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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
(Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012/2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
(Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 11 weeks
- Housing: Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm). Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm). Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature ( °C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
- Amount of vehicle: 5 mL/kg bw
Details on mating procedure:
Following a minimum of 14 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 occurred, the males and females were separated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. Samples remained on dry ice until receipt at ABL, The Netherlands, where samples were stored at 70 °C until analysis. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
In the Group 1 formulation, no test substance was detected. The concentrations analysed in the formulations of Group 2, 3 and Group 4 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of Group 2, 3 and Group 4 were homogeneous (i.e. coefficient of variation = 10%). Analysis of Group 2, 3 and Group 4 formulations after storage yielded an absolute relative difference = 10%. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 5 hours.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41 - 54 days, i.e. during 2 weeks prior to mating, during mating, during gestation, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 48 (Group 1), 52, 55, 58 (Group 2), 76 and 77 (Group 4) were not dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on results of the dose range finding study where dose levels of 0, 100, 300 and 1000 mg/kg bw/day were assessed. At 1000 mg/kg bw/day
Parental animals: Observations and examinations:
- Mortality / Viability: At least twice daily.
- Clinical signs: Daily, detailed clinical observations were made in all animals
- Body weights: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.
- Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.
Sperm parameters (parental animals):
From the selected 5 males of the control and high dose group (see Allocation), and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis.
Litter observations:
- Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera (please refer to repeated dose toxicity study)
Postmortem examinations (offspring):
- Necropsy pups: Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.
Statistics:
The following statistical methods were used to analyze 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 differences
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:
For each group, the following calculations were performed:
- Mating index (%) = (Number of females mated / Number of females paired) x 100
- Fertility index (%) = (Number of pregnant females / Number of females paired) x 100
- Conception index (%) = (Number of pregnant females / Number of females mated) x 100
- Gestation index (%) = (Number of females bearing live pups / Number of pregnant females) x 100
- Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
For each group, the following calculations were performed:
- Percentage live males at First Litter Check = (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check = (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
- Percentage of postnatal loss Days 0-4 of lactation = (Number of dead pups on Day 4 of lactation / Number of live pups at First Litter Check) x 100
- Viability index = (Number of live pups on Day 4 post partum / Number of pups born alive) x 100
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
slightly increased leucocyte counts in males and females at 100 mg/kg bw/day, primarily due to increased neutrophil counts. Increased leucocyte counts were also present in females at 30 mg/kg bw/day, and the percentage of neutrophils was increased in both sexes at this dose level.
Clinical biochemistry findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related microscopic findings were noted in the mesenteric lymph nodes and adrenal gland cortex (all dose groups). Findings in the mesenteric lymph nodes consisted of:
- foamy macrophages with/without fibrosis (up to a marked degree), which was correlated with enlargement of the mesenteric lymph nodes at necropsy.
- Sinus ectasia (up to a moderate degree) 10, 30 and 100 mg/kg bw/day, which was considered secondary to the occurrence of foamy macrophages.
- Focal necrosis within the aggregates of foamy macrophages with/without fibrosis of males at 10, 30 and 100 mg/kg bw/day (up to a moderate degree) and in females at 30 and 100 mg/kg bw/day (up to a marked degree).
- Macrophage foci up to a moderate degree (females) or marked degree (males) at 10, 30 and 100 mg/kg bw/day.

Findings in the adrenal cortex consisted of inflammatory lymphocytic cells (up to a slight degree) in females at 10, 30 and 100 mg/kg bw/day.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: See "Remarks"
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproduction toxicity was observed up to the highest dose level tested (100 mg/kg bw/day).
Critical effects observed:
not specified
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No developmental toxicity was observed up to the highest dose level tested (100 mg/kg bw/day).
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Conclusions:
No reproduction and developmental toxicity was observed for treatment up to 100 mg/kg bw/day. Therefore, a reproduction and developmental NOAEL of at least 100 mg/kg bw/day was derived.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates
EC Number:
279-632-6
EC Name:
Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates
Cas Number:
80939-62-4
Molecular formula:
not applicable
IUPAC Name:
Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 11 weeks
- Housing: Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm). Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm). Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. Samples remained on dry ice until receipt at ABL, The Netherlands, where samples were stored at 70 °C until analysis. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
In the Group 1 formulation, no test substance was detected. The concentrations analysed in the formulations of Group 2, 3 and Group 4 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of Group 2, 3 and Group 4 were homogeneous (i.e. coefficient of variation = 10%). Analysis of Group 2, 3 and Group 4 formulations after storage yielded an absolute relative difference = 10%. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 5 hours.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41 - 54 days, i.e. during 2 weeks prior to mating, during mating, during gestation, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
Frequency of treatment:
Once daily, 7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on results of the dose range finding study where dose levels of 100, 300 and 1000 mg/kg bwday were assessed.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
PARENTAL ANIMALS
- Mortality / Viability: At least twice daily.

