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Administrative data

Description of key information

Oral (OECD 407), rat: NOAEL = 50 mg/kg bw/day

Oral (OECD 422), rat: NOAEL ≥ 75 mg/kg bw/day

WoE conclusion: NOAEL = 75 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
4 June - 2 July 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, UK
- Age at study initiation: 28 ± 2 days
- Weight at study initiation: 133 - 151 g (males), 127 - 149 g (females)
- Housing: 5 animals of the same sex per cage in stainless steel cages with stainless steel grid floors
- Diet: pelleted SDS Rat and Mouse No. 1 modified maintenance diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.5 - 27
- Humidity (%): 48 - 74
- Air changes (per hr): approx. 12 -15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: All formulations were prepared freshly each week and stored at 4 °C in the dark.

VEHICLE
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analysed concentrations of the test substance in corn oil, for the formulations prepared in Week 1 of the study, were within -2 to -3% of the nominal concentrations and were considered to be within acceptable limits for use on the study. These results also confirm that specimen formulations were homogeneous and stable during ambient temperature storage for 2 days and refrigerated storage for 15 days.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily, 7 days/week
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous dose range-finding toxicity study. Groups of 6 rats (3 males and 3 females) were treated with test substance in corn oil at dose levels of 50, 150 and 250 mg/kg bw/day for 7 days. A control group received the vehicle alone. Significant reduced body weight gain (-40 and -25% in males and females, respectively) was recorded in both sexes at 250 mg/kg bw/day. Based on these observations, dose levels of 15, 50 and 150 mg/kg bw/day were considered to be suitable for the main study.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included: mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed at intervals after dosing each day for signs of ill health, behavioural changes, reaction to treatment or ill health for the first two weeks of treatment. As the clinical signs followed a constant pattern the detailed check was then made twice each week over the remainder of the treatment period. Any observed changes were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed one week prior to dosing when allocated to treatment groups (Week -1), prior to dosing on Day 1 (Week 0) and subsequently at twice weekly intervals throughout the study. The last measurement was performed on Day 28.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: The quantity of food consumed in each cage was measured at weekly intervals throughout the study.

FOOD EFFICIENCY:
- Food conversion ratio calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily monitoring by visual appraisal was maintained throughout the dosing period. However, visual assessment revealed a higher apparent intake of water among animals receiving 150 mg/kg bw/day, thus an accurate assessment of water consumption was undertaken over seven consecutive days during Weeks 3 and 4 of the treatment period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to study termination on Day 29
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters examined: packed cell volume (PCV), haemoglobin (Hb), red blood cell count (RBC), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), total white cell count (WBC total), differential WBC counts (neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M), large unstained cells (LUC)), cell morphology (anisocytosis (anis), micro/macrocyosis (micro/marco), variation in colour (var), hypo/hyperchromasia (hypo/hyper), left shift (LS), atypical/blast cells (atyp/blast)), prothrombin time (PT), activated partial thromboplastin time (APTT); In case of atypical/blast cells or other abnormalities, confirmation or a written description from a blood film was made.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to study termination on Day 29
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters examined: glucose (hexokinase mediated), total protein, albumin (Alb), albumin/globulin ratio (A/G), urea nitrogen (urea nitr), creatinine, alkaline phosphatase (AP), alanine transaminase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (γ-GT), total bilirubin, sodium (Na), potassium (K), calcium (Ca), chloride (Cl), inorganic phosphorus (P), cholesterol (Chol), triglycerides (Tri-glyc)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: before initiation of treatment and during Week 4 of treatment (complete assessment); during Week 1, 2 and 3 (shortened assessment)
- Dose groups that were examined: all animals from each dose group
- Battery of functions tested: presence and occurrence of convulsions, tremors, twitches, ease of removing the animal from the cage, ease of handling the animal, salivation/lacrimation, palpebral closure, exophthalmus, piloerection, vocalisation on handling, level of activity in the arena, level of arousal, rearing count, grooming, assessment of gait, record presence of faecal boluses, urine; the following manipulations were only made during complete assessment: approach response, touch response, startle response, righting reflex, tail pinch response, pupil reflex, grip strength (fore and hindlimb), landing foot splay, body temperature
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The macroscopic appearance of the tissues of all rats was recorded and samples of the following tissues were preserved: adrenals, brain (medullary, cerebellar and cerebral sections), caecum, colon, duodenum, epididymides, femur (with joint), head#, heart, ileum (incl. Peyer's patches), jejunum, kidneys, liver, lungs (all lobes, mainstem bronchi), lymph nodes (mandibular and mesentric), oesophagus, ovaries, pancreas#, prostate, rectum, sciatic nerve, seminal vesicles, spinal column (to preserve a sample of spinal cord from the cervical region), spleen, sternum (for bone and marrow sections)#, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus (with cervix), vagina, other macrocopically abnormal tissue.

