was confirmed, the males and females were separated. For Group 1-3 animals, a maximum of 15 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males. After 10 days of pairing, any Group 1, 2 and 3 females not showing evidence of mating, were re-mated once with a proven male of the same group for a maximum of 4 days. For Group 4 females, pairing was discontinued after 10 days since most females showed an acyclic/irregular estrous cycle and no evidence of mating was obtained. Therefore, it was not to be expected that offspring would be born at this dose level, and as such insufficient data would be obtained from these females for adequate assessment of reproductive and developmental toxicity. Additionally, veterinairy examination of these animals indicated that their clinical condition was such that collecting daily vaginal smears was ethically not favourable. Therefore, in consultation with the sponsor, it was decided to discontinue both mating and collection of vaginal smears for these females. After the mating period, all males were returned to their home cage and females remained housed in the same cage.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
see attached background material
Duration of treatment / exposure:
Animals received test diet up to the day prior to necropsy.
F0-generation:
Males were exposed for 91 or 92 days, i.e. 10 weeks prior to mating, during mating, and up to the day prior to scheduled sacrifice. F0 females of Groups 1-4 that delivered were exposed for 115-121 days (most females) or 127-128 days (one or two females of Groups 1-3), i.e. during 10 weeks prior to mating, during mating, during post-coitum, and during at least 21 days of lactation (up to the day prior to scheduled necropsy). F0 females which failed to deliver healthy offspring were treated for 99 days (non-pregnant females of Groups 1-3) or 94 days (all 20 non-pregnant females of Group 4).
F1-generation (F0-pups):
The F1-generation could potentially have been exposed to the test item in utero, via maternal milk, from exposure to maternal urine/faeces or via spilled diet from the food hopper, and directly when pups begin eating solid food. After weaning, pups were treated for 77 days prior to mating and continuing until euthanasia; F1 animals received test diet for a total of 113 or 114 days for males and 120-126 days for females.
F2-generation (F1-pups):
The F2-generation animals were not dosed directly but could potentially have been exposed to the test item in utero, via maternal milk, from exposure to maternal urine/faeces or via spilled diet from the food hopper.

Frequency of treatment:
continuously, ad libitum
Details on study schedule:
See above.
Doses / concentrationsopen allclose all
Dose / conc.:
1 500 ppm
Remarks:
F0 group 2, days 1-35
Dose / conc.:
5 000 ppm
Remarks:
F0 group 3, day 1-35
Dose / conc.:
15 000 ppm
Remarks:
F0 group 4, day 1-35
Dose / conc.:
1 000 ppm
Remarks:
Group 2: F0 day 36 - end and F1 parents
Dose / conc.:
3 000 ppm
Remarks:
Group 3: F0 day 36 - end and F1 parents
Dose / conc.:
10 000 ppm
Remarks:
Group 4: F0 day 36 - end
No. of animals per sex per dose:
24
Control animals:
yes, plain diet
Details on study design:
Dose selection rationale:
Initial dose levels as used until Day 35 were based on results of the 14-d dose range finding study (Test Facility Study No. 512946). In this DRF study, 3 females were given 15000 ppm in the diet for 14 days (test item intake 1292 mg/kg bw/d). No mortality, no clinical signs, normal food consumption, no macroscopic abnormalities and normal liver and kidney weight were noted. A slightly lower weight gain for 1/3 females was noted.
From Day 36 of treatment onwards, dose levels in all test item treated groups were lowered in agreement with the Sponsor due to the reduced bodyweight gain of animals in Groups 3 and 4.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
prior to first administration, and at least once daily from start of administration onwards. For all F0 parental animals this was also performed outside the home cage in a standard arena once prior to start of administration and at weekly intervals during the treatment phase .

BODY WEIGHT: Yes
- Time schedule for examinations:
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, 4, 7, 14 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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 during lactation on Days 1, 4, 7, 14 and 21.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

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: Yes
- Time schedule for examinations:
at pre-test: First 12 F0 males of each group and all females (including spare animals) Week 13: First 12 F0 males of Groups 1 and 4. First 10 F0 non-pregnant Group 4 females. During lactation: Selected 10 F0 females of Groups 1 and 3.

Blood samples collected for haematology and clinical biochemistry using isoflurane as anaesthetic:
Week 7 (premating) - 10 F0 high dose males and 10 F0 control males3 (these selected animals were not fasted overnight). Samples were collected in support of the severely lower body weight gains in the high dose group (15,000/10,000 ppm).
Week 13 (end of treatment): first 12 F0 males per group and first 10 F0 non-pregnant Group 4 females (collected from the retro-orbital sinus as part of the necropsy procedure).
End of lactation (end of treatment): - selected 10 F0 Group 1-3 females.

HAEMATOLOGY: Yes
- Animals fasted: Yes, overnight
- How many animals:
10-12/sex/group
- Parameters according to OECD 408 were examined.

CLINICAL CHEMISTRY: Yes
- Animals fasted: Yes, overnight
- How many animals:
10-12/sex/group
- Parameters according to OECD 408 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Week 12-13 of treatment: First 12 F0 parental males/group and first 10 F0 non-pregnant Group 4 females, and last week of lactation: First 10 F0 selected Groups 1-3 females.
- Battery of functions tested: hearing ability, pupillary reflex and static righting reflex, grip strength, locomotor activity

OTHER: no other general toxicity parameters were included.
Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed to determine the stage of determination estrous beginning 21 days prior to initiation of the mating period and until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period.
For ethical reasons, no vaginal lavage was collected for Group 4 non-mated females from Day 12 of the mating period onwards and at necropsy. Pairing of these females was discontinued after 10 days based on estrous cycle irregularity/acyclicity and absence of evidence of copulation for most Group 4 females.
For mated females, a vaginal smear was collected at necropsy.
Sperm parameters (parental animals):
All surviving males: sperm samples were taken from the proximal part of the vas deferens (right):
1. Sperm motility and progressive motility was assessed from all samples.
2. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal were recorded. Evaluation was performed for all samples of the control and high dose group in first instance. Since no treatment-related effect was found, the intermediate dose groups were not assessed.
Litter observations:
STANDARDISATION OF LITTERS
To reduce variability among the litters, on PND 4-6 eight pups from each litter of equal sex distribution (if possible) were selected. For litters consisting of fewer than eight pups, adjustments for litter size was not performed.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
Mortality / Viability: The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check are presented in the respective tables.
Body weights: Live pups were weighed on PND 1, 4 and 7 and weekly thereafter.
Sex: Sex was determined for all pups on PND 1 and 4.
Sex ratio (% male pups / % female pups) was calculated per group.
Balanopreputial separation: Each selected male F1-pup (24 rats/group) was observed for balanopreputial separation (prepuce opening) beginning on postnatal Day 35. Examination of the males was continued daily until balanopreputial separation was present. The body weight of each male was recorded on the day of acquisition of balanopreputial separation.
Vaginal perforation: Each selected female F1-pup (24 rats/group) was observed for vaginal perforation (vaginal opening) beginning on postnatal Day 25. Examination of the females was continued daily until vaginal perforation was present. The body weight of each female was recorded on the day of acquisition of vaginal perforation.

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
Postmortem examinations (parental animals):
Parameters examined for haematology and clinical biochemistry are described under 7.5.1 (90-day study).

F1 females were not deprived of food overnight prior to scheduled necropsy.
All other animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy.
Necropsy was conducted on the following days:
Condition Day of necropsy
Parental Males : After at least 90 days of administration of F0-parental males and as soon as possible after delivery of litters for F1-parental males.
Females which delivered: F0: Lactation Day 22-24 (i.e. one day after necropsy of pups as dams needed to be fasted for blood sampling).
F1: Lactation Day 21-23.
Females which failed to deliver: Post-coitum Day 25-27 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).
Dead pups: Within 24 hours of death.
Euthanized in extremis: When pain, distress or discomfort is considered not transient in nature or is likely to become more severe.
All non-pregnant F0 females of Group 4: After 94 days of administration (20 October 2016)

GROSS PATHOLOGY: Yes, for the first 12 F0 males of all groups, the first 10 F0 non-pregnant Group 4 females, the selected 10 Group 1-3 F0 generation females, and all animals that were killed in extremis; organs and tissue according to OECD 408. For all other F0 animals and all F1 animals: adrenal glands, brain (cerebellum, mid-brain and cortex), cerivx, clitoral gland, coagulation gland, epididymidis, female mammary gland area, heart, kidneys, liver, ovaries, pituitary gland, preputial gland, seminal vesicles, spleen, testis, thymus, thyroid incl parathyroid, uterus, vagina, mesenteric lymph nodes, all gross lesions.
HISTOPATHOLOGY: Yes, according to OECD 408, i.e. preserved organs and tissues of all group 1 F0 and F1 males and females, the selected 10 F0 females of Group 3, all group 3 F1 males and females, and all group 4 F0 males and females, all gross lesions of all dose groups and preserved organs and tissues from animals killed in extremis.

