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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 September 2016 - 15 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
yes
Remarks:
There were no prostate and seminal vesicles weighing following a confusion between guideline versions 2015 and 2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3,3,5-trimethylcyclohexyl acrylate
EC Number:
289-200-9
EC Name:
3,3,5-trimethylcyclohexyl acrylate
Cas Number:
86178-38-3
Molecular formula:
C12H20O2
IUPAC Name:
3,3,5-trimethylcyclohexyl prop-2-enoate
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: the males were approximately 10 weeks old and the females were approximately 11 weeks old
- Mean body weight at study initiation: the males had a mean body weight of 459 g (range: 417 g to 500 g) and the females had a mean body weight of 274 g (range: 236 g to 304 g)
- Housing: F0 animals were individually housed, except during mating and lactation, in polycarbonate cages (Tecniplast 2154, 940 cm²) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing of F0 animals was chosen in order not to jeopardize gestations and lactations and to avoid aggressive behavior between males around mating. Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were provided to females and their litter as nesting material.
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: the males were acclimated to the study conditions for 7 days before treatment anf the females were acclimated to the study conditions for 7 days before the beginning of estrous cycle monitoring during the pre-treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 11 October 2016 to 15 December 2016.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected since it is a common route of administration which is recommended by the Regulatory Authorities for this type of study.
The dose formulations were administered by gavage using a plastic syringe fitted with a plastic gavage tube, once a day, at approximately the same time of day.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: GC-FID (Gas Chromatography with FID detection)
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Preparation process validated at 2 and 200 mg/mL
Duration of treatment / exposure:
In the males:
- 2 weeks before mating,
- during the pairing period,
- until sacrifice (4 weeks in total),

In the females:
- 2 weeks before mating,
- during the mating period,
- during gestation,
- during lactation until Day 14 post-partum inclusive,
- until sacrifice for females with no evidence of mating.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose-levels were selected in agreement with the Sponsor, on the basis of the results of a previous 2 week study where groups of five Sprague Dawley rats per sex received the test item in corn oil at 100, 300 or 1000 mg/kg/day by gavage.
There were no premature deaths. Test item-treated animals had ptyalism (all animals at 300 and 1000 mg/kg/day) and sometimes chromodacryorrhea. Mean food consumption was in high-dose males slightly lower than controls (-17%) in the last days of treatment. At necropsy, mean liver and kidneys weights were in males higher than controls (up to about +17% at the high-dose). Accentuated lobular pattern was seen in the liver of a few animals of the high-dose mainly.
Therefore, 1000 mg/kg/day was selected as the high dose-level and was expected to induce some toxicity. The low-dose and mid dose were selected using a ratio representing approximately a 3-fold interval (i.e. 300 and 100 mg/kg/day) and the low-dose was expected to be devoid of adverse effects.

- Rationale for animal assignment: stratification procedure.
Positive control:
no (not required)

Examinations

Observations and examinations performed and frequency:

MORTALITY/MORBIDITY:
- Time schedule: Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Detailed clinical examinations were performed on all animals at least once before the beginning of the treatment period and then once a week until the end of the study.

CLINICAL SIGNS:
- Time schedule: From arrival, each animal was observed at least once a day as part of routine examinations. From the start of the treatment period, each animal was observed at least once a day, at approximately the same time of day.

BODY WEIGHT:
- Time schedule: The body weight of each male was recorded on the first day of treatment (Day 1), then once a week until euthanasia including 1 or 2 days before euthanasia.
The body weight of each female was recorded on the first day of treatment (Day 1), then once a week until mated (or until euthanasia for the female with no evidence of mating) and on Days 0, 7, 14 and 20 post coitum (p.c.) and Days 1, 4, 8 and 13 p.p.