#: Tissue not required for histopathology.

Testes/epididymides were fixed in Bouin's solution and then transferred to 70% industrial methylated spirits. All other tissues were preserved in buffered 10% formalin.

The following ogans from each animal were dissected free of fat and weighed: adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus.

HISTOPATHOLOGY: Yes
Microsccopic examination of prepared slides (from tissues indicated under gross pathology) was carried out for all animals of the control and high-dose group. Microscopic examination was extended to the liver, thymus and thyroid for all animals of the low- and middle-dose group following treatment related changes to these tissues being observed among the high-dose group. Macroscopic abnormalities were processed and examined for any animal.

Fixed tissue samples required for microscopic examination were prepared by embedding in paraffin way; sections were cut at 4 µm and stained with heamatoxylin and eosin. A transverse section of each testis (left and right) and a full longitudinal section of each epididymis (left and right) were stained with Periodic Acid Schiff (PAS)-heamatoxylin.
Statistics:
All statistical analyses were carrid out separately for males and females. For all parameters, the analyses were carried out using the individual animal as the basic experimental unit. Body weight data were analysed using weight gains. Food consumption data could not be analysed statistically due to the small group size (one cage/sex/group).
If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75%), the proportion of animals with values different from the mode was analysed (Fisher, 1950 and Mantel, 1963).
Otherwise: A test was applied to test for heterogeneity of variance between treatments (Bartlett, 1973). Where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-was analysis of variance was carried out. If significant heterogeneity of variance was present and could not be removed by a transformation, an analysis of ranks was used (Kruskal-Wallis, 1952/3).
Analyses of variance were followed by Student's t-test and Williams' test (Williams, 1971/2) for a dose related response, although only the one thought most appropriate for the response pattern observed was reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of these tests (Shirley, 1977).
Where appropriate, analysis of covariance was used in place of analysis of variance in the above sequence. For organ weight data, analysis of variance was performed using terminal body weight as covariate when the within group relationship between organ weight and body weight was significant at the 10% level (Angervall and Carlstrom, 1963), in an attempt to allow for differences in body weight which might influence the organ weights.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Transient post dose salivation was observed in animals of both sexes in all treated groups, principally from the second week of treatment. The incidence was dosage-related with all animals affected at 150 mg/kg bw/day. Paddling forepaws were also noted after dosing from Week 2, affecting both males and females at 50 and 150 mg/kg bw/day, and was considered most likely to be grooming in response to the salivation. Wet coat was observed one hour after dosing on Day 1, amongst three males and two females treated at 150 mg/kg bw/day, also considered to be associated with post dose salivation.
Other findings observed on one or more occasions after dosing at 150 mg/kg bw/day included piloerection, walking on toes and hunched posture.
Mortality:
no mortality observed
Description (incidence):
No unscheduled deaths occurred during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the first week of treatment, a significant and dose-related reduction in group mean body weight gain was noted amongst males at 50 and 150 mg/kg bw/day. During the remaining treatment period, body weight gain among males at 150 mg/kg bw/day continued to be significantly reduced, while body weight gain for males at 50 mg/kg bw/day was only marginally below that of the controls. Overall body weight gain among all treated groups of females was not statistically affected.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Group mean food consumption was not statistically affected by the treatment.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Analysis of the food conversation ratios for the treatment period revealed slightly reduced efficiency of food utilisation for males at 150 mg/kg bw/day (FCR = 4.3 treated vs. 3.4 control group), indicating that the reduction in weight gain for males at this dosage could not be wholly explained by the reduction in food consumption and may indicate some degree of toxicity.

FCR (weeks 1-4): males: 3.4, 3.6, 3.6, 4.3 for 0, 15, 50, 150 mg/kg bw/day group
FCR (week 1-4): females: 5.2, 5.5, 6.0, 5.8 for 0, 15, 50, 150 mg/kg bw/day group