Organ weights:
The following organ weights and terminal body weight were recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands Prostate
Brain Seminal vesicles with coagulating glands
Epididymides (total and cauda separately) Spleen
Heart Thyroid including parathyroid
Kidneys Testes
Liver Uterus (including cervix)
Ovaries Thymus
Pituitary gland (after fixation)
Reproductive organs:
For all paired females, the number of former implantation sites in the uterus was recorded.
All surviving males: One testis and one epididymis (left) were removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing the left testis and epididymis were weighed, homogenized and evaluated for sperm numbers.
In the case of any abnormalities in the testes and/or epididymidis (left), the left side organ(s) was/were fixed in modified Davidson's, and the right side organ(s) was/were used for evaluation of sperm numbers.
If abnormalities were found in testes and/or epididymides (both sides), both these organs were fixed in modified Davidson's solution. No evaluation of sperm numbers was performed.
Evaluation was performed for all samples of the control and high dose group in first instance. Since no treatment-related effect was found, the intermediate dose groups were not assessed. Any remaining testis and epididymis stored in the freezer at ≤-15°C for possible sperm enumeration were discarded at finalization of the study report.
Postmortem examinations (offspring):
SACRIFICE
Pups, younger than 7 days were killed by decapitation. All remaining pups were sacrificed using Euthasol®20% (AST Farma B.V., Oudewater, The Netherlands) by ip injection.
Pups found dead during the weekend were fixed in identified containers containing 70% ethanol (Klinipath, Duiven, The Netherlands) as they were not necropsied on the same day.
Culling was performed on Day 4 of lactation or shortly thereafter. The remaining pups (including the few Group 4 litters, but excluding F1-pups of Groups 1-3 selected for mating) were killed on Day 21 of lactation or shortly thereafter.

GROSS NECROPSY
Stillborn pups and pups dying between birth and Day 4 of lactation were sexed both internally and externally and externally examined.
Pups found dead or sacrificed in extremis from Day 5 of lactation to weaning and all non-selected F1- and F2-weanlings were sexed and subjected to external examination of the cranium, and macroscopic examination of the thoracic and abdominal tissues and organs (including sex determination). Descriptions of all macroscopic abnormalities were recorded. Selected gross lesions were collected and placed in 10% buffered formalin (see also Study Plan Deviations).
Culled pups were sexed both internally and externally and externally examined.

HISTOPATHOLOGY
Organ weights: Brain, spleen, thymus. Organ weights (and terminal body weight) were recorded from one pup/sex/litter (both generations) at scheduled necropsy on Day 21 of lactation or shortly thereafter.

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.
-Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup difference.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
Percentage mating males = (Number of males mated x 100)/ Number of males paired

Percentage mating females = (Number of females mated x 100) / Number of females paired

Fertility index males = (Number of pregnant females x 100) / Number of males paired

Fertility index females = (Number of pregnant females x 100) / Number of females paired

Conception index = (Number of pregnant females x 100) / Number of females mated

Gestation index = (Number of females with living pups on Day 1 x 100) / Number of pregnant females

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 x 100) / Number of live pups at First Litter Check

Percentage live females at First Litter Check = (Number of live female pups at First Litter Check x 100) / Number of live pups at First Litter Check

Percentage of postnatal = (Number of dead pups on Day 4 of lactation x 100) / Number of live pups at First Litter Check
Loss lactation Days 0-4

Percentage of breeding = (Number of dead pups between Days 5 and 21 of lactation x 100) / Number of live pups on Day 4 of lactation
loss Day 5 until weaning

Percentage live males at weaning = (Number of live male pups on Day 21 of lactation x 100) / Number of live pups on Day 21 of lactation

Percentage live females at weaning = (Number of live female pups on Day 21 of lactation x 100) / Number of live pups on Day 21 of lactation

Viability index = (Number of live pups on Day 4 of lactation x 100) / Number of pups born alive

Lactation (weaning) index = (Number of live pups on Day 21 of lactation x 100) / Number of live pups on Day 4 of lactation

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related clinical signs of toxicity were noted at 10,000 ppm in both sexes and, to a lesser extent, at 3000 ppm in females.
Findings at 10,000 ppm in surviving animals primarily included hunched posture, piloerection and a pale appearance in up to most or all animals, and a lean appearance in up to about one third of the animals. Additional findings in 10,000 ppm females consisted of chromodacryorrhoea in about one third of the females, lethargy in four females, and abnormal gait and swelling of the legs in two females. These clinical signs were mostly noted during the last few weeks of the treatment period, except for abnormal gait and swelling of the skin which were noted only on the last few days of the study.
Treatment-related findings in surviving females at 3000 ppm consisted of piloerection and hunched posture in several females during the last few weeks of the treatment period, and a lean appearance, abnormal gait and swelling of the legs in a single female on the last few days of the study.
No additional clinical signs of toxicity were noted during the weekly arena observations.
Any other clinical signs noted incidentally 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, showed no dose-related trend and/or incidences were similar to those encountered in controls. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two males at 10,000 ppm and one female at 3000 ppm were sacrificed in extremis, which was considered to be related to treatment.
The moribundity of both males (sacrificed at Days 58 and 65, respectively) and of the female (sacrificed at Day 102) was considered to be treatment-related. At the day of sacrifice (or one or more days before), these animals showed clinical signs including but not limited to hunched posture, abnormal gait, piloerection, a lean appearance (one male), ptosis (one male) and/or swelling of the skin of the hindlegs (female). In the week prior to sacrifice, both males lost weight. Major gross lesions consisted of enlarged mesenteric lymph nodes and/or spleen, many nodules/foci of the mesenteric lymph nodes and/or spleen and liver, and/or thickened skin of hindleg(s). Main correlating microscopic findings consisted of necrotizing granuloma(s) of the mesenteric lymph node and/or spleen and liver, extranodal inflammation of the mesenteric lymph node and arthritis of the hindleg(s). These pathology findings were considered to be related with the moribundity of the animals. As similar clinical signs of toxicity, gross lesions and microscopic changes were noted in surviving animals at 3,000 and 10,000 ppm, the moribundity of both males and the female was considered to be related to treatment with the test item.
The moribundity of another female at 3000 ppm (sacrificed at Day 43) was not considered related to treatment with the test item. At the day of sacrifice, she showed tremors, piloerection, abdominal swelling, squeaking and hypothermia. Her body weight development was normal. The cause of her moribundity was a yolk sac carcinoma, a malignant tumor with many metastasis in many organs, correlating with the nodules in many abdominal organs and tissues observed at necropsy. Based on the single occurrence and character of the tumor, this condition was considered to be spontaneous.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights were dose-dependently reduced at 3000 and 10,000 ppm in both sexes throughout the treatment period (statistically significant on most occasions). In males, the differences from controls gradually increased to about 17% (3,000 ppm) and 40% (10,000 ppm) at the end of the treatment period. Mean body weights of females were reduced by about 10% (3000 ppm) or 25% (10,000 ppm) at the end of the pre-mating period and about 15% (3000 ppm) or 30% (10,000 ppm) at the end of the post-coitum period. In the course of the lactation period, the differences from controls remained approximately the same (approximately 9%) at 3000 ppm and decreased to about 16% at 10,000 ppm.
Body weight gain was dose-dependently reduced at 3000 and 10,000 ppm in both sexes throughout the pre-mating period. Body weight gain in males remained reduced at 3,000 and 10,000 ppm until the end of the study. Body weight gain of pregnant females was reduced only at 10,000 ppm during the last week of the gestation period. Higher body weight gain was noted during the second week (10,000 ppm) and third week (3000 and 10,000 ppm) of the lactation period. These variations achieved a level of statistical significance on most occasions.
Note: At 10,000 ppm, evaluation of body weight development during the gestation and lactation periods was based on only four females. The remaining 20 females of this dose group were not pregnant.
Body weight and body weight gain at 1000 ppm were not affected by treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before allowance for body weight was reduced (generally statistically significantly) at 10,000 ppm in both sexes during the pre-mating period, and in females during the post-coitum period. No remarkable differences were noted during the lactation period. Food consumption after allowance for body weight (relative food consumption) at this dose was on most occasions higher than controls (generally statistically significant) throughout the treatment period in males (but not always clearly dose-dependently during the pre-mating period). For females at this dose, relative food consumption was higher during the lactation period and more occasionally during the post-coitum period (not always statistically significant), and was generally similar to that of controls during the pre-mating period.
Relative food consumption was also higher (generally statistically significantly) throughout the treatment period in males and females at 3000 ppm (not clearly dose-dependently during the pre-mating period), which was ascribed to the lower body weights at this dose.
Any other statistically significant differences in absolute or relative food consumption were minor and/or occurred in the absence of a clear dose-related trend and were therefore not considered to be related to treatment.
Note: At 10,000 ppm, evaluation of food consumption during the gestation and lactation periods was based on only four females. The remaining 20 females of this dose group were not pregnant.

The mean daily intake of the test item per kg body weight during the different phases of the study is given in the table below.
Test item intake (mg test item/kg bw/day)(1)

Group 2 Group 3 Group 4
1000 ppm(2) 3000 ppm(2) 10,000 ppm(2)
Males
Pre-mating 96 326 1025
Mating 60 201 953
Mean of means(3) 88 299 1010

Females
Pre-mating 104 360 996
Post-coitum 76 252 836
Lactation 73 253 890
Mean of means(3) 90 309 930

