Registration Dossier

Administrative data

Description of key information

The available data from OECD 422 study in rats involving exposures up to 54 days by gavage, did not show significant effects up to highest dose of 1000 mg/kg bw. A conservative NOAEL of 100 mg/kg was selected based on an increase of macrophage foci in mesenteric lymph nodes and macrophage foam cells in the lamina propia of small intestines. In a combined 90-day/2-generation study with dosing via diet, toxicity was observed at the higher dose groups, considered to be mediated by malnutrition from interaction with food. The study resulted to a NOAEL < 1000 ppm (72 – 90 mg/kg bw/day in males and females over both generations), based on minimal to slight necrotizing granulomas in mesenteric lymph nodes observed in some animals at the lowest dose group of 1000 ppm.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
72 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Well performed study according GLP. The LOAEL is derived from a combined 90-day/2-generation from dosing at 1000 ppm (LOAEL = 90-88 mg/kg bw in parental males and females, and 79-72 mg/kg bw in F1 males and females)
System:
gastrointestinal tract
Organ:
mesenteric lymph node

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

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

A mode of action has not been established but it is possible to suspect that the known corrosivity/irritation to be at least partially involved. The observed effects are local and they are by some interpreted as phospholipidosis, something commonly observed following treatment with cationic amphiphilic material, including marketed pharmaceuticals, and generally considered to be non-adverse.

Phospholipidosis is a plausible mechanism. In physiological circumstances, the polyamines have a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar tails easily dissolve in the membranes, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule will not easily pass membrane structures. Noteworthy in this respect is that recent research shows that the log distribution coefficient for cationic surfactants between water and phospholipid are possibly several orders of magnitude higher than between water and oil. The complex of cationic surfactant and phospholipids are difficult to digest by the macrophages, and they accumulate with the lysosomes. Recent (unpublished) studies have shown that these cationic surfactants are lysosomotropic, and scored positive for phospholipidosis in in vitro screening studies with HepG2 cells.

Additional information

ORAL

A combined 28day repeated dose study with screening for reproductive and developmental effects was performed according to OECD/EC guidelines and GLP principles. Di-C16-C18 (evennumbered) alkyl tripropylenetetramine was administered in corn oil by daily oral gavage to male and female rats at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41-54 days). Analysis of the formulations confirmed target exposure concentrations and showed that formulations were stable.

 

No test substance related mortality occurred during the study and no treatment related toxicologically relevant clinical signs were observed. Functional observations of treated animals were normal. Only in the males of the highest dose group body weight gain was decreased from day 15 on, resulting in a lower mean body weigh at the end of the study (appr. -6.2% compared to controls).

Food consumption (absolute and relative) was slightly increased for females at 1000 mg/kg. (esp. relative during pregnancy & lactation) This increase did not correlate with any body weight change as it remained at control level throughout the study. As these changes were only minimal, these were not considered adverse.

There were no differences noted in haematological parameters between control and treated animals that were considered test substance related.

ALAT levels were increased in males and females at 300 and 1000 mg/kg bw/day, however biological relevance is questionable as the increase was highest at the mid-dose of 300 mg/kg. ASAT levels were increased in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day. As these increases were marginal and liver morphology was unaffected, also here effects not of large biological significance.

Males at 300 and 1000 mg/kg bw/day showed statistically significantly higher adrenal weights (absolute (with respectively +26% and +23% for the 300 and 1000 mg/kg bw/ day groups and relative to body weight (+18% for both mid and high dose groups respectively)) when compared to control values). This seems significant, but at 1000 mg the BW is most affected, whereas effects on adrenal are not different to from the 300 mg dose group. Besides, the values for the historical control (± 1 SD) are exactly around all the values.

Organ weights and organ to body weight ratios of treated females up to and including 1000 mg/kg bw/day were considered to be similar to those of control animals.

Histopathology showed diffuse hypertrophy of the cortex of the adrenal glands of males at minimal degrees in males treated at 300 (3/5) and 1000 (5/5) mg/kg bw/day.

Also an increase of macrophage foci in mesenteric lymph nodes and macrophage foam cells in the lamina propia of small intestines (minimal to slight) has been observed from 300 mg onwards, which is probably a local effect following the absorption of test material.

On overall, the administration of di-C16-C18 (evennumbered) alkyl tripropylenetetramine showed very limited toxicity up to 1000 mg/kg bw/day. Although 1000 mg/kg could possibly be selected for NOAEL, based on effects on adrenal glands of male rats (marginal increases weight, minimal diffuse hypertrophy) observed at 300 and 1000 mg/kg, a No Observed Adverse Effect Level (NOAEL) for di-C16-C18 (evennumbered) alkyl tripropylenetetramine of 100 mg/kg bw/day was established.