FCR: food conversion ratio = food consumption (g) / bodyweight (g)
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Following visual assessment of water bottle residues in which a higher water intake was suspected among animals treated at 150 mg/kg bw/day, accurate recording of water consumption was introduced during weeks 3 to 4. Group mean water consumption was increased (+16 and +20% for males and females, respectively) at 150 mg/kg bw/day. Group mean water consumption for all other treated groups was considered to be essentially similar to controls throughout the 7-day period of measurement.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
A number of parameters attained statistical significance in comparison with concurrent controls but intergroup differences were generally slight and showed no consistent trend between the sexes or relationship to dosage.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Biochemical analysis revealed a significant increase in cholesterol values for males and females at 150 mg/kg bw/day.
Minor changes were observed in Alb, ALT, AST, urea nitrogen and total protein levels either in males or females without statistical significance. The differences were considered to be of little toxicological importance.
Electrolyte concentrations among treated groups occasionally attained statistical significance but intergroup differences were generally slight and showed no dosage-relationship or consistent trend between the sexes and were therefore considered to be of no toxicological importance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Treatment with the test substance for 28 days was not associated with any behavioural changes which were considered indicative of neurotoxicity.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Significantly decreased mean thymus weights among all treatment groups in both sexes were observed. The decrease for males was dose-related. However, differences from control were slight for both sexes at 15 mg/kg bw/day.
Mean liver weights were slightly increased without statistical significance among males treated with 150 mg/kg bw/day and females at 50 and 150 mg/kg bw/day.
Slightly decreased adrenal and epididymides weights were observed in males at 150 mg/kg bw/day.
All other intergroup variations were generally slight, inconsistent between the sexes and were not dose-related. Therefore, the findings were considered not to be of toxicological significance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination did not reveal any changes that were considered to be attributable to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examination revealed minimal/slight apparent cell loss and/or inflammatory cell infiltration in the centrilobular regions accompanied with hypertrophy of the centrilobular hepatocytes in both sexes at 150 mg/kg bw/day. These changes were considered to be degenerative, although the lack of other hepatic changes in the enzyme biochemistry profile may indicate an early change. The slight reduction in colloid in the follicles of the thyroid noted among both sexes at 150 mg/kg bw/day, may indicate an early stage of hypertrophy of the follicular epithelium and as such could be a secondary effect of the changes induced by treatment in the liver. Hepatic microsomal enzyme induction in rats can result in an imbalance in the feedback control of thyroid secretion causing the change observed in the thyroids.
Decreased thymus weight together with involution/atrophy of the thymus was also noted among all treated animals, although the toxicological significance of this change is unclear.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1. Body weight gain in males (g/animal).

 

Dose level (mg/kg bw/day)

0

15

50

150

Week 0 - 1

56.4 ± 5.5

53.3 ± 4.2

47.2 ± 7.1*

40.3 ± 4.1**

Week 1 - 4

161.7 ± 20.2

141.3 ± 13.2

146.3 ± 21.0

122.7 ± 11.8**

Week 0 – 4

218.1 ± 25.2

194.6 ± 15.3

193.5 ± 27.6

163.0 ± 10.6**

* (p < 0.05); ** (p < 0.01) significantly different from controls

Table 2. Cholesterol values in males and females at study termination (mean values).

 

Dose level (mg/kg bw/day)

0

15

50

150

 

males

females

males

females

males

females

males

females

Cholesterol (mg/dL)

67

76

66

91

79

87

88*

101*

* (p < 0.05) significantly different from controls

Table 3. Thymus weights in males and females at study termination (mean values ± standard deviation).

 

Dose level (mg/kg bw/day)

0

15

50

150

males

females

males

females

males

females

males

females

Thymus (g)

Unadjusted

0.553±0.202

0.457±0.073

0.382±0.031

0.357±0.082*

0.281±0.04

0.148±0.035**

0.171±0.058

0.235±0.072**

Adjusted

0.49

-

0.37*

-

0.27**

-

0.19**

-

* (p < 0.05); ** (p < 0.01) significantly different from controls

Table 4. Incidences of histopathological findings.

 

Dose level (mg/kg bw/day)

0

15

50

150

males

females

males

females

males

females

males

females

Liver

Apparent cell loss/inflammatory cell infiltration

0/5

0/5

0/5

0/5

0/5

0/5

5/5**

5/5**

Centrilobular hypertrophy of hepatocytes

0/5

0/5

0/5

0/5

0/5

0/5

5/5**

5/5**

Thymus

Involution/atrophy

0/5

0/5

5/5**

5/5 slight

5/5**

5/5 slight

5/5**

5/5 slight

5/5**

5/5 slight

5/5**

3/5 slight

2/5 moderate

5/5**

5/5 slight

** (p < 0.01) according to Fisher’s Exact Test significantly different from controls

Conclusions:
Based on the results of this study, the NOAEL was 50 mg/kg bw in males and females due to cell loss or inflammatory infiltration and hypertrophy of the centrilobular hepatocytes accompanied by changes in biochemistry noted among both sexes at 150 mg/kg bw/day.
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
01 Dec 2016 - 19 Jun 2017
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)
Version / remarks:
Jul 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
Jul 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han), SPF
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 12 (males) and 13 (females) weeks
- Housing: Animals were housed in groups of five in Makrolon type-4 cages (height 18 cm) for pre-test and pre-mating period and for recovery animals throughout the complete study period. During the mating period, females were housed with sexually mature males (1:1) in Macrolon type-3 cages. 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). Sterilized sawdust was used as bedding with paper enrichment.
During locomotor activity monitoring (1h 44 min), animals were housed individually in a Hi-temp polycarbonate cage (48.3 x 26.7 x 20.3 cm) without cage-enrichment and bedding material.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum; during motor activity measurements, animals did not have access to food for a maximum of 1 h and 44 min
- Water: tap-water, ad libitum; during motor activity measurements, animals did not have access to water for a maximum of 1 h and 44 min
- Acclimation period: at least 5 days

DETAILS OF FOOD, WATER AND BEDDING QUALITY: Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test item and the vehicle. No correction was made for the purity/composition of the test item.