1 Values are the overall group means in the periods indicated.
2 These dietary concentrations were used from Day 36 of the study (lower concentrations were used during the lactation period, see section 5.5). Before Day 36, higher concentrations were used: 1,500 (Group 2), 5,000 ppm (Group 3) and 15,000 ppm (Group 4).
3 Mean of means of all periods, weighed for number of measurement intervals per period:
Males: ((11 x mean premating) + (3 x mean mating)) / 14
Females: ((11 x mean premating) + (6 x mean post-coitum) + (4 x mean lactation)) / 21
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
At 10,000 ppm, mean food conversion efficiency (g body weight gained per g food consumed) of males was reduced with statistical significance during most of the pre-mating period. For females at this dose, mean food conversion efficiency was reduced during the initial 4 weeks of the premating period. Females at 10,000 ppm also showed lower mean food conversion efficiency on several occasions during the post-coitum period.
At 3000 ppm, mean food conversion efficiency was reduced for males over Days 1-36 of the premating period and during a single occasion during the mating period, and for females only over Days 1-8 and 15-22 of the premating period.
At 1000 ppm, mean food conversion efficiency was only reduced during the first week of treatment for males.
Overall, mean over mean food conversion efficiency during the premating phase (males and females) and post-coitum phase showed an apparent downward trend over the dose groups. During lactation however, there was no apparent dose-related trend in mean over mean food conversion efficiency. It should be noted that the concentration in the test diets was lowered during lactation.
The statistically significantly lower mean food conversion efficiency of males at 1000, 3000 and 10,000 ppm over Days 8-15 of the mating period was not considered to be related to treatment since this difference occurred in the absence of a dose related trend and was not consistently noted with continuing treatment.
The higher mean food conversion efficiency of females at 10,000 ppm over Days 14-21 of lactation was attributed as secondary to the lower mean litter size.
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):
Note: The 10,000 ppm females selected for haematology were non-pregnant (selected females of the other groups were lactating) and they were treated 3-4 weeks shorter compared to females of the other groups. When assessing the possible relation with treatment of differences in haematology values between 10,000 ppm females and concurrent controls, it was taken into consideration that physiological status may influence haematology parameters (Ref 1, Ref 2).
Ref. 1 Urasoko Y., He X.J., Masao T., Kinoshita Y., Edamoto H., Hatayama K., Asano Y., Tamura K, Mochizuki M.. Changes in blood parameters and the expression of coagulation-related genes in lactating Sprague-Dawley rats. Journal of the American Association of Laboratory Animal Science 51, 144-149 (2012).
Ref. 2 Menke A., Wolberbeek A., Snel C., Bruijntjes J., Groot D. de, Oostrum L. van, Waalkens I., Kuper C.F.. Potentially increased sensitivity of pregnant and lactating female rats to immunotoxic agents. Toxicologic Pathology 40, 255-260 (2012).

The following (mostly statistically significant) changes in haematology parameters distinguished treated animals from control animals:
• Higher total white blood cell counts (WBC) at 3000 and 10,000 ppm in both sexes. Values in 10,000 ppm females were higher (not statistically significantly) relative to concurrent (lactating) and historical (nulliparous) controls.
• Higher percentage of neutrophils and lower percentage of lymphocytes at 3,000 and 10,000 ppm in males and from 1000 ppm onwards in females. Values in 10,000 ppm females differed from concurrent (lactating) controls (neutrophils not statistically significantly) and historical (nulliparous) controls.
• Lower percentage of eosinophils at 1000 and 3000 ppm in females.
• Higher percentage of reticulocytes in females at 3000 and 10,000 ppm. Values in 10,000 ppm females were higher relative to concurrent (lactating) and historical (nulliparous) controls.
• Lower haemoglobin concentration and haematocrit at 10,000 ppm in both sexes. Values for haemoglobin in 10,000 ppm females were lower relative to concurrent (lactating) and historical (nulliparous) controls, values for haematocrit were lower only relative to concurrent controls.
• Lower mean corpuscular volume (MCV) in males at 3000 and 10,000 ppm, and in females at 10,000 ppm (relative to concurrent (lactating) and historical (nulliparous) controls).
• Lower mean corpuscular haemoglobin (MCH) in males from 1000 ppm onwards, and in females at 10,000 ppm (relative to concurrent (lactating) and historical (nulliparous) controls).
• Higher platelets at 10,000 ppm in both sexes. Values in 10,000 ppm females were higher relative to concurrent (lactating) and historical (nulliparous) controls.
• Lower activated partial thromboplastin time (APTT) at 10,000 ppm in males.

The lower percentage of monocytes (statistically significant) noted in 10,000 ppm females (compared to concurrent controls) was considered to be due to the difference in physiological status. Monocyte values in 10,000 ppm females were in the normal range for nulliparous control females.
Other statistically significant variations noted in haematology parameters at the end of the treatment period were considered unrelated to treatment as they occurred in the absence of a dose-related trend.
Haematology conducted in Week 7 of the pre-mating period in males of the control and 10,000 ppm groups showed similar changes in haematology parameters as noted at the end of the treatment period, with the following exceptions:
• Higher percentage of monocytes in Week 7 (no change at the end of treatment).
• Higher number of red blood cells (RBC) in Week 7 (no change at the end of treatment).
• Higher haemoglobin and haematocrit in Week 7 (lower at the end of treatment).
• Higher prothrombin time (PT) in Week 7 (no change at the end of treatment).
• No change in APTT in Week 7 (lower at the end of treatment).

Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Note: The 10,000 ppm females selected for clinical biochemistry were non-pregnant (selected females of the other groups were lactating) and they were treated 3-4 weeks shorter compared to females of the other groups. When assessing the possible relation with treatment of differences in clinical biochemistry values between 10,000 ppm females and concurrent controls, it was taken into consideration that physiological status may influence clinical biochemistry parameters (Ref. 1).

Ref. 1 Urasoko Y., He X.J., Masao T., Kinoshita Y., Edamoto H., Hatayama K., Asano Y., Tamura K, Mochizuki M.. Changes in blood parameters and the expression of coagulation-related genes in lactating Sprague-Dawley rats. Journal of the American Association of Laboratory Animal Science 51, 144-149 (2012).

The following (mostly statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
• Higher activity of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) at 3000 and 10,000 ppm in males.
• Lower (not statistically significant) alkaline phosphatase activity (ALP) at 10,000 ppm in both sexes. Values in 10,000 ppm females were in the normal range for nulliparous control females.
• Lower total protein at 10,000 ppm in males and females (not statistically significant for females; the mean was lower relative to concurrent (lactating) and historical (nulliparous) controls).
• Lower albumin at 3000 ppm in males and at 3000 and 10,000 ppm in males and females (not statistically significant for females; the mean was lower relative to concurrent (lactating) and historical (nulliparous) controls).
• Higher urea at 3000 ppm in females.
• Lower glucose at 10,000 ppm in males.
• Lower total cholesterol at 10,000 ppm in both sexes. Although values in 10,000 ppm females were normal compared to historical (nulliparous) controls, it cannot be ruled out that the lower cholesterol in these females was related to treatment taken in the context of the treatment-related reduction of cholesterol in males.
• Higher potassium at 10,000 ppm in males.
The statistically significantly lower values for ALAT and urea, and the higher values for sodium and chloride noted in 10,000 ppm females (compared to concurrent controls) were considered to be due to the difference in physiological status. Values for these parameters in 10,000 ppm females were in the normal range for nulliparous control females.
Clinical chemistry conducted in Week 7 of the pre-mating period in males of the control and 10,000 ppm groups showed similar changes in clinical chemistry parameters as noted at the end of the treatment period, with the following exceptions:
• Higher urea and creatinine in Week 7 (no change at the end of treatment).
• Statistically significantly lower ALP (not statistically significant at the end of treatment).
• No statistically significant difference in potassium in Week 7 (higher at the end of treatment).
• No statistically significant difference in albumin in Week 7 (lower at the end of treatment).
• Lower inorganic phosphate in Week 7 (no change at the end of treatment).
Other statistically significant variations noted in clinical biochemistry parameters were considered unrelated to treatment or not toxicologically relevant due to the slight magnitude and/or direction of the differences.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
In males, statistically significantly lower fore- and hindlimb grip strength values were noted at all dose levels. The differences from controls showed a dose-related trend.
In females, the statistically significantly lower fore- and hindlimb grip strength noted at 10,000 ppm was ascribed to the difference in physiological status and time of testing of 10,000 ppm females and concurrent controls (10,000 ppm females: non-pregnant, tested in Week 12-13 of the treatment period; controls: lactating, tested towards the end of the lactation period after approximately 17 weeks of treatment). Compared to historical control results for female rats of this strain and age, grip strength values in females at 10,000 ppm were within normal limits.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Due to the large number of tables for microscopy, details are presented under "Any other information on results incl. tables.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
At 10,000 ppm length and regularity of the estrous cycle were affected. Only one female at this dose level (no. 174) had regular cycles of 5 days (with extended di-estrus during pairing). The other females at this dose level for which cycle lengths could be determined had maximum estrous cycle lengths ranging from 6 to 20 days. Most 10,000 ppm females (18/24) were acyclic, 16 of which also had extended di-estrus. A total of 5/24 females had irregular cycles, four of which also had extended di-estrus.
Length and regularity of the estrous cycle were not affected by treatment up to 3000 ppm. The percentage of females classified as having regular cycles of 4-5 days was 75% in the control group, 92% at 1000 ppm and 96% at 3000 ppm. In the control group, 4/24 females had an irregular cycle, one had extended estrus, and one had extended di-estrus during pairing. At 1000 ppm, 2/24 females had an irregular cycle and one had extended di-estrus during pairing. At 3000 ppm, one female had an irregular cycle and 2/23 had extended di-estrus during pairing.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Sperm motility, concentration and morphology were considered not to be affected by treatment up to 10,000 ppm.
A statistically significantly higher epididymal sperm count was noted at 10,000 ppm. This finding was considered not to be related to treatment due to the direction and small magnitude of the difference (mean value in treated males remained within the normal control range), and the absence of corroborative morphological alterations in the epididymides.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
There were 20/24 couples at 10,000 ppm without offspring (these females were sacrificed early, during the mating period, due to acyclic/irregular estrus cycles or absence of evidence of mating).
After 13 weeks of treatment at 10,000 ppm, examination of the male reproductive organs (including testes with stage awareness), did not reveal test item-related microscopic findings, and sperm parameters were normal (see section 7.2.14). Changes in male reproductive organ weights were limited to a decrease in prostate gland weight (absolute and relative to body weights) at 10,000 ppm which occurred in the presence of a considerable decrease in body weight (mean terminal body of 10,000 ppm males was 40% lower compared to controls).
In females (most of which were treated for 16-17 weeks), a test item-related ovarian change, characterized by foamy cell aggregates, was noted at 3000 and 10,000 ppm. These microscopic finding at the recorded low degrees (up to slight) and unilateral presence for the granulomas could not explain the lack of offspring. During the in-life phase, most females at 10,000 ppm were acyclic or had irregular cycles (examined after seven weeks of treatment). Like males, they had considerably reduced body weights (mean body weights were about 25% and 30% lower compared to controls at the end of the pre-mating period and scheduled termination, respectively).
It was considered that the decreased body weight gain, early in the study, combined with the young age at the start of treatment (6 weeks) and marked toxicity in many organs, such as necrotizing granulomas of mesenteric lymph node/spleen/liver and/or extranodal inflammation of the mesenteric lymph node (peritonitis) might have resulted in retarded maturation of the males and females at 10,000 ppm. The poor condition of the rats during the mating period and/or retarded maturation most likely resulted into unsuccessful mating.
For the few couples without offspring in the control group (female/male nos. 112/16 without offspring; 117/21, male did not mate), the 1000 ppm group (female/male nos. 137/41, female not pregnant, male produced offspring with another female; 136/40, male did not mate) and the 3000 ppm group (female/male nos. 149/53, female sacrificed before mating; 159/63, female not pregnant), no abnormalities were seen in the reproductive organs which could account for their lack of offspring.