 

A further 90-day sub-chronic toxicity study was performed as part of a combined 90-day/2-gerenation study according to OECD 408 and 416.The test substance was administered during two generations by dietary administration to SPF-bred Wistar Han rats. Dose levels were: 1500, 50000 and 15,000 ppm in feed to groups of 24 animals per sex. After day 36 (wk 5) the levels were lowered to 0, 1000, 3000 and 10000 ppm in diet, in view of serious lagging behind of BW in the HD (High Dose) groups. As result of malnutrition resulting to very low BW, the HD females were almost all acyclic. As result, only 4 females produced a litter (which were besides low fetal BW otherwise without any adverse reproductive effects). Consequently, the HD was dropped for the next generation (F1). The F1 dose levels were 0, 1000 and 3000 ppm in diet.

The mean intake was (mg/kg bw/day):

Group

Dose level ppm

F0-males

mg/kg bw/d

F0-females

mg/kg bw/d

F1-males

mg/kg bw/d

F1-females

mg/kg bw/d

1

0

0

0

0

0

2

(1500) 1000

88

90

72

79

3

(5000) 3000

299

309

245

261

4

(15000)10000

1010

930

n.a.

n.a.

 

Duration of dosing was for 91-121 days for parental males resp. females, and 113-126 days in F1 males resp. females.

Results:

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).

In-life findings in the F0 and F1 generation included clinical signs of toxicity from 3000 ppm (incidental piloerection in a few animals and hunched posture in a few females of F0; In F1 only higher incidence of piloerection in 3000 ppm). The F0 HD group displayedpiloerection, hunched posture, Chromodacryorrhoea and pale during mating and post mating, and lean appearance.

Parental body weights were severely reduced in both sexes in F0 at 10000 ppm (40% in males and up to 30% in females) and in both F0 and F1 at 3000 ppm (15% in F0 males and females and 17% and 14% in resp. F1 males and females). The reduced body weight gain was accompanied by a higher relative food consumption and lower food conversion efficiency, indicative for a test substance related malabsorption. Most F0 HD females wereacyclic as consequence to the malabsorption with severe BW decrease.

Key findings were further the occurrence of accumulation of foamy macrophages within the villi of small intestines at 3000 and 10,000 ppm in parental rats of the F0- and F1-generation. The draining mesenteric lymph nodes showed an increased incidence of small foci with non-foamy and foamy macrophages at 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. 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. Occasionally, arthritis of a leg was noted at 3000 and 10,000 ppm. The increased neutrophils at haematological examination at 3000 and 10000 ppm is considered to be related to the inflammatory reactions.

 

With the knowledge from previous OECD 422 gavage study showing little effects up to 1000 mg/kg bw, and the kinetics study showing hardly any absorption at all, effects in this study at higher dose levels are more severe than expected. In F0 (parents) the relative food consumption for the HD does not look much affected. However, food efficiency is dropped remarkably, explaining the rapid and severe decreased BW gain. Concluded is that the effects seen in this study at the high dose levels, except for effects on the mesenteric lymph nodes, foamy macrophages and granulomas and related inflammatory reactions, which are also seen in the earlier OECD 422 study by gavage, are likely secondary to malnutrition caused by the presence of the substance in the food. 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 NOEL of around 20 mg/kg bw/day, a 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.

(In this respect, the effects seen in mesenteric lymph nodes by di-C16-C18 (evennumbered) alkyl tripropylenetetramine are similar to those seen for various other polyamines, expect that the dose levels for it to occur are much higher for di-C16-C18 (evennumbered) alkyl tripropylenetetramine)

 

DERMAL

Manufacture and use are highly controlled. Its use is limited to industrial and professional users where its severe irritating properties will provide for sufficient protection measures to prevent exposure. Furthermore, dermal uptake can be expected to be very limited (mw ~ 700; Pow > 10), and as consequence, effects will be characterised by local irritation rather than by systemic toxicity following dermal uptake. Animal testing via dermal application is therefore not indicated.

 

INHALATION

Likelihood of exposures via inhalation is low considering that di-C16-C18 (evennumbered) alkyl tripropylenetetramine is a viscous fluid with a high boiling point (> 400 °C) and very low vapour pressure (1.3 x 10-6 Pa at 20°C). Its use is limited to industrial and professional users and does not involve the forming of aerosols, particles or droplets of an inhalable size. So exposure to humans via the inhalation route will be unlikely to occur. Furthermore, the substance is classified as corrosive, whereas repeated dose studies by oral route indicated low systemic toxicity. This means that effects by inhalation will rather be characterised by local irritation than systemic toxicity.

Justification for classification or non-classification

Available data shows low systemic toxicity for di-C16-C18 (evennumbered) alkyl tripropylenetetramine. The effects observed at LOAEL of 1000 ppm (72-90 mg.kg bw) are not considered significant health effects that can impair function. No STOT-RE classification is therefore warranted.