VEHICLE
- Amount of vehicle: 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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%.

Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 5 hours at room temperature.
Duration of treatment / exposure:
Males of main groups and recovery groups were treated for 32 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females of main group that delivered were treated for 52-66 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for 41-45 days. Females of recovery groups were exposed during the same period as females of main group, until at least the first scheduled necropsy of main group high dose females (i.e. 41 days). Routinely, females that are littering are left undisturbed. In this study, female nos. 51, 52, 59 (control group), 66, 67, 71 (low-dose group), 80, 83 and 85 (mid-dose group) were left out from treatment for one day as they were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.
Frequency of treatment:
once daily, 7 days/week, approx. the same time each day with a max. of 6 h difference between the earliest and latest dose
Dose / conc.:
3 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 (main groups)
5 (recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previously conducted 28-day repeated dose toxicity study dose levels of 15, 50 and 150 mg/kg bw /day were tested and a No Observed Adverse Effect level could not be determined. In this study decreased thymus weights (dose related in males) and slight (15-50 mg/kg bw/day) to moderate (150 mg/kg bw/day) thymus involution/atrophy were observed. At 50 mg/kg bw/day increased liver weights were observed in both sexes. In the 150 mg/kg bw/day group several signs of systemic toxicity were noted. Therefore, based on results of 28-day repeated dose toxicity study, in present study dose levels of 3, 15 and 75 mg/kg bw/day were selected.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily mortality/viability
- Cage side observations included: All animals were observed for general condition and clinical signs at least once daily throughout the study.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to start of treatment and at weekly intervals during treatment period

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weight on Day 1 (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

FOOD EFFICIENCY: No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy; for recovery animals additionally at the end of the treatment period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight fast
- How many animals: 5 males and females were randomly selected from the main study groups in addition to all animals from the recovery groups.
- Parameters examined: white blood cells (WBC), neutrophils, lymphocytes, monocytes, eosinophils, basophils, red blood cells, reticulocytes, red blood cell distribution width (RDW), haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelets, prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy; for recovery animals additionally at the end of the treatment period
- Animals fasted: Yes, overnight fast
- How many animals: 5 males and females were randomly selected from the main study groups in addition to all animals from the recovery groups.
- Parameters examined: total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate (inorg. phos)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Selected males of main group were tested once during Week 4 of treatment and the selected females of main group were tested once during the last week of lactation, and all recovery females were tested on Day 2 of the recovery period and again (only grip strength test) at the and of the recovery treatment.
- Dose groups that were examined: 5 males and 5 females were randomly selected from the main study groups in addition to all recovery animals
- Battery of functions tested: hearing ability, reflex, pupillary reflex, static righting reflex, fore- and hind-limb grip strength test, locomotor activity test

IMMUNOLOGY: No

OTHER:
THYROID HORMONE ANALYSIS:
- Time schedule for collection of blood: F0-generation, males and females: Samples were collected on the day of necropsy from main group females on Day 14-16 of lactation, non-mated main group females, non-pregnant main group females and all main group males after at least 4 weeks of treatment. For the recovery animals, samples were taken at the end of the treatment period and on the day of scheduled necropsy. No samples were collected from animals that were sacrificed in extremis.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: T4: all male animals of main and recovery groups, females of vehicle control and high dose group of main and recovery group; TSH: males and females of vehicle control and high dose groups of main and recovery groups
- Parameters examined: thyroxine (T4), thyroid stimulation hormone (TSH)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All males of the main group were sacrificed after mating (min. of 28 days of dose administration) and females of the main group were sacrificed on Day 14-16 post-partum. All animals of the recovery group were sacrificed 2 weeks after final dosing. Animals were sacrificed in extremis if pain, distress or discomfort was considered not transient in nature or was likely to become more severe. After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

ORGAN WEIGHTS: Yes
Animals were fasted overnight prior to necropsy and body weights were recorded on the day of necropsy. Organs were weighed and organ-to-body weight ratios were calculated. 5 males and 6 females were randomly selected from the main study animals in addition to all recovery animals for necropsy. Following organs were weighed for selected animals: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles (including coagulating glands), spleen, testes, thymus, thyroid (including parathyroid if detectable), uterus (including cervix). For all remaining animals: epididymides, prostate, seminal vesicle (including coagulating glands), testes, thyroid (including parathyroid if detectable)