Details on results (P0)

Mating index
At 10,000 ppm, mating index was markedly decreased. Only 6/24 females showed evidence of mating (smear positive for sperm); no second mating period was initiated since after 10 days of pairing only 6 out of 24 females had shown evidence of mating. Pairing was discontinued from Day 11 onwards based on consideration of their health condition and acyclicity/irregularity of the estrous cycle for most of the Group 4 females.
Mating index was not affected by treatment up to 3000 ppm. All paired females at 1000 and 3000 ppm showed evidence of mating.

Precoital time
At 10,000 ppm, precoital time was increased to an average duration of 5.3 days (based on only six females) versus 2.4 days in the control group.
Precoital time was not affected by treatment up to 3000 ppm.

Implantations
At 10,000 ppm, the mean number of implantation sites (based on only four females) was lower (not statistically significant; 7.8 versus 11.5 in controls).
The number of implantation sites was not affected by treatment up to 3000 ppm.
For a few females (nos. 108 and 116 of the control group; nos. 124 and 138 at 1000 ppm; no. 170 at 10,000 ppm) the number of pups born was slightly higher than the number of implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation. No toxicological relevance was attached to this finding in this study.

Fertility and conception index
At 10,000 ppm, fertility index was decreased to 18 and 17% for males and females, respectively (versus 96% in the other groups). Four of the six mated 10,000 ppm females were pregnant. At 10,000 ppm, conception index was decreased to 67% (versus 96% in the in the other groups). Four of the six mated 10,000 ppm females were pregnant.

Gestation index and duration
Gestation index and duration of gestation were not affected by treatment up to 10,000 ppm. All pregnant females had live offspring, resulting in a gestation index of 100% in all groups.

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

Litter size
At 10,000 ppm, litter size at birth was lower (not statistically significant; 8.0 versus 10.6 in controls). This correlated with the lower number of implantation sites at 10,000 ppm.
Litter size was unaffected by treatment up to 3,000 ppm.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
LOAEL
Effect level:
90 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
88 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
299 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 ppm
System:
cardiovascular
Organ:
mesenteric lymph node
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 3000 ppm, a higher incidence of piloerection was noted in females during the post-mating period compared to controls (11/21 versus 2/21 control dams). This clinical sign was only noted on one or a few days for each of these females. One male showed swelling of the left foreleg with focal erythema during post-mating.
Other clinical signs noted incidentally 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 showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No treatment-related mortality occurred during the study period.
One male at 1000 ppm (no. 231) was sacrificed in extremis on study Day 44. In the week prior to sacrifice, this animal showed hunched posture, piloerection, a pale appearance, dehydration and weight loss. Macroscopic findings consisted of emaciation, enlarged heart, spleen and caudate lobe of the liver, pale discoloration of the liver, and dark red discoloration of the mesenteric lymph node. Main microscopic findings were moderate erythrophagocytosis in the mesenteric lymph node, massive hematopoiesis of the spleen and moderate oval cell proliferation of the liver. Since these findings occurred in only a single low-dose male, they were not considered to be related to treatment with the test item.

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 3000 ppm, mean body weights of males and females were statistically significantly lower compared to controls throughout the treatment period. In males, the relative differences gradually increased from 10% at Day 1 of the pre-mating period to 17% at the end of the post-mating period, but body weight gain of 3000 ppm males did not differ statistically significantly from that of controls.
In females, the relative differences in mean body weight were about 10% during the pre-mating period, 14% at the end of the gestation period, and slightly attenuated during the lactation period. Body weight gain of 3000 ppm females did not differ statistically significantly from that of controls, except at Days 14 and 21 of the lactation period when it was higher compared to controls.
The statistically significantly lower body weight gain of females on Days 17 and 21 of post-coitum occurred in the absence of a dose-related trend and were therefore not considered to be affected by treatment.
At 1000 ppm, body weights were not considered to be affected by treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before allowance for body weight was not affected by treatment up to 3000 ppm. Any statistically significances differences were considered unrelated to treatment as they were minor and not consistently observed.
Food consumption after allowance for body weight was higher (statistically significantly) in males and females at 3000 than in controls throughout the treatment period. This difference in relative food consumption was due to the lower body weights at 3000 ppm which occurred in the absence of a change in absolute food consumption.
Occasional statistically significantly higher relative food consumption values noted at 1000 ppm were considered to be unrelated to treatment as they were small (generally less than 10%) and not corroborated by changes in body weight or absolute food consumption.

The mean daily intake of the test item per kg body weight during the different phases of the study is given in the table below.
Test item intake (mg test item/kg bw/day)1
Group 2 Group 3
1000 ppm(2) 3000 ppm(2)
Males
Pre-mating 79 264
Mating 58 211
Post-mating 55 194
Mean of means(3) 72 245

Females
Pre-mating 84 276
Post-coitum 74 250
Lactation 74 235
Mean of means3 79 261
1 Values are the overall group means in the periods indicated.
2 Lower dietary concentrations were used during the lactation period, see section 5.5.
3 Mean of means of all periods, weighed for number of measurement intervals per period:
Males: ((11 x mean premating) + (3 x mean mating) + (2 x post-mating)) / 16
Females: ((11 x mean premating) + (6 x mean post-coitum) + (4 x mean lactation)) / 21


Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
At 3000 ppm, mean food conversion efficiency (g body weight gained per g food consumed) was reduced in males with statistical significance during most of treatment period.
Food conversion efficiency in females showed no consistent changes during the pre-mating, post-coitum and lactation period; only on post-coitum days 7-11 and 17-20 a statistically significantly lower food conversion efficiency was recorded.
Other statistically significant variations in food conversion efficiency noted across the dose groups were considered unrelated to treatment as they occurred in the absence of a dose-related trend and were only occasionally noted during the treatment period.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related organ weight changes were noted at 3000 ppm and consisted of higher spleen weights in both sexes, and higher liver weights (relative to body weight) in males. The relative differences from controls are shown in the table below.
Males Females
Dose level (ppm): 1000 3000 1000 3000

SPLEEN
Absolute 3 9* 10 12*
Relative to body weight 6 34** 12** 23**

LIVER Not applicable
Absolute 1 -11**
Relative to body weight 4 9**
* P<0.05; ** P<0.01

Some statistically significant organ weight differences noted at 3000 ppm were considered to be due to the test item-related decrease in body weight, particularly in 3000 ppm males which had on average 18% lower terminal body weights than concurrent controls.
Any other differences, including those that reached statistical significance were considered not to be test item-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.

Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At scheduled necropsy, test item-related macroscopic findings were observed in F1-males at both dose levels and in F1-females at 3000 ppm, as listed below. The number of animals examined was 24/sex/group, except in the 1000 group of males which included 23 survivors.
• Mesenteric lymph node: enlarged in two males at 1000 ppm and in all males and all females at 3000 ppm. Microscopic correlates were necrotizing granulomas, extranodal inflammation and/or foamy macrophages.
• Spleen: grown together with peritoneum in one male at 3000 ppm. The microscopic correlate was serosal inflammation.
• Skin/subcutis of a foreleg: nodule in one male at 3000 ppm. The microscopic correlate was edema of the subcutis/skin.
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.

Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Due to the large number of tables for microscopy, details are presented under "Any other information on results incl. tables.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment up to 3000 ppm.
Most females had regular cycles of 4 days. Extended estrus occurred in one control female and one female at 1000 ppm. Another female at 1000 ppm had an irregular cycle. One The female with the irregular cycle was not pregnant and the other two females had normal litters. These findings were considered to be unrelated to treatment because their incidence was within normal limits and showed no dose-related trend.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
At 3000 ppm, testicular sperm count was statistically significantly lower (relative difference from controls: about 20%).
Epididymal sperm count, sperm motility and sperm morphology were not affected by treatment up to 3000 ppm.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The following couples had no offspring: 3/24 control couples (male/female nos. 307/214, 312/209, 317/204, female not pregnant), 3/24 couples at 1000 ppm (male/female nos. 328/241 and 336/233, female not pregnant; 334/235 no offspring) and 3/24 couples at 3000 ppm (male/female nos. 351/266, 354/263, 364/253, female not pregnant). No abnormalities were seen in the reproductive organs which could account for their lack of offspring. Primordial and primary oocyte counts in the ovaries of F1-females were not affected by treatment up to 3000 ppm as shown in the table below:  

Females
Dose level (ppm): 0 1000 3000        
OVARIESa 10 10 10       
Total number/Group of primordial and primary oocytes 26 32 35
a = Number of tissues examined from each group.

Details on results (P1)

Mating index and precoital time
Mating index was not affected by treatment up to 3000 ppm. All paired females showed evidence of mating. Precoital time was not affected by treatment up to 3000 ppm.