FOOD CONSUMPTION:
- Time schedule: The quantity of food consumed by each male was measured once a week from the first day of treatment until the start of the mating period.
The quantity of food consumed by each female was measured once a week from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1-4, 4-8 and 8-13 p.p.
During the mating period, food consumption was not measured for males or females.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule: The first five males and the first five females euthanized on Day 15 p.p. from each group were evaluated with a Functional Observation Battery once at the end of the treatment period. For females, this was performed on Day 14 p.p. after euthanasia of the pups.

HAEMATOLOGY:
The parameters were determined from the first five males and from five to seven females euthanized on Day 15 p.p. from each group on the day of euthanasia, after food fasting.

CLINICAL CHEMISTRY:
The parameters were determined from the first five males and the first five females euthanized on Day 15 p.p. from each group on the day of euthanasia, after food fasting.

URINALYSIS:
The parameters were determined from the first five males and the first five females euthanized on Day 15 p.p. from each group on the day of euthanasia, after food fasting.

THYROID HORMONES:
Blood samples were taken, in the first half of the morning (generally before 10 a.m.), at termination from all F0 animals, after food fasting.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
A complete macroscopic post-mortem examination was performed on all F0 animals including the female prematurely euthanized.

HISTOPATHOLOGY: Yes (see table)
A microscopic examination was performed on:
- all tissues listed in the study protocol from the first five euthanized as scheduled males and the first five females euthanized on Day 15 p.p. of the control and high-dose groups (groups 1 and 4),
- adrenal glands (males only), liver and kidneys (both sexes) from the first five euthanized as scheduled males and the first five females euthanized on Day 15 p.p. of the low- and intermediate-dose groups (groups 2 and 3),
- all macroscopic lesions of all groups,
- reproductive organs from the pair that did not mate, to investigate possible causes.
Statistics:
Body weight, food consumption: Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).
Hematology, blood biochemistry, urinalysis, hormones: Citox software was used to perform the statistical analyses.
Organ weight: PathData software was used to perform the statistical analysis

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only test item-related clinical sign was ptyalism noted at all dose-levels in a dose-related manner (incidence and duration).
The other clinical signs observed in test item groups (chromodacryorrhea, area of hair loss, cutaneous lesion, scab, desquamation) were considered to be incidental (noted in isolated animals and commonly observed in this species and strain at this age of breeding).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no unscheduled deaths in test item-treated groups.
In the control group, one male was prematurely sacrificed on Day 14. Prior to sacrifice, piloerection, dyspnea, swollen and hard abdomen were observed. This animal had the highest body weight gain in the first week of treatment which may also be related to its prostatic sarcoma.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 1.
There were no adverse effects in any sex.

In males at 1000 mg/kg/day and compared with controls, there was a slightly statistically significantly lower mean body weight gain over the treatment period which was considered to be test item-related but of limited toxicological significance as mean body weights were comparable with controls.

In females at 1000 mg/kg/day and compared with controls, there was a statistically significantly higher mean body weight change in the first days of the lactation period which was considered to be test item related but of limited toxicological significance as mean body weights during lactation were comparable with controls. There were no effects on mean body weight or mean body weight change during premating and gestation periods at any dose-levels.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See table 2.
There were no adverse effects in any sex.

In males at 1000 mg/kg/day and compared with controls, there was a slightly statistically significantly higher mean food consumption in the second week of treatment which was considered to be test item-related but of limited toxicological significance as the magnitude of difference from controls was minimal.

In females at 1000 mg/kg/day and compared with controls, there was a statistically significantly higher mean food consumption in the last days of gestation and in the first week of lactation which was considered to be test item-related but of minor toxicological significance in view of the magnitude of difference from controls.

There were no relevant effects on mean food consumption at 100 and 300 mg/kg/day.
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):
See table 5.
There were no effects in females and no adverse effects in males.