HISTOPATHOLOGY: Yes
Tissue preservation and slide preservation:
Samples of the following tissues and organs were collected for selected 5 animals per group and sex of recovery and main groups and were fixed in 10% buffered formalin or modified Davidson's solution (epididymides and testes): adrenal glands, brain (cerebellum, mid-brain and cortex), caecum, cervix, clitoral gland, colon, coagulation gland, duodenum, epididymides, esophagus, eyes (with optic nerve and Harderian gland), heart, ileum, jejunum, kidneys, liver, lung (infused with formalin), lymph nodes (mandibular, mesenteric), ovaries, Peyer´s patches, pituitary gland, preputial gland, prostate gland, rectum, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, mid-thoracic, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid (including parathyroid), trachea, urinary bladder, uterus, vagina, all gross lesions

5 males and 5 females were randomly selected from the main vehicle control and high dose group in addition to all recovery animals for tissue preparation and examination by an pathologist. Additionally, the follwing organs/tissues were examined:
- slides of testes of the selected 5 main group males of vehicle control and high dose groups and all main group males that failed to sire to examine staging of spermatogenesis
- preserved organs of animals euthanized in extremis and of all males of main group that failed to sire to examine staging of spermatogenesis
- all gross lesions of all dose groups
- thymus and spleen of all selected 5 main group animals of low- and mid-dose group and all recovery animals (males and females) and liver and mesenteric lymph nodes of all selected 5 main group animals of low- and mid-dose group and all recovery animals (males), based on (possible) treatment-related changes in these organs in high dose group
- the reproductive organs of all main group males that failed to sire and all main group females that failed to deliver healthy pups


Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. 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):
At 75 mg/kg bw/day, rales were observed in 5 out of 15 males, ranging from one day to 20 days during the treatment period.
Salivation seen after dosing among animals of the 3 (one male for one day only), 15 and 75 mg/kg bw/day dose groups during the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
Incidental findings that were noted included focal erythema, scales, scabs and alopecia. Rales were noted for individual males in the vehicle control and 15 mg/kg bw/day dose groups for one day only. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered to be signs of toxicological relevance.
No findings were noted during the arena observations in this study
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test item. One female at 3 mg/kg bw/day was sacrificed due to delivery difficulties on Day 23 post-coitum.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No adverse changes in body weights and body weight gain were noted up to 75 mg/kg bw/day.
Treatment related reduced body weight gain was observed at 75 mg/kg bw/day in males from the first week of treatment onwards and in females starting in the second week of treatment. Body weights of 75 mg/kg bw/day treated males were approximately 6% and 8% lower compared with concurrent controls at the end of treatment and end of recovery, respectively. In 75 mg/kg bw/day treated recovery females body weights were approximately 8% lower compared with concurrent vehicle controls at the end of the mating period (partially due to body weight gain of one female which mating was overlooked). At the end of recovery, the difference was approximately 2%.
During the post-coitum phase, females treated at 75 mg/kg bw/day had lower body weights compared to the control group starting from Day 0 post coitum (statistically significant from Day 4 post-coitum onwards). Females in this dose group had similar body weight gain as concurrent controls up to Day 11 post-coitum, which subsequently stagnated from Day 14 post-coitum onwards. The lower body weights from Day 0 post-coitum onwards were resulting from the reduced body weight gain during the first weeks of treatment, whereas the reduced body weight gain during the last part of the post-coitum phase was considered to be due to the pregnancy status of these females (i.e. implantation sites only).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No adverse effect on food consumption was observed up to 75 mg/kg bw/day.
Food consumption was reduced in the first week of treatment at 75 mg/kg bw/day compared with the concurrent controls (not statistically significant).
During the post-coitum phase food consumption before or after allowance for body weight was slightly reduced in 75 mg/kg bw/day treated females compared to the concurrent controls. This was considered not adverse but due to the pregnancy status of these females (i.e. implantation sites only). Values at 75 mg/kg bw/day were in line with that of untreated, nulliparous females of this strain and age.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Note: Females at 75 mg/kg bw/day had no live offspring and the recovery females were not mated. Several haematology parameters are known to be influenced by physiological status (lactating versus no former pregnancy or former pregnancy but no offspring). Therefore, results for non-lactating 75 mg/kg bw/day treated females were compared to non-mated recovery females.
The following (statistically significant) changes in haematology parameters distinguished treated animals from control animals at the end of treatment:
- (Slightly) lower percentage of reticulocytes at 75 mg/kg bw/day at the end of treatment (statistically significant in males only) and at the end of recovery (statistically significant in females only)
- Lower mean corpuscular volume (MCV) in 75 mg/kg bw/day treated males (end of treatment) and lower mean corpuscular haemoglobin concentration (MCHC) in 75 mg/kg bw/day treated females at the end of treatment and after recovery
The above changes remained within the normal range for rats of this age, strain and applicable physiologically status and were considered not adverse.
Values for the percentages of neutrophils and lymphocytes in 3 and 15 mg/kg bw/day treated females achieving a level of statistical significance when compared to controls, were considered to have arisen as a result of slightly high or low control values and in the absence of a treatment-related distribution not considered to be toxicologically relevant.
The statistically significantly decrease in red blood cells in 15 mg/kg bw/day treated females was within the normal limits and at 75 mg/kg bw/day red blood cell counts appeared unaffected by treatment, therefore the decrease at 15 mg/kg bw/day was not attributed to treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Note: Females at 75 mg/kg bw/day had no live offspring and the recovery females were not mated. Several clinical biochemistry parameters are known to be influenced by physiological status (lactating versus no former pregnancy or former pregnancy but no offspring). Therefore, results for non-lactating 75 mg/kg bw/day treated females were compared to non-mated recovery females at the end of treatment.
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Increased total bilirubin levels at 75 mg/kg bw/day in males
- Increased levels of total protein and albumin in 75 mg/kg bw/day treated females
These statistically significant changes at 75 mg/kg bw/day were not considered to be toxicologically significant as they remained within the range considered normal for rats of this age and strain.
Values at the end of recovery achieving a level of statistical significance when compared to controls, were considered of no toxicological relevance in the absence of a treatment related effect at the end of treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease in thymus weight was recorded in females at 15 mg/kg bw/day (relative to body weight) and in both sexes at 75 mg/kg bw/day (absolute and relative to body weight). The microscopic correlate for this finding was lymphoid atrophy. The weight decrease was still present after a 14-day treatment free period. A statistically significant increase in liver weight was noted in males at 75 mg/kg bw/day (relative to body weight only). The microscopic correlate for this finding was centrilobular hepatocellular hypertrophy. This was not present after the 14-day recovery period.
The statistically significant decrease in liver weight in females at 75 mg/kg bw/day is considered to be related to the physiological state of these animals: Females at 75 mg/kg bw/day were not lactating in contrast to the females of the control group. Therefore this weight decrease was regarded unrelated to the treatment with the test item.
Some organ weight differences were statistically significant when compared to the control group but were considered to be the result of a lower final body weight or were considered not to be test item-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values. There were no other test item-related organ weight changes.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was one test item-related macroscopic finding: A reduced size of the thymus was recorded in 1 out of 10 females at 75 mg/kg bw/day at the end of treatment (microscopic correlate: lymphoid atrophy).
All of the remaining macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of treatment, a potential treatment related decrease of the hind leg grip strength was observed in the 75 mg/kg bw/day treated recovery females, consequently grip strength assessment was repeated at the end of recovery for these animals. However, as this decrease was not observed in (combination with) the main 75 mg/kg bw/day treated females and the effect was not present at the end of recovery, this was considered to be a chance finding and not toxicologically relevant.
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected main and recovery animals.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period a decreasing trend in activity over the duration of the test period.
The slight increase of ambulations in the 75 mg/kg bw/day treated males was not statistically significant and remained within the normal variation.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with the test substance were noted in the thymus of both sexes at 75 mg/kg bw/day and in the liver and spleen of males at 75 mg/kg bw/day.
Thymus: An increased incidence and/or severity of lymphoid atrophy was present in the thymus of males and females at 75 mg/kg bw/day. After a 14-day recovery period this finding showed complete recovery.
Spleen: A minor increase in incidence and severity of pigmentation, yellow-brown was recorded in males at 75 mg/kg bw/day. After a 14-day recovery period this finding showed complete recovery.
Liver: An increased incidence and/or severity of inflammatory cell infiltrate (slight), macrovesicular vacuolation (minimal) and hepatocellular hypertrophy (minimal) was recorded in the liver of males at 75 mg/kg bw/day. After a 14-day recovery period these findings showed complete recovery.
There were no other test item-related histologic changes. 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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
THYROID HORMONE ANALYSES:
Note: Females at 75 mg/kg had no live offspring and the recovery females were not mated. To account for possible differences in thyroid hormone levels due to the physiological status of these females, results for non-lactating 75 mg/kg bw/day treated females were compared to non-mated recovery females at the end of treatment.
Serum levels of T4 in F0 males were statistically significantly decreased at 75 mg/kg bw/day at the end of treatment. At the end of recovery T4 serum levels remained reduced compared with concurrent controls (not statistically significant). At the end of treatment serum levels of TSH were unaffected in 75 mg/kg bw/day treated males, therefore the significantly reduced serum levels at the end of recovery in males of this dose group were considered not toxicologically relevant.
The T4 serum levels in F0 females were unaffected by treatment at 75 mg/kg bw/day. At the end of treatment, serum TSH levels of 75 mg/kg bw/day treated females were slightly increased compared to the non-mated recovery females of the control group. As this effect was only slight and not statistically significant, this was considered not treatment related. The lower TSH serum levels for both controls and 75 mg/kg bw/day treated recovery females at the end of recovery were considered to be a chance finding.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at 75 mg/kg bw/day (highest dose tested)
Critical effects observed:
no