Implantations
The mean number of implantation sites was considered unaffected by treatment..
For a few females (three of the control group; two at 1000 ppm; three at 3000 ppm) the number of pups born was slightly higher than the number of implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation. No toxicological relevance was attached to this finding in this study.

Fertility and conception index
Fertility and conception index were not affected by treatment.
Except for three control females (nos. 307, 312 and 317), two females at 1000 ppm (nos. 328 and 336), and three females at 3000 ppm (nos. 351, 354 and 364) all mated females were pregnant. These cases of non-pregnancy, all without related histopathology changes in reproductive organs, were not considered to be related to treatment as their incidence showed no dose-related trend.

Gestation index and duration
Gestation index and duration of gestation were not affected by treatment.
Except for one female at 1000 ppm (no. 334), all pregnant females had live offspring. This incidental failed pregnancy without related histopathology changes in reproductive organs was not considered to be related to treatment.

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

Litter size
At 3000 ppm, litter size at birth was lower (statistically significant; 9.5 versus 11.8 in controls), which correlated with the lower number of implantation sites at this dose level.
Litter size at 1000 ppm was unaffected by treatment.

Effect levels (P1)

open allclose all
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Key result
Dose descriptor:
LOAEL
Effect level:
72 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
79 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
245 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P1)

Key result
Critical effects observed:
yes
System:
cardiovascular
Organ:
mesenteric lymph node
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: F1 generation

General toxicity (F1)

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.
The 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 index was 100% in all groups.
At first litter check, one pup of the control group (litter no. 116), one pup at 1000 ppm (litter no. 125) and one pup at 3000 ppm (litter no. 150) were found dead. This incidental pup mortality was considered to be unrelated to treatment because the mortality incidence showed no dose-related trend and remained within normal limits.

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 across the groups were 99-100%.
Three pups at 1000 ppm (of three litters) and one pup at 3000 ppm went missing (presumably cannibalized) or were found dead (one of the 3000 pups) at PND 2. This post-natal loss was considered to be unrelated to treatment as the incidence showed no dose-related trend and remained within the range considered normal for pups of this age.

Lactation index (number of live offspring on PND 21 as percentage of number of live offspring after culling on PND 4) was not affected by treatment. The lactation index was 96% at 3000 ppm (all six pups of litter no. 148 were necropsied on PND 8 since the dam was sacrificed in extremis) and 100% in the other groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 3000 and 10,000 ppm, body weight gain of pups was dose-dependently decreased. Mean body weights of 10,000 ppm pups of both sexes were approximately 10% lower (not statistically significant) at birth (PND 1) and approximately 25% lower at PND 21 (statistical significance was achieved in both sexes primarily from PND 7 onwards). Mean body weights of 3000 ppm pups of both sexes were lower from PND 4 onwards (statistically significant from PND 7 onwards). At PND 21, male and female pups at 3000 ppm had on average 10% lower body weights than controls.
Body weights of pups at 1000 ppm were not considered affected by treatment.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 10,000 ppm, male and female pups had lower absolute weights of the brain, thymus and spleen (approximately 5, 30 and 35% lower than control mean, respectively). Organ to body weight ratios of thymus and spleen were also lower (approximately 10% and 15-20%, respectively), except for relative brain weight (approximately 20-30% higher). These differences occurred in the presence of about 25% lower terminal body weights.
Minor, statistically non-significant variations in absolute organ weights at 3000 ppm, were ascribed to slightly lower terminal body weights, since organ weights corrected for terminal body weights were similar to controls.
Organ weights of pups at 1000 ppm were not considered affected by treatment.
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.
The nature and incidence of the macroscopic findings noted incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Description (incidence and severity):
Sex ratio
Sex ratio was not considered to be affected by treatment.
At 10,000 ppm, sex ratio appeared to indicate more male than female pups (63% male pups versus 50% in the other groups). However, evaluation of sex ratio at 10,000 ppm was hampered by the low number of litters at this dose level. All four 10,000 ppm litters had more male than female pups (% of males/females: 56/46, 57/43, 70/30 and 67/33 in litter nos. 170, 174, 180 and 184, respectively). It was considered that the sex ratios at 10,000 ppm reflected normal biological variation rather than a test item-related change.

Balanopreputial separation
The day of balanopreputial separation was similar between pups of the control group and those of the 1000 and 3000 ppm groups (on average 41- 42 days). Balanopreputial separation in the 10,000 ppm group (including only four litters) was not examined as these litters were sacrificed on Day 21 or 23 of lactation.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
88 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Results: F2 generation

General toxicity (F2)

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.
The clinical signs observed incidentally remained within the range considered normal for pups of this age and showed no dose-related trend. They 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 up to 3000 ppm. The live birth indices across the groups were 99-100%.
At first litter check, three pups at 1000 ppm (two litters) and two pups at 3000 ppm (two litters) were found dead. This pup mortality was considered to be unrelated to treatment because the mortality incidence was within normal limits and showed no dose-related trend.

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 across the groups were 94-99%.
Two pups of the control group (of two litters), 13 pups at 1000 ppm (of four litters, one of which lost 9/10 pups), and eight pups 3000 ppm (of two litters, one of which lost 6/10 pups) were found dead or went missing (presumably cannibalized) at PND 2-4 (mostly PND 2). The number of pups lost between PND 1-4 in the two treated groups was statistically significantly higher than that in the control group. However, this post-natal loss was not considered to be related to treatment as the incidence showed no dose-related trend and remained within the range considered normal for pups of this age.

Lactation index (number of live offspring on PND 21 as percentage of number of live offspring after culling on PND 4) was not affected by treatment up to 3000 ppm. The lactation index was 99% at 3000 ppm (one pup was euthanized in extremis on PND 18) and 100% in the other groups
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 3000 ppm, mean male and female pup body weights appeared slightly lower during the lactation period. The differences from controls were small (up to 10% at PND 21) and not statistically significant.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically relevant changes in organ weights of pups.
The few statistically significant differences noted were considered to be unrelated to treatment due to the absence of a dose-related trend (about 10% higher absolute and relative thymus weight in 1000 ppm males) or related to slightly lower final body weights (4% lower absolute brain weight in 3000 ppm males).
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.
The nature and incidence of the few findings noted remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Sex ratio was not affected by treatment.
In the absence of a dose-related trend, the statistically significant difference noted at 1000 ppm (61% males versus 44% in controls and 51% at 3000 ppm) was considered unrelated to treatment.

Effect levels (F2)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
72 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Overall reproductive toxicity

open allclose all
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
3 000 ppm
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
245 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

P0 histopathology

Note: 20/24 females of the 10,000 ppm group were not pregnant and treated for three to about five weeks shorter than females of the other groups (most of which had offspring).

Test item-related microscopic findings were noted in the small intestines, mesenteric lymph node, spleen, liver, adrenal glands, kidneys, lungs, thymus (males only), sternal bone marrow, ovaries and skin/subcutis of the hindleg. These changes are summarized and described in the following table.

Summary Test Item-Related Microscopic Findings F0– Small Intestines

 

Males

Females

Dose level (ppm):

0

1000

3000

10000

0

1000

3000

10000

 

 

 

 

 

 

 

 

 

DUODENUMa

12

12

12

14

10

10

13

10

   Foamy macrophages villi

 

 

 

 

 

 

 

 

      Minimal

-

-

-

11

-

-

-

6

      Slight

-

-

-

3

-

-

-

1

JEJUNUMa

12

12

12

14

12

10

13

11

   Foamy macrophages villi

 

 

 

 

 

 

 

 

      Minimal

-

-

3

-

-

-

2

1

      Slight

-

-

-

4

-

-

3

4

      Moderate

-

-

-

9

-

-

-

4

      Marked

-

-

-

1

-

-

-

2

ILEUMa

12

12

12

14

10

10

13

10

   Foamy macrophages villi

 

 

 

 

 

 

 

 

      Minimal

-

-

5

10

-

-

1

4

      Slight

-

-

-

3

-

-

1

-

      Moderate

-

-

-

-

-

-

-

1

a = Number of tissues examined from each group.

In the small intestines, the following changes were observed:

·    Foamy macrophages of the duodenum at 10,000 ppm in both sexes, up to slight degree.

·    Foamy macrophages of the jejunum from 3000 ppm onward in both sexes, up to marked degree.

·    Foamy macrophages of the ileum from 3000 ppm onward in both sexes, up to moderate degree.

Summary Test Item-Related Microscopic Findings F0– Mesenteric lymph node

 

Males

Females

Dose level (ppm):

0

1000

3000

10000

0

1000

3000

10000

 

 

 

 

 

 

 

 

 

MESENTERIC LYMPH NODEa

12

12

12

14

10

11

15

16

   Necrotizing granuloma(s)

 

 

 

 

 

 

 

 

      Minimal

-

3

2

-

-

-

1

1

      Slight

-

2

5

3

-

3

5

3

      Moderate

-

-

1

3

-

-

6

9

      Marked

-

-

-

2

-

-

-

1

   Extranodal inflammation

 

 

 

 

 

 

 

 

      Minimal

-

-

-

3

-

-

5

2

      Slight

-

-

1

-

-

1

2

3

      Moderate

-

-

-

2

-

-

-

4

   Foamy macrophages

 

 

 

 

 

 

 

 

      Minimal

-

3

-

1

-

4

1

1

      Slight

-

9

4

6

-

6

6

6

      Moderate

-

-

7

4

-

1

8

8

      Marked

-

-

1

3

-

-

-

1

   Macrophage foci

 

 

 

 

 

 

 

 

      Minimal

1

2

1

-

2

2

-

-

      Slight

-

1

-

-

-

7

1

-

      Moderate

-

1

-

-

-

1

-

-

   Lymphangectasia

 

 

 

 

 

 

 

 

      Slight

-

-

-

1

-

-

1

-

a = Number of tissues examined from each group.