In males at 1000 mg/kg/day when compared with controls, there was a slightly higher mean hemoglobin concentration, associated with a trend towards slightly higher mean red blood cell count and mean hematocrit (PCV). In view of the slight magnitude of variation from controls, the individual data within the Historical control data in Sprague-Dawley rats of approximately the same age and in the absence of correlating pathology findings, this test item effect was considered non-adverse and may be indicative of mild dehydration together with mean urea, creatinine, proteins and albumin data (see next paragraph).

In the same group, 4/5 males tended to have a prolonged prothrombin time and a higher fibrinogen level when compared with most control data. However, the differences from controls were slight, comparable to Historical control data for prothrombin time mainly, and one control male had a higher fibrinogen level than three out of these four males given 1000 mg/kg/day. Moreover, there were no correlating findings at pathology and no signs of inflammation. An effect of the test item was considered to be unlikely.

The statistically significantly lower mean platelets count in males at 100 mg/kg/day vs. controls was considered incidental (not dose-related).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See table 6.
There were no adverse effects in any sex.

When compared with controls, mean urea concentration was higher in a dose-related manner at 300 and 1000 mg/kg/day in males and females, with statistical significance (except for females at 300 mg/kg/day). In males, mean creatinine level had a trend to be higher at both doses (one high-dose male, with the highest creatinine and urea data, had only one kidney and therefore its data may be related to this congenital malformation). Mean total proteins level was statistically significantly higher at 1000 mg/kg/day, the same tendency was observed at 300 mg/kg/day, correlating with the trend towards slightly higher mean albumin level.
These effects were considered to be test item-related but non-adverse in the absence of associated changes in pathology, as most individual data were included in Historical control data and as the effects were slight. Together with the effects on mean red blood cell parameters discussed earlier, these findings may be indicative of a mild dehydration in males.

When compared with controls, there was a trend towards higher mean triglyceride concentration at 1000 mg/kg/day in females. A relationship with the test item was unclear in the absence of correlates in pathology, in the absence of statistical significance and in absence of this effect in males (the slightly lower mean triglyceride levels in test item-treated males vs. controls was due to one high control individual data and no test item treatment effect was considered).

The statistical significance noted in mean sodium, calcium and glucose levels and mean ASAT and ALAT activities were considered to be incidental in view of the absence of dose-relationship, of the opposite effect in males and females and/or of the absence of biological/pathological significance.
The trend towards higher mean cholesterol level in males at 1000 mg/kg/day was due to 1/5 males in particular (which also had the highest mean protein and albumin levels); a relationship with the test item was considered to be doubtful.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
See table 7.
There were no adverse effects in any sex.

In males when compared with controls, there was a dose-related lower mean urine pH, reaching statistical significance at 300 and 1000 mg/kg/day. Besides, there were less magnesium ammonium phosphate crystals in males at the high-dose, which was likely linked to the lower mean pH. These effects were considered to be test item-related and non-adverse (non-severe decrease of urine pH).

Urine of males treated at 1000 mg/kg/day had the tendency to be turbid when compared with control urines. A relationship with the test item remained unclear in the absence of correlating findings in urine (white blood cells, phosphates, epithelial cells).

In females when compared with controls, most urines had a few to several calcium oxalate crystals while there were none in controls. In the absence of other urine findings or associated changes (blood calcium) in females and as these crystals are common, a relationship with the test item treatment was considered to be unlikely.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Detailed clinical examination and reactivity to manipulation and different stimuli
At 1000 mg/kg/day when compared with controls, males had a trend towards lower mean landing foot splay (59 mm vs. 73 mm). However, there were no other signs indicating problems of gait or equilibrium during FOB, there was a moderate inter-variability and mean data were poorly dose-related. This finding was considered to be unrelated to the test item treatment.
There were no effects in females.