CLINICAL SIGNS: Daily, detailed clinical observations were made in all animals, immediately after dosing (0-15 minutes after dosing). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.
FOB: The following tests were performed on the selected 5 animals/sex/group: hearing ability, pupillary reflex, static righting reflex, grip strength, locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA).

BODY WEIGHT: 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 of gestation and during lactation on Days 1 and 4.
- Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 of gestation and on Days 1 and 4 of lactation.

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters were examined: White blood cells, Differential leucocyte count: Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets. The following clotting parameters were determined in plasma:
Prothrombin Time, Activated Partial Thromboplastin Time.

CLINICAL CHEMISTRY: Yes
- Animals fasted: Yes
- How many animals: all
The following clinical biochemistry parameters were determined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline Phosphatase, Total protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.
Sacrifice and pathology:
- Necropsy: Samples of the following tissues and organs were collected from all animals and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution: Adrenal glands, (Aorta), Brain
- cerebellum, mid-brain, cortex, Caecum, Cervix, Clitoral gland, Colon, Coagulation gland, Duodenum, Epididymides, Eyes (with optic nerve (if detectable) and Harderian gland), (Male and Female mammary gland area), Femur including joint, Heart, Ileum, Jejunum, Kidneys, (Lacrimal gland, exorbital), (Larynx), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), (Esophagus), Ovaries, (Pancreas), Peyer's patches [jejunum, ileum] if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid including parathyroid if detectable, (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions. The epididymides, eyes and testes were fixed in modified Davidson's solution and Milli-Ro water and transferred to formalin after fixation for at least 24 hours. Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
- Organ weights: The following organ weights and terminal body weight were recorded from the selected 5 animals/sex/group on the scheduled day of necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus (including cervix), Prostate (weighed when fixed for at least 24 hours), Seminal vesicles including coagulating glands (weighed when fixed for at least 24 hours), Thyroid including parathyroid (weighed when fixed for at least 24 hours). All remaining males: Epididymides, Testes.
- Histopathology: The following slides were examined by a pathologist: (1) The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4. (2) The additional slides of the testes of all males of Groups 1 and 4 and all males suspected to be infertile or which died before mating to examine staging of spermatogenesis. (3) All gross lesions of all animals (all dose groups). (4) Mesenteric lymphnodes (males and females) and adrenal glands (females) of all selected 5 animals of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4. (5) The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina) from all animals of Groups 1 and 4 and from male no.17 (Group 2) that failed to sire and female no. 57 (Group 2) that failed to deliver healthy pups. (6) The preserved organs and tissues of the animals of all dose groups which died spontaneously. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Other examinations:
PUPS: Each litter was examined to determine the following, if practically possible:
- Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation.
- Necropsy pups: Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.
Statistics:
The following statistical methods were used to analyze 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 differences

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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
slightly increased leucocyte counts in males and females at 100 mg/kg bw/day, primarily due to increased neutrophil counts. Increased leucocyte counts were also present in females at 30 mg/kg bw/day, and the percentage of neutrophils was increased in both sexes at this dose level.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Spleen weights were moderately increased in both sexes at 100 mg/kg bw/day.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Enlarged spleen was observed in one male and 4 females at 100 mg/kg bw/day. Enlarged mesenteric lymph nodes were present in 5 males and all females at 100 mg/kg bw/day, in 6 females at 30 mg/kg bw/day and in 2 females at 10 mg/kg bw/day.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related microscopic findings were noted in the mesenteric lymph nodes and adrenal gland cortex (all dose groups). Findings in the mesenteric lymph nodes consisted of:
- foamy macrophages with/without fibrosis (up to a marked degree), which was correlated with enlargement of the mesenteric lymph nodes at necropsy.
- Sinus ectasia (up to a moderate degree) 10, 30 and 100 mg/kg bw/day, which was considered secondary to the occurrence of foamy macrophages.
- Focal necrosis within the aggregates of foamy macrophages with/without fibrosis of males at 10, 30 and 100 mg/kg bw/day (up to a moderate degree) and in females at 30 and 100 mg/kg bw/day (up to a marked degree).
- Macrophage foci up to a moderate degree (females) or marked degree (males) at 10, 30 and 100 mg/kg bw/day.

Findings in the adrenal cortex consisted of inflammatory lymphocytic cells (up to a slight degree) in females at 10, 30 and 100 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
LOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Treatment with the test substance by oral gavage in male and female Wistar Han rats at dose levels of 10, 30 and 100 mg/kg bw/day revealed parental toxicity at all dose levels. These findings were confined to histopathological lesions in the mesenteric lymph nodes (foamy macrophages with/without fibrosis, sinus ectasia, focal necrosis within the aggregates of foamy macrophages with/without fibrosis and macrophage foci) and adrenal gland cortex (inflammatory lymphocytic cells). The higher neutrophil counts (and white blood cell counts) at 30 and 100 mg/kg bw/day were considered to be due to these inflammatory findings. Therefore, the LOAEL was set at 10 mg/kg bw/day.