In the mesenteric lymph node, the following changes were observed:

·    Necrotizing granuloma(s) (i.e. granuloma with central necrosis) from 1,000 ppm onward in both sexes, up to marked degree.

·    Extranodal inflammation (peritonitis) from 3000 ppm onward in both sexes from, up to moderate degree.

·    Foamy macrophages (foci and in sinusoids) from 1000 ppm onward in both sexes, up to marked degree.

·    Macrophage foci (not foamy) at increased incidence and severity (up to moderate) at 1000 ppm in both sexes.

- Lymphangectasia at slight degree in one 3000 ppm female and one 10,000 ppm male.

Summary Test Item-Related Microscopic Findings F0– Kidneys

 

Males

Females

Dose level (ppm):

0

1000

3000

10000

0

1000

3000

10000

 

 

 

 

 

 

 

 

 

KIDNEYSa

12

12

12

14

10

10

12

11

   Vacuolation glomerular podocytes, foamy

 

 

 

 

 

 

 

 

      Minimal

-

-

-

1

-

-

-

-

      Slight

-

-

-

3

-

-

-

2

      Moderate

-

-

-

10

-

-

-

9

   Intranuclear inclusion bodies, tubular

 

 

 

 

 

 

 

 

      Minimal

-

-

-

7

-

-

-

2

      Slight

-

-

-

1

-

-

-

5

      Moderate

-

-

-

-

-

-

-

2

a = Number of tissues examined from each group.

In the kidneys, the following changes were observed:

·    Foamy vacuolation of glomerular podocytes at 10,000 ppm in both sexes, up to moderate degree.

·    Intranuclear inclusion bodies, tubular at 10,000 ppm in both sexes, up to moderate degree.

Summary Test Item-Related Microscopic Findings F0– Bone Marrow (sternum)

 

Males

Females

Dose level (ppm):

0

1000

3000

10000

0

1000

3000

10000

 

 

 

 

 

 

 

 

 

BONE MARROWa

12

12

12

14

10

10

12

10

   Increased myelopoiesis

 

 

 

 

 

 

 

 

      Minimal

-

6

11

6

2

2

9

3

      Slight

-

-

-

3

-

-

-

4

a = Number of tissues examined from each group.

In the bone marrow (sternal), the following change was observed:

·   Increased incidence ofincreased myelopoiesisin males starting at 1,000 ppm and in females starting at 3000 ppm, up to slight degree.

Summary Test Item-Related Microscopic Findings F0– Ovaries

 

Females

Dose level (ppm):

0

1000

3000

10000

 

 

 

 

 

OVARIESa

24

11

13

24

   Necrotizing granuloma(s)

 

 

 

 

      Slight

-

-

-

3

   Foamy cell aggregates

 

 

 

 

      Minimal

-

-

5

5

      Slight

-

-

4

4

a = Number of tissues examined from each group.

In the ovaries, the following changes were observed:

·    Necrotizing granuloma(s) (i.e. granuloma with central necrosis) at 10,000 ppm, at slight degree.

·    Foamy cell aggregates starting at 3000 ppm, up to a slight degree.

Summary Test Item-Related Microscopic Findings F0– Spleen and Liver

 

Males

Females

Dose level (ppm):

0

1000

3000

10000

0

1000

3000

10000

 

 

 

 

 

 

 

 

 

SPLEENa

12

12

12

14

10

11

12

11

   Necrotizing granuloma(s)

 

 

 

 

 

 

 

 

      Marked

-

-

-

2

-

-

-

-

   Increase in granulocytes

 

 

 

 

 

 

 

 

      Present

-

-

9

13

-

4

8

9

LIVERa

12

12

12

14

10

12

12

12

   Necrotizing granuloma(s)

 

 

 

 

 

 

 

 

      Minimal

-

-

-

1

-

-

-

1

      Slight

-

-

-

1

-

-

-

1

      Moderate

-

-

-

1

-

-

-

3

   Serosal inflammation

 

 

 

 

 

 

 

 

      Minimal

-

-

-

-

-

-

-

1

   Increased mitosis

 

 

 

 

 

 

 

 

      Slight

-

-

-

1

-

-

-

-

      Moderate

-

-

-

1

-

-

-

-

   Sinusoidal changes

 

 

 

 

 

 

 

 

      Present

-

11

12

14

-

3

9

12

a = Number of tissues examined from each group

In the spleen, the following changes were observed:

·    Necrotizing granuloma(s) (i.e. granuloma with central necrosis) at 10,000 ppm in males, at marked degree.

·    Increase in granulocytes in the vascular sinus in females from 1000 ppm onward and in males from 3000 ppm onward.

In the liver, the following changes were observed:

·    Necrotizing granuloma(s) (i.e. granuloma with central necrosis) at 10,000 ppm of both sexes, up to moderate degree.

·    Increased mitosis in two 10,000 ppm males, up to moderate degree.

·    Serosal inflammation in one 15000/10000 ppm female (minimal).

·    Sinusoidal changes from 1000 ppm onward in both sexes. The main observation was a multifocal, scattered sinusoidal dilation, with sometimes eosinophilic contents. Occasionally, there was some Kupfer cell hypertrophy and/or increase in number of granulocytes in the bloodstream.

Summary Test Item-Related Microscopic Findings F0– Thymus

 

Males

Females

Dose level (ppm):

0

1000

3000

10000

0

1000

3000

10000

 

 

 

 

 

 

 

 

 

THYMUSa

12

12

12

14

11

4

2

10

   Lymphoid Atrophy

 

 

 

 

 

 

 

 

      Minimal

1

-

1

9

1

-

-

1

a = Number of tissues examined from each group

In the thymus, the following change was observed:

·   Lymphoid atrophy at increased incidence at 10,000 ppm in males, at minimal degree.

Summary Test Item-Related Microscopic Findings F0– Adrenal glands

 

Males

Females

Dose level (ppm):

0

1000

3000

10000

0

1000

3000

10000

 

 

 

 

 

 

 

 

 

ADRENAL GLANDSa

12

12

12

14

10

10

12

10

   Infiltrate inflamm.cells,

 

 

 

 

 

 

 

 

      Minimal

-

-

-

3

-

2

-

3

      Slight

-

-

-

2

-

-

-

-

   Vacuol.z. fasc. multifocal

 

 

 

 

 

 

 

 

      Minimal

-

1

9

9

-

4

9

5

      Slight

-

-

3

1

-

-

-

-

   Degeneration. z. reticul.

 

 

 

 

 

 

 

 

      Slight

-

-

-

-

-

-

-

1

   Serosal inflammation

 

 

 

 

 

 

 

 

      Minimal

-

-

-

1

-

-

-

-

      Slight

-

-

-

1

-

-

-

-

a = Number of tissues examined from each group

 

In the adrenal glands, the following changes were observed:

·    An increased incidence and severity of inflammatory cell infiltrate, mainly lymphocytic, at 10,000 ppm in males, up to slight degree.

·    Multifocal scattered vacuolation of the zona fasciculata from 1,000 ppm onward in both sexes, up to slight degree.

·    Degeneration of the zona reticularis in one 10,000 ppm female, at slight degree.

·    Serosal inflammation in two 10,000 ppm males, up to slight degree.

Summary Test Item-Related Microscopic Findings F0– Lung

 

Males

Females

Dose level (ppm):

0

1000

3000

10000

0

1000

3000

10000

 

 

 

 

 

 

 

 

 

LUNGa

12

12

12

14

10

10

12

11

   Alv.macroph. aggregation

 

 

 

 

 

 

 

 

      Minimal

2

4

5

4

-

1

4

5

      Slight

-

-

-

2

-

-

-

2

   Inflammation pleura

 

 

 

 

 

 

 

 

      Moderate

-

-

-

-

-

-

-

1

a = Number of tissues examined from each group

In the lung, the following changes were observed:

·    Alveolar macrophage aggregation at increased incidence and severity at 10,000 ppm in both sexes, up to slight degree.

·    Inflammation of the pleura in one 10,000 ppm female, at moderate degree.

Summary Test Item-Related Microscopic Findings F0– Skin/Subcutis Hindleg

 

Males

Females

Dose level (ppm):

0

1000

3000

10000

0

1000

3000

10000

 

 

 

 

 

 

 

 

 

SKIN/SUBCUTIS HINDLEGa

0

0

0

1

0

0

2

2

   Arthritis

 

 

 

 

 

 

 

 

      Slight

-

-

-

1

-

-

-

-

      Moderate

-

-

-

-

-

-

1

1

      Marked

-

-

-

-

-

-

1

1

   Edema subcutis

 

 

 

 

 

 

 

 

      Moderate

-

-

-

1

-

-

1

1

      Marked

-

-

-

-

-

-

1

1

a = Number of tissues examined from each group

In the skin/subcutis of the hindleg, the following changes were observed:

·    Arthritis in females starting at 3000 ppm and in one male at 10,000 ppm, up to marked degree.

·    Edema of the subcutis in females starting at 3000 ppm and in one male at 10,000 ppm, up to marked degree.

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.

P1 histopathology

Test item-related microscopic findings were noted in the ovaries (examined in all females) and in a few organs (mesenteric lymph node, foreleg, spleen) which were examined because of the presence of gross lesions. The latter organs were examined only in the animal(s) showing gross lesions in the respective organ.

 

Females

Dose level (ppm):

0

1000

3000

 

 

 

 

OVARIESa

24

13

24

  Foamy cell aggregates

 

 

 

      Minimal

-

2

7

      Slight

-

-

17

a = Number of tissues examined from each group

In the ovaries, foamy cell aggregates located in the ovarian cortex were observed starting at 1000 ppm, up to slight degree. A few 3000 ppm females showed also foamy cells in the interstitial gland and/or corpora lutea.