Motor activity
In absence of correlating clinical signs and FOB findings and in view of the slight to marked inter-variability, no test item related effects were evidenced.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See table 9.
There were dose-related higher absolute and relative-to-body mean liver weights in males treated at 300 mg/kg/day and in males and females treated at 1000 mg/kg/day (up to +28% in males treated at 300 mg/kg/day in relative-to-body mean weights; p<0.01). These differences correlated with the microscopic hepatocellular hypertrophy.
Higher absolute and relative-to-body mean kidney weights were noted in males treated at 300 or 1000 mg/kg/day and correlated with the microscopic hyaline droplets and tubular dilation. No effects on the kidney weights were observed in females.
Higher absolute and relative-to-body mean adrenal gland weights were noted in males treated at 300 mg/kg/day and in males and females treated at 1000 mg/kg/day. These differences correlated with the microscopic cortical hypertrophy in males treated at 1000 mg/kg/day.
Although poorly dose-related, these findings were considered to be related with the test item administration.
All other weight differences were minimal, did not correlate with macroscopic or microscopic findings and were considered no to be test item treatment-related.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Unscheduled death
One control male sacrificed on Day 14 had a large 12 x 10 cm red mass in the prostate with red discharge which correlated with a sarcoma.

Terminal sacrifice
There were no test item-related gross findings.
All the macroscopic findings were considered to be unrelated to test item administration because they were seen in both control and test item-treated animals, were not dose-related and/or were consistent with spontaneous background findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See table 10.
Unscheduled death
One control male sacrificed on Day 14 had a large prostatic sarcoma (12 x 10 cm) which was considered to be the cause of moribundity.

Terminal sacrifice
There were dose-related findings in the liver (hepatocellular hypertrophy), in the kidneys (hyaline droplets, tubular dilation and basophilia) and in the adrenal glands (cortical cell hypertrophy) in males and females treated from 100 mg/kg/day.

. liver
Minimal dose-related centrilobular hepatocelullar hypertrophy was seen in the liver from males treated at 100 mg/kg/day, and from males and females treated at 300 or 1000 mg/kg/day.
This correlated with the increased mean liver weights and was considered to be non-adverse in view of the low magnitude of this change (i.e. grade 1) and of the absence of associated degeneration/necrosis.

. kidneys
There were dose-related minimal to moderate diffuse hyaline droplets in the tubular epithelium from the outer cortex together with tubular dilation and basophilia seen with increased incidence and severity in males treated at 300 or 1000 mg/kg/day. In females treated at all dose-levels, minimal to slight tubular dilation was observed.
These findings correlated with the increased mean kidney weights and were considered to be non-adverse in view of their low magnitude and of the absence of associated degeneration/necrosis.

. adrenal glands
Minimal cortical cell hypertrophy was noted in 3/5 males treated at 1000 mg/kg/day and correlated with the increased mean weights.
This finding was considered to be non-adverse in view of its low magnitude and of the absence of associated degeneration/necrosis.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
See table 8.
Thyroid hormones
There were no effects on mean T4 levels in F0 males.
In view of the variability in the data and taking into account that individual data were comparable with concurrent controls or with control data of similar studies, a test item effect on the slightly higher mean TSH level at 1000 mg/kg/day was considered unlikely.
Details on results:
Table 1: Body weight

Sex Male Female
Dose-level (mg/kg/day) 0 100 300 1000 0 100 300 1000
Pre-mating or whole study
Day 1 461 457 460 459 276 270 268 282
Day 8 511 509 500 497 280 278 277 293
Day 15 541 548 537 530 291 286 287 299
Day 29 576 584 575 559 / / / /
Days 1-8 +50 +52 +40 +38* +4 +7 +9 +11
Days 1-15 +83 +92 +77 +70 +14 +15 +19 +17
Days 15-29 +35 +35 +38 +29 / / / /
Days 1-29 +118 +127 +115 +99* / / / /
Gestation
Day 0 p.c. / / / / 300 297 289 312
Day 20 p.c. / / / / 465 464 454 473
Days 0-20 p.c. / / / / +165 +167 +165 +161
Lactation
Day 1 p.p. / / / / 373 362 355 369
Day 4 p.p. / / / / 370 367 363 383
Day 13 p.p. / / / / 397 396 384 410
Days 1-4 p.p. / / / / -4 +5 +8 +14**
Days 1-13 p.p. / / / / +24 +34 +30 +40*
/: not applicable, statistical significance *: p<0.05, **: p<0.01.