Table 1. Mean percent organ weight differences from control groups

 

main group males (%)

 

recovery males (%)

main group females (%)

recovery females (%)

Dose level (mg/kgbw/day):

3

15

75

75

3

15

75

75

 

 

 

 

 

 

 

 

 

Thymus

 

 

 

 

 

 

 

 

Absolute

10

-16

-47**

-43**

-20

-33

-49**

-36**

Relative to body weight

12

-18

-42**

-37**

-19

-33*

-44**

-35**

 

 

 

 

 

 

 

 

 

Liver

 

 

 

 

 

 

 

 

Absolute

-3

8

4

-12

-2

1

-22**

-3

Relative to body weight

-4

5

12**

-3

1

1

-15**

-2

*: P<0.05, **: P<0.01

Table 2. Test substance related findings in the thymus (males)

 

main group

recovery group

Dose level

(mg/kg bw/day):

0

3

15

75

0

75

 

 

 

 

 

 

 

Thymus (males)a

5

5

5

5

5

5

  Lymphoid atrophy

 

 

 

 

 

 

    Minimal

-

-

-

1

-

-

    Slight

-

-

-

4

-

-

Thymus (females)a

5

7

5

5

5

5

  Lymphoid atrophy

 

 

 

 

 

 

    Minimal

1

1

-

4

-

-

a = Number of tissues examined from each group.

Table 3.Test substance related findings in the spleen (males)

 

main group

recovery group

Dose level

(mg/kg bw /day):

0

3

15

75

0

75

 

 

 

 

 

 

 

Spleen (males)a

5

5

5

5

5

5

  Pigmentation yellow-brown

 

 

 

 

 

 

    Minimal

5

1

1

2

2

-

    Slight

-

-

-

3

-

-

a = Number of tissues examined from each group.

Table 4.Test substance related findings in the liver (males)

 

main group

recovery group

Dose level

(mg/kg bw/day):

0

3

15

75

0

75

 

 

 

 

 

 

 

Liver (males)a

5

5

5

5

5

5

  Inflammatory cell infiltrate

 

 

 

 

 

 

    Minimal

2

2

4

-

4

3

    Slight

-

-

-

3

-

-

  Vacuolation macrovesicular

 

 

 

 

 

 

    Minimal

-

-

-

2

-

-

  Hypertrophy hepatocellular centrilobular

 

 

 

 

 

 

    Minimal

-

-

-

3

-

-

a = Number of tissues examined from each group.

Conclusions:
Based on the results of this study, the NOAEL for systemic toxicity was ≥ 75 mg/kg bw/day for males and females since no systemic adverse effects were observed up to the highest dose tested.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises adequate and reliable studies (Klimisch score 1), and are thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.
System:
hepatobiliary
Organ:
liver

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

Additional information

The test substance was tested in a 28-day repeated dose oral toxicity study according to OECD Guideline 407 and in compliance with GLP (Chambers, 1998). The test substance was administered oral by gavage, once daily, to groups of 5 male and 5 female rats for 28 consecutive days at dosage levels of 15, 50 or 150 mg/kg/day. The test substance was used as supplied and administered as a solution in corn oil at concentrations of 3, 10 and 30 mg/mL. A further group of rats (five males and five females) was held as a concurrent control receiving the vehicle (corn oil) only at the same dose volume (5 mL/kg bw/day).

Transient post dose salivation was observed among all animals receiving 150 mg/kg bw/day from the second week of treatment, and to a lesser extent, among animals of both sexes receiving 15 or 50 mg/kg bw/day. Piloerection, walking on toes, and hunched posture were also occasionally observed at 150 mg/kg bw/day. Bodyweight gain was reduced in a generally dosage related manner among all treated male groups and females receiving 50 or 150 mg/kg bw/day. Food consumption was generally considered to have been unaffected by treatment apart from an initial slight, dosage related reduction occurring among males receiving 50 or 150 mg/kg bw/day during Week 1 of treatment. A slight impairment of food utilisation was apparent among males receiving 150 mg/kg bw/day, reflecting the reduced bodyweight gain at this dose level. Measurement of water consumption during Weeks 3 and 4 of treatment revealed increased intake among both sexes receiving 150 mg/kg bw/day. Treatment with the test substance was not associated with any behavioural changes considered to be indicative of neurotoxicity. Pathological examination revealed that the liver was the principal target organ. The examination showed increased cholesterol levels in blood, increased liver weight, and minimal/slight apparent cell loss and/or inflammatory cell infiltration in the centrilobular region, together with hypertrophy of centrilobular hepatocytes observed among both sexes receiving 150 mg/kg bw/day. These changes were considered to be degenerative changes, although the lack of other hepatic changes in the enzyme biochemistry profile may indicate an early change. The slight reduction in colloid in the follicles of the thyroid noted among both sexes receiving 150 mg/kg bw/day, may indicate an early stage of hypertrophy of the follicular epithelium and as such could be a secondary effect of the changes induced by treatment in the liver. In rats, hepatic microsomal enzyme induction can result in an imbalance in the feedback control of thyroid secretion causing the change observed in the thyroids. Decreased thymus weight together with involution/atrophy of the thymus was also noted among all treated animals, although the toxicological significance of this change is unclear.