 

Males

Females

Dose level (ppm):

0

1000

3000

0

1000

3000

 

 

 

MESENTERIC LYMPH NODEa

0

3

24

0

0

24

   Necrotizing granuloma(s)

 

 

 

 

 

 

      Minimal

-

1

2

-

-

4

      Slight

-

-

8

-

-

4

      Moderate

-

1

8

-

-

4

      Marked

-

-

2

-

-

-

   Extranodal inflammation

 

 

 

 

 

 

      Minimal

-

-

6

-

-

7

      Slight

-

1

6

-

-

1

   Foamy macrophages

 

 

 

 

 

 

      Minimal

-

1

1

-

-

-

      Slight

-

1

8

-

-

7

      Moderate

-

-

13

-

-

15

      Marked

-

-

2

-

-

2

   Increased macroph. foci

 

 

 

 

 

 

      Minimal

-

-

-

-

-

1

      Slight

-

-

3

-

-

-

      Marked

-

1

-

-

-

-

   Lymphangectasia

 

 

 

 

 

 

      Slight

-

-

2

-

-

1

      Moderate

-

-

 

-

-

1

a = Number of tissues examined from each group.

    

In the mesenteric lymph node, the following changes were noted:

·    Necrotizing granuloma(s) (i.e. granuloma with central necrosis) in males from 1000 ppm onward and in females at 3000 ppm, up to marked degree.

·    Extranodal inflammation (peritonitis) in males from 1000 ppm onward and in females at 3000 ppm, up to slight degree.

·    Foamy macrophages (foci and in sinusoids) in males from 1000 ppm onward and in females at 3000 ppm, up to marked degree.

·    Macrophage foci (not foamy) at increased incidence and/or severity (up to marked) in males from 1000 ppm onwa

- Lymphangectasia at slight or moderate degree in two males and two females at 3000 ppm.

The remaining test item-related microscopic findings occurred at 3000 ppm in two males. In theskin/subcutis of the forelegof male no. 252 (macroscopic finding: nodule) edema of the subcutis and arthritis were seen at moderate degree. In thespleenof male no. 261 (macroscopic finding: grown together with peritoneum) slight serosal inflammation was noted.

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.

Applicant's summary and conclusion

Conclusions:
In conclusion, based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: <1000 ppm, based on adverse morphologic changes in the mesenteric lymph nodes of F0 females starting at 1000 ppm (necrotizing granulomas with extranodal inflammation).
Reproduction NOAEL: 3000 ppm, based on unsuccessful mating in most couples resulting to acyclic females following malnutrition and severe low BW at 10,000 ppm in the diet.
Developmental NOAEL: 1000 ppm, based on reduced pup body weight gain in both generations at 3000 ppm.

1000 ppm corresponds to 88 and 72 mg/kg bw/day in F0 and F1 males, respectively, and to 90 and 79 mg/kg bw/day in F0 and F1 females, respectively.
3000 ppm corresponds to 299 and 245 mg/kg bw/day in F0 and F1 males, respectively, and to 309 and 261 mg/kg bw/day in F0 and F1 females, respectively.
10,000 ppm corresponds to 1010 and 930 mg/kg bw/day in F0 males and females, respectively.
Executive summary:

A combination of a two-generation reproduction and 90 -day toxicity study was performed according to guidelines and GLP.

The test substance was administered during two generations by dietary administration to SPF-bred Wistar Han rats. In the F0-generation one control group and three treated groups were tested, each consisting of 24 males and 24 females (Groups 1-4, respectively). The dose levels (dietary concentrations) were 1000 (low dose), 3000 (mid dose) and 10,000 ppm (high dose). Up to and including Day 35, dose levels were 1500 (low dose), 5000 (mid dose) and 15,000 ppm (high dose). These initial dose levels were reduced on account of reduced body weight gain at the mid (5000 ppm) and high (15,000 ppm) dose. The F1-generation consisted of one control group and two treated groups (1000 and 3000 ppm) with 24 animals/sex/group. In the F1-generation, no 10,000 ppm group was included since the F0-generation produced only four litters at this dose level.

Males were exposed for 91 or 92 days, i.e. 10 weeks prior to mating, during mating, and up to the day prior to scheduled sacrifice. F0females of Groups 1-4 that delivered were exposed for 115-121 days (most females) or 127-128 days (one or two females of Groups 1-3), i.e. during 10 weeks prior to mating, during mating, duringpost-coitum, and during at least 21 days of lactation (up to the day prior to scheduled necropsy). F0females which failed to deliver healthy offspring were treated for 99 days (non-pregnant females of Groups 1-3) or 94 days (all 20 non-pregnant females of Group 4). Between Days 4 and 6 of lactation, litters were reduced in size to eight pups by random culling of F1-pups. After weaning, one F1-male and one F1-female of each litter of the control group and the 1,000 and 3,000 ppm groups were selected for mating with a pup of another litter of the same dose group to produce an F2-generation. F1-females were allowed to produce and rear a litter until Days 21-23 of lactation. After weaning, pups were treated for 77 days prior to mating and continuing until euthanasia; F1animals received test diet for a total of 113 or 114 days for males and 120-126 days for females.

Evaluated parameters

The following observations and examinations were evaluated: mortality / viability, clinical signs, functional observations, body weight and food consumption, food conversion efficiency, clinical biochemistry, macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio, sexual maturation and postnatal pup development (mortality, clinical signs, body weight, macroscopy and organ weights). Chemical analyses of diets were conducted on several occasions to assess accuracy, homogeneity and stability.

Results/discussion

Accuracy, homogeneity and stability of diet preparations were considered acceptable for the purpose of this study.

Formulation analysis showed that the accuracy, homogeneity and stability of diet preparations were acceptable for the purpose of this study.

Parental results:

In both generations parental toxicity was observed from 1000 ppm onwards.

Three animals of the F0-generation, i.e. one female at 3000 ppm and two males at 10,000 ppm, were prematurely sacrificed for humane reasons (between study Days 58 and 102). Their moribund condition was related to the presence of necrotizing granuloma(s) within the mesenteric lymph node, spleen and/or liver and extranodal inflammation of the mesenteric lymph node and/or arthritis of the hindleg(s). Several of these morphological findings were also noted as adverse findings in surviving animals as discussed below.

One of the key findings in this study was the occurrence of accumulation of foamy macrophages within the villi at 3000 and 10,000 ppm in parental rats of the F0- and F1-generation. This was considered to be due to absorption of the test item in the small intestines. The draining mesenteric lymph node, however, already showed an increased incidence of small foci with non-foamy and foamy macrophages (foci and in sinusoids) at the low dose (1000 ppm). At the higher doses (3000 and 10,000 ppm), those foci developed into small to medium sized foamy macrophage aggregates (foci and in sinusoids) that most likely merged into large necrotizing granulomas (i.e. a centre of necrosis, surrounded by a rim of inflammatory cells like neutrophils and (giant) macrophages) and/or extranodal inflammation into the abdominal cavity (i.e. peritonitis). This was regarded to have resulted in serosal inflammation as noted in a few other organs. Besides in the mesenteric lymph nodes, foamy cell aggregations and/or necrotizing granulomas were also noted in other organs like lung, liver, spleen and ovaries.

Secondary effects of these inflammatory processes were noted in the liver and spleen, in the form of trafficking of especially granulocytes through the vasculature of spleen and/or liver. The increased myelopoiesis observed in the sternal bone marrow (from 1000 ppm in males, from 3000 ppm in females) was believed to be secondary due to increased demand of inflammatory cells (neutrophils and monocytes), as supported by hematological data.

Occasionally, arthritis of a leg was noted at 3000 and 10,000 ppm. Since there were only a few macrophages at the site of the arthritis, there might be another pathway for this, like direct exposure after absorption and transport through blood or lymph stream, or by immune mediated processes.

The cytoplasmic foamy vacuolation of glomerular podocytes of the kidneys, observed at 10,000 ppm in both sexes, suggest that the test-item or related degradation products was/were able to pass the glomerular basement membrane. This cytoplasmic change could potentially have its effect on the filtration process.

Based on cases of moribundity at 3000 and 10,000 ppm, and severity and degenerative character of microscopic findings (granulomas/necrotizing inflammation, serosal inflammation suggestive of peritonitis/pleuritis) or passage through basement membranes (kidney)), the following findings were considered adverse for the F0-generation:

Adverse findings at 1000 ppm:

·      Mesenteric lymph node: necrotizing granulomas with extranodal inflammation in a female.

Adverse findings starting at 3000 ppm:

·      Mesenteric lymph node: necrotizing granulomas at increased incidence and severity and/or extranodal inflammation in both sexes.

·      Hindleg: arthritis in a few females.

Adverse findings at 10,000 ppm:

·      Spleen: necrotizing granulomas in males.

·      Liver: necrotizing granulomas in both sexes.

·      Kidneys: the presence of foamy cells in the podocytes of the glomeruli in both sexes.

·      Ovaries: necrotizing granulomas in females.

·      Adrenal glands: serosal inflammation in males.

·      Lung: inflammation pleura in a female.

·      Hindleg: arthritis with marked edema in a male.

Histopathological changes in parental rats of the F1-generation consisted of a non-adverse ovarian change at 1000 and 3000 ppm (described below under reproductive results). In addition, there were treatment-related macroscopic findings, the main finding being enlargement of the mesenteric lymph nodes (in two males at 1000 ppm and all animals at 3000 ppm). The other findings were for the spleen of a 3000 ppm male (grown together with peritoneum) and the skin/subcutis of a foreleg (nodule) of another 3000 ppm male. The microscopic findings in these organs with gross lesions were generally similar in nature as in the F0-generation. Based on severity and degenerative character (inflammation and/or necrosis), the following findings (all gross lesions) were considered adverse for the F1-generation:

Adverse findings at 1000 ppm:

·      Mesenteric lymph node: necrotizing granulomas with extranodal inflammation in a male.