Table 2: Food consumption

Sex Male Female
Dose-level (mg/kg/day) 0 100 300 1000 0 100 300 1000
Pre-mating or whole study
Days 1-8 32 32 31 33 18 18 18 19
Days 8-15 32 32 34 36* 20 19 20 21
0 +6 +13 -5 0 +5
Gestation
Days 0-7 p.c. / / / / 23 22 23 25
Days 7-14 p.c. / / / / 25 25 26 28
Days 14-20 p.c. / / / / 29 28 29 34**
-3 0 +17
Lactation
Days 1-4 p.p. / / / / 34 38 38 44#
+12 +12 +29
Days 4-8 p.p. / / / / 49 52 52 59**
+6 +6 +20
Days 8-13 p.p. / / / / 63 68 67 69
/: not applicable, statistical significance: *: p<0.05, **: p<0.01, #: p<0.001.
in italic: differences from controls (%).

Table 5: Haematology and coagulation

Sex Male Female
Dose-level (mg/kg/day) 0 100 300 1000 0 100 300 1000
Red blood cell (T/L) 8.74 8.83 9.05 9.46 8.00 8.33 8.07 8.15
Hemoglobin (g/dL) 15.6 16.0 15.9 16.9* 15.3 15.4 15.3 15.3
PCV (L/L) 0.47 0.48 0.48 0.50 0.45 0.46 0.46 0.45
Platelets (G/L) 947 710* 840 861 813 880 959 848
Fibrinogen (g/L) 3.24 3.31 3.27 3.64 3.23 3.38 2.96 3.21
Prothrombin time (s) 19.6 21.3 20.5 20.1 24.0 22.2 24.6 23.4
Statistical significance: *: p<0.05.

Table 6: Blood biochemistry

Sex Male Female
Dose-level (mg/kg/day) 0 100 300 1000 0 100 300 1000
Urea (mmol/L) 4.1 4.6 5.6* 6.7** 7.5 7.8 9.0 9.9**
Creatinine (µmol/L) 33.55 34.95 36.50 37.64 37.58 38.51 38.97 36.84
Proteins (g/L) 60.0 60.6 63.1 66.3* 57.5 58.7 59.2 57.3
Albumin (g/L) 35 35 36 37 34 35 35 34
Triglycerides (mmol/L) 1.13 1.01 1.07 0.99 2.11 1.87 2.99 3.88
Cholesterol (mmol/L) 1.98 2.09 2.13 2.31 2.79 3.01 3.14 2.99
Sodium mmol/L) 145 144 145 145 141 140 139* 141
Calcium (mmol/L) 2.69 2.72 2.79* 2.77 2.63 2.61 2.62 2.59
Glucose (mmol/L) 6.91 6.33 5.54* 5.94 6.08 8.07** 6.42 6.43
ASAT (U/L) 82 86 72 67* 140 136 104* 103*
ALAT (U/L) 42 38 26* 32 92 83 74 69
Statistical significance: *: p<0.05, **: p<0.01.

Table 7: Urinalysis

Sex Male Female
Dose-level (mg/kg/day) 0 100 300 1000 0 100 300 1000
pH 7.5 6.7 6.3* 6.0** 6.4 6.4 6.1 6.5
Urine turbidity a 0 0 0.2 1.2 0 0 0 0
Magnesium ammonium phosphate crystals a 2.0 2.0 1.8 0.6 0.6 0.6 0.2 0.2
Calcium oxalate crystals a 0 0 0 0 0 0.6 0.4 2.0
Statistical significance: *: p<0.05, **: p<0.01; a: mean of grades from 0 to 4.