In conclusion, due to adverse effects found on the liver at the highest dose level a NOAEL of 50 mg/kg bw/day was derived.

 

The test substance was tested in a combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study according to OECD Guideline 422 and in compliance with GLP (Hartman-Van Dyck, 2017). Based on the previously conducted 28-day repeated dose toxicity study (Chambers, 1998) groups of 10 male and 10 female Han Wistar rats were treated once daily by gavage with the test substance at 3, 15 and 75 mg/kg bw/day or the vehicle (corn oil) only. For recovery,5 additional animals/sex were added to the each of the control and high dose group.Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 32 days). The females that delivered were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 52-66 days). Females that failed to deliver healthy offspring were treated for 41-45 days. The recovery males and females were not subjected to mating and were allowed a 2-week treatment-free period. The recovery females were treated for 41 days before start of recovery. Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 5 hours at room temperature.

At 75 mg/kg bw/day, rales were observed in 5 out of 15 males, ranging from one day to 20 days during the treatment period. As the rales were observed frequently, a treatment related effect could not be excluded, however, in the absence of additional findings, this was considered not adverse.

Treatment related reduced body weight gain was observed at 75 mg/kg bw/day in both sexes from the first week of treatment onwards. It was noted that individual males and females showed slight body weight loss after one week of treatment at 75 mg/kg bw/day, which subsequently recovered. As similar body weight loss was also observed in individual concurrent control females, and overall no body weight loss was observed in both sexes, this was considered not to be adverse. At the end of treatment, body weights of the Recovery females were approximately 6-8% lower compared with concurrent vehicle controls. However, as these effects were only slight and there were no clinical signs of toxicity in the high dose group, the reduced body weight gain was considered not to be adverse.

An increased incidence and/or severity of lymphoid atrophy was present in the thymus of males and females at 75 mg/kg bw/day. It should be noted that lymphoid atrophy of the thymus can be seen in non-treated lactating females, and that in this study, none of the females treated at 75 mg/kg bw/day was lactating, in contrast to the females of the control group. The lower weight of the thymus with correlating lymphoid atrophy as recorded in males and females at 75 mg/kg bw/day was considered to be test item-related. This microscopic finding showed complete recovery and was considered non-adverse at the incidences and severities recorded. The thymus weight at 75 mg/kg bw/day was still decreased after the recovery period, in the absence of concurrent microscopic findings this was considered not to be adverse. The increase in incidence and severity of pigmentation in the spleen of males at 75 mg/kg bw/day/day was only mild and without any additional indication for inflammation, degeneration or proliferation. Therefore, this finding was considered to be non-adverse. The minimal centrilobular hepatocellular hypertrophy in the liver of males at 75 mg/kg bw/day was accompanied by low grades of inflammatory cell infiltrates and macrovesicular vacuolation. These liver findings showed complete recovery after a 14-day treatment-free period. These reversible liver findings at the incidences and severities recorded were considered to be non-adverse. The significantly decreased liver weights in high dose females were considered to have arisen from the difference in physiological status between 75 mg/kg bw/day treated and control females. Serum levels of T4 and TSH in F0females were unaffected by treatment at 75 mg/kg bw/day. Serum levels of T4 in F0males were statistically significantly decreased at 75 mg/kg bw/day at the end of treatment and remained reduced after the recovery period (not statistically significant). In the absence of treatment related effects on the TSH serum levels, no microscopic findings in the thyroid and unaffected weights of the thyroids, this was considered non-adverse.

In conclusion,no parental toxicity was observed up to the highest dose level tested (75 mg/kg bw/day). Therefore, a NOAEL of 75 mg/kg bw/day for systemic toxicity was derived in male and female rats in this study.

 

Considering both studies as reliable and in a weight of evidence approach a final NOAEL of 75 mg/kg bw/day can be derived because at the OECD 422 no adverse effects were observed at 75mg/kg bw/day whereas in the OECD 407 the lowest dose with adverse effects was 150 mg/kg bw/day and no adverse effects were observed at 50 mg/kg bw/day.

Justification for classification or non-classification

The available data on repeated dose toxicity of the test substance meet the criteria for classification as STOT RE 2 (H 373, liver) according to Regulation (EC) No 1272/2008 due to cell loss or inflammatory infiltration and hypertrophy of the centrilobular hepatocytes accompanied by changes in biochemistry noted among both sexes at 150 mg/kg bw/day.