Adverse findings at 3,000 ppm:

·      Mesenteric lymph node: necrotizing granulomas at increased incidence and severity and/or extranodal inflammation in both sexes.

·      Spleen: serosal inflammation in a male.

·      Foreleg: arthritis and edema in a male.

In-life findings in the F0and F1generation included clinical signs of toxicity, lower body weights, food intake and food conversion efficiency. For theF0generation, clinical signs were noted at 3000 and 10,000 ppm, mostly during the last few weeks of the treatment period, and mainly consisted of hunched posture, piloerection and a pale appearance.Abnormal gait and swelling of the hindlegs were occasionally observed in a few of these animals, one of which was sacrificed for humane reasons. In theF1-generation, only piloerection was observed at increased frequency at 3000 ppm, but only on one or a few days during treatment. 

Parental body weights were reduced in both sexes at 3000 (F0and F1generation)and 10,000 ppm (F0-generation)throughout the treatment period. In males, the differences from controls were about 15% (3000 ppm) and 40% (10,000 ppm) at the end of the treatment period. Body weights of females were reduced by approximately 10% (3000 ppm) or 25% (10,000 ppm) at the end of the pre-mating period and approximately 15% (3000 ppm) or 30% (10,000 ppm) at the end of the post-coitum period. During the lactation period, the differences from controls became smaller. The reduced body weight gain was accompanied by a higher relative food consumption at 10,000 ppm and lower food conversion efficiency (g body weight gain per g food consumed) at 3000 and 10,000 ppm.

Fore- and hindlimb grip strength were dose-dependently reduced inF0-males starting at 1000 ppm. Values in these animals remained in the normal range for male rats of this strain and age. Moreover, there were no corroborative clinical signs or changes in other measures in the neuromuscular domain (including gait, air righting reflex and motor activity). Therefore, these changes in grip strength were considered not to reflect impaired neuromuscular function. A relationship to the observed arthritis/edema in several animals at 3000 and 10000 ppm was not considered likely since these morphological changes were also noted in females for which mean grip strength appeared unaffected by treatment.

Several macroscopic findings at 10,000 ppm were considered to be related to the significant reduction in body weight gain at this dose level. These findings included emaciation in both sexes, pituitary changes in females (discoloration and/or reduced in size and/or gelatinous contents, without histologic correlate), reduced size and weight of the prostate gland, and reduced size of the seminal vesicles and preputial gland (without histologic correlate).

Haematology, conducted in F0parental rats at the end of the treatment period, showed treatment-related changes at 10,000 ppm and, to a lesser extent, at the lower dose levels. The 10,000 ppm animals showed changes in white blood cell parameters (higher total white blood cell count, higher percentage of neutrophils and lower percentage of lymphocytes in both sexes; lower percentage of eosinophils in females), red blood cell parameters (lower haemoglobin, haematocrit, mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) in both sexes, higher percentage of reticulocytes in females) and coagulation parameters (higher platelet count and activated partial prothrombin time in both sexes). At 3000 ppm, total white blood cells, neutrophils, lymphocytes (both sexes), MCV and MCH (males) and reticulocytes (females) were affected. Changes at 1000 ppm were limited to higher neutrophils and lower lymphocytes in females, and lower MCH in males.

The increases in neutrophils and total white blood cells were considered to be related to morphologic changes involving inflammatory cells as described above. The changes in MCV, MCH and reticulocytes at 1000 and 3000 ppm occurred in the absence of changes in main red blood cell parameters (haemoglobin, number of red blood cells) or supportive morphological changes and were therefore considered non-adverse.    

Clinical biochemistry values in F0parental rats at the end of the treatment period were affected at 3000 and 10,000 ppm, particularly in males. F0-males had higher plasma activities of alanine and aspartate aminotransferase and lower albumin from 3,000 ppm onwards, and higher potassium and lower total protein, glucose, total cholesterol and alkaline phosphatase activity at 10,000 ppm. Females at 10,000 ppm had lower values for total protein, albumin and total cholesterol.  

Treatment-related organ weight changes in F0parental rats consisted of higher relative weights of the spleen in both sexes and of the liver, kidneys and adrenal glands in males from 3000 ppm onward, and lower prostate weights at 10,000 ppm (the latter considered being secondary to the lower body weights). Test item-related higher spleen weights (absolute and relative to body weights) were noted in the 1000 ppm F1females (relative to body weights) and 3000 ppm F1males and females (absolute and relative to body weights) and higher liver weights (relative to body weights) were noted in 3000 ppm F1males. In F0-rats there were histologic correlates for the increases in liver and spleen weightsin the form of necrotizing granulomas (in F1-rats liver and spleen were not examined microscopically, except for one spleen with a gross lesion).

Reproductive results:

At 10,000 ppm, only 6/24 F0-females showed evidence of mating, four of which were pregnant (mating, fertility and conception indices were reduced to 25, 17 and 67%, respectively). Precoital time of the six mated females was prolonged (mean 5.3 days versus 2.4 in controls) and the pregnant females had less implantation sites (mean 7.8 versus 11.5 in controls). Nearly all 10,000 ppm females were acyclic (18/24) or had irregular cycles (5/24), mostly with extended di-estrus. Microscopic examination of reproductive organs showed a non-adverse treatment-related ovarian change, characterized by foamy cell aggregates. A few females had necrotizing granulomas in the ovaries, which was an adverse change. Changes in male reproductive organs were limited to a decrease in prostate weight at 10,000 ppm. Sperm parameters of treated F0males were normal. 

It was considered that the decreased body weight gain, early in the study and marked toxicity in many organs, such as necrotizing granulomas of mesenteric lymph node/spleen/liver and/or extranodal inflammation of the mesenteric lymph node (peritonitis) might have resulted in retarded maturation of the males and females at 10,000 ppm leading to unsuccessful mating.

As only four F0-females at 10,000 ppm had offspring, no F1-pups were used for mating to produce a F2-generation.

At 3000 ppm, F0- and F1-females also showed foamy cell aggregates in the ovaries, a non-adverse change. F1-males had a lower (about 20%) testicular sperm count. However, the mean testicular sperm count in these males was similar to the mean recorded in F0males at 10,000 ppm. Also, there were no adverse testicular histology findings in F0males at 10,000 ppm and F1males at 3000 ppm, and other sperm parameters showed no apparent treatment-related changes. Therefore, the lower mean testicular sperm count in F1-males at 3000 ppm was not considered to be adverse, and most likely unrelated to treatment with the test item.

Estrus cycle, morphology of male reproductive organs and female reproductive organs other than the ovaries, number of primordial and primary oocytes in the ovaries (F1only), precoital time, and mating, fertility and conception indices were not affected by treatment at 3000 ppmfor F0- and F1-females.

At 1000 ppm, there were no treatment-related changes in any of the F0and F1reproductive parameters (i.e. mating, fertility and conception indices, precoital time, number of implantations, estrous cycle, sperm parameters, and histopathological examination of reproductive organs).

Developmental results:

Since there were only four litters at 10,000 ppm, all pups of these litters were sacrificed at PND 21 or 23 as it was considered that this very limited number of litters would not result in a meaningful evaluation of the potential of this dose level to affect growth and development of the F1-offspring from conception to maturity, and the development of their offspring (F2) to weaning.

The data available from the four 10,000 ppm litters showed the following developmental changes: reduced litter size (8.0 versus 10.6 in controls); reduced pup body weights (decreases from 10% at birth to 25% at PND 21); lower organ weights (absolute brain weight; absolute and relative weights of the thymus and spleen). No changes were noted ingestation index and duration, parturition, maternal care, viability and lactation indices, sex ratio, clinical signs, macroscopy, vaginal opening and balanopreputial separation.

At 3000 ppm, F1-pups showed reduced body weight gain. Their body weights at birth were similar those of controls, but at PND 21 they had 10% lower body weights (statistically significant). In the F1-generation, but not in the F0generation, litter size was decreased at 3,000 ppm (9.5 versus 11.8 in controls - however, in comparison to control of F0 not significant!). F2-pups showed slightly reduced body weight gain, but their mean body weights did not differ statistically significantly from those of controls (body weights at PND 21 were 10 or 7% lower in male and female pups, respectively). No treatment-related changes were noted in gestation index and duration, parturition, maternal care, viability and lactation indices, sex ratio, clinical signs, macroscopy, organ weights, vaginal opening and balanopreputial separation.

At 1000 ppm, there were no treatment-related changesin any of the F1and F2pup developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care,viability and lactation indices, litter size, sex ratioand postnatal pup development consisting of mortality, clinical signs, body weight, vaginal opening, balanopreputial separation, organ weights and macroscopy).

Based on these results, the parental NOAEL was <1000 ppm based on adverse morphologic changes (necrotizing granulomas with extranodal inflammation) in the mesenteric lymph nodes of F0 females starting at 1000 ppm(corresponds to 90 and 79 mg/kg bw/d in F0 and F1 females, resp.). The NOAEL for reproduction toxicity was established at 3000 ppm based on unsuccessful mating in most couples at 10,000 ppm (3000 ppm corresponds to 299 and 245 mg/kg bw/d in F0 and F1 males, resp. and to 309 and 261 mg/kg bw/d in F0 and F1 females, resp.). The NOAEL for developmental toxicity was established at 1000 ppm based on reduced pup body weight gain in both generations at 3000 ppm (1000 ppm corresponds to 88 and 72 mg/kg bw/d in F0 and F1 males, resp., and to 90 and 79 mg/kg bw/d in F0 and F1 females, resp.). However, the lower BW is comparable for F0, F1 and F2 and remains the same over all dosing periods, so it cannot considered to be a specific developmental effect. (See atached graph).