Table 8: Thyroid hormones

Dose-level (mg/kg/day) 0 100 300 1000
T4 (ng/mL) 34.9
±5.01 37.8
±5.72 36.4
±4.47 32.6
±4.44
TSH (pg/mL) 1083
±874 1284
±1361 1145
±547 1579
±934

Table 9: Organ weights

Sex Male Female
Dose-level (mg/kg/day) 100 300 1000 100 300 1000
- Final body weight +1 0 -4 +1 -3 -1
- Liver
.absolute +4 +19 +19 +7 +1 +12*
.relative-to-body +4 +21* +28** +8 +6 +15**
- Kidneys
.absolute +7 +16* +16* -2 -7 -1
.relative-to-body +7 +18* +24** -2 -2 +2
- Adrenals
.absolute +1 +27* +17 +2 -7 +14
.relative-to-body +1 +30** +25* +2 -3 +18*
Statistically significant from controls: *: p<0.05; **: p<0.01.

Table 10: Microscopic examination

Test item-related microscopic findings in the liver (incidence and severity*)
Sex Male Female
Dose-level (mg/kg/day) 0 100 300 1000 0 100 300 1000
Number of examined animals 5 5 5 5 5 5 5 5
Hypertrophy; hepatocellular
. grade 1 - 2 2 5 - - 2 3
-: not recorded.
*: out of five grades (minimal; slight; moderate; marked; severe).

Test item-related microscopic findings in the kidneys (incidence and severity*)
Sex Male Female
Dose-level (mg/kg/day) 0 100 300 1000 0 100 300 1000
Number of examined animals 5 5 5 5 5 5 5 5
Hyaline droplets; tubular epithelium
. grade 1 - 1 - - - - - -
. grade 2 - 4 3 1 - - - -
. grade 3 - - 2 4 - - - -
Dilation; tubule
. grade 1 1 2 2 - - 4 3 1
. grade 2 - - 2 5 - - 2 3
Basophilia; tubule
. grade 1 3 5 3 2 - - - -
. grade 2 - - 1 2 - - - -
-: not recorded.
*: out of five grades (minimal; slight; moderate; marked; severe).

Test item-related microscopic findings in the adrenal glands (incidence and severity*)
Sex Male Female
Dose-level (mg/kg/day) 0 100 300 1000 0 100 300 1000
Number of examined animals 5 5 5 5 5 0 0 5
Hypertrophy; cortex
. grade 1 - - - 3 - na na -
na: not applicable.
-: not recorded.
*: out of five grades (minimal; slight; moderate; marked; severe).

Effect levels

Dose descriptor:
NOAEL
Remarks:
systemic and local toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The test item was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, gestation and until Day 14 p.p., at dose levels of 100, 300 and 1000 mg/kg/day.
Based on the experimental conditions and results of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity (systemic and local) was considered to be 1000 mg/kg/day based on the absence of adverse effects at this dose and the non adverse findings in clinical signs, body weight change, food consumption and hematology, blood biochemistry, urinalysis and pathology,
Executive summary:

The objective of this GLP study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until Day 14 post-partum (p.p.).

This study provides information on the possible health hazards (including neurological and immunological effects) likely to arise from repeated exposure over a relative limited period of time.

Method

Three groups of ten male and ten female Sprague-Dawley rats (F0 animals) received the test item daily, by oral administration (gavage), 2 weeks before mating, during mating and, for the males, until sacrifice, for the females, throughout gestation until Day 14 post-partum(p.p.) inclusive. Another group of ten males and ten females received the vehicle (corn oil) and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used.

The concentration of the dose formulation was checked in study Weeks 1 and 6.

 

The F0 animals were checked daily during the dosing period for mortality, morbidity and clinical signs. Detailed clinical observations were performed once a week. Body weight and food consumption were recorded once a week during premating, mating (food consumption not during mating), gestation (0, 7, 14 and 20p.c.) and lactation (Days 1, 4, 8 and 13p.p.)periods.

The F0 animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until Day 14p.p.The total litter sizes and the sex of each pup were recorded after birth. The pups were observed daily for clinical signs, abnormal behaviour and external abnormalities and weighed on Days 1, 4, 8 and 13p.p.

A Functional Observation Battery (FOB) including touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, rectal temperature and motor activity was performed on five F0 animals per sex and group at the end of the treatment period. Analysis of hematology, blood biochemistry and urine parameters were performed from blood and urine samples taken prior to sacrifice from at least five F0 animals per sex and group. Thyroid hormones (T4 and TSH) plasma levels were determined from all F0 males and Day 14p.p.pups.

The F0 males were sacrificed after 4 weeks of treatment and the F0 females on Day 15p.p.Final body weights and selected organs weights were recorded and a complete macroscopicpost-mortemexamination was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on long list of organs from five F0 animals per sex in the control and high-dose groups and on all macroscopic lesions.

Pups not selected on Day 4p.p.were sacrificed and discarded without further examination.

Pups selected were sacrificed on Day 14p.p.and submitted to a detailed external examination with particular attention to the external genital organs.


Results

Chemical analyses

The test item concentrations in the analyzed dose formulations were within an acceptable range of variations (± 10% of the nominal concentrations required) and no test item was observed in the control dose formulations analyzed.

 

F0 animals

There were no unscheduled deaths in test item-treated groups andthe only test item-related clinical sign was ptyalism noted at all dose-levels in a dose-related manner (incidence and duration). There were no test item-related functional effects at FOB.

 

There were no relevant effects on mean body weights. At 1000 mg/kg/day when compared with controls, mean body weight change was lower in males over the treatment period (+99 gvs.+118 g in controls, p<0.05; limited toxicological significance) or higher in females on Days 1-4p.p.(+14 gvs.-4 g, p<0.01; limited toxicological significance). There were no effects on mean body weight change in females during premating and gestation periods.

 

At 1000 mg/kg/day when compared with controls, mean food consumption was slightly higher in males in the second week of treatment (+13%, p<0.05; limited toxicological significance) and higher in females from Day 14p.c.to Day 8p.p.(up to +29% on Days 1 -4p.p.; minor toxicologically significant).

There were no relevant effects on mean food consumption at 100 and 300 mg/kg/day.

 

At laboratory investigations, high mean urea concentration was noted in both sexes at 300 mg/kg/day (males: 5.6vs.4.1 mmol/L in controls, p<0.05; females: 9.0vs.7.5 mmol/L) and 1000 mg/kg/day (males: 6.7 mmol/L, p<0.01; females: 9.9 mmol/L, p<0.01). In males, together with high mean protein level [63.1 or 66.3 (p<0.05) g/Lat 300 and 1000 mg/kg/day respectively,vs.60.0 g/Lin controls], trend towards high mean creatinine level (36.5 or 37.6 µmol/L at 300 and 1000 mg/kg/day respectively,vs.33.6 µmol/L in controls) and albumin level (37 g/L at 1000 mg/kg/day,vs.35 µmol/L in controls), high mean hemoglobin concentration (16.9 at 1000 mg/kg/dayvs.15.6 g/dL in controls, p<0.05), trend towards slightly high mean red blood cell count and mean hematocrit (respectively 9.46 T/L and 0.50 L/L at 1000 mg/kg/dayvs.8.74 T/L and 0.47 L/L in controls), this may be indicative of a mild dehydration in males.

At urinalysis, there was a dose-related lower mean urine pH in males, statistically significant at 300 and 1000 mg/kg/day, and with the probable subsequent decrease inmagnesium ammonium phosphate crystalsat 1000 mg/kg/day.

 

There were no effects considered as test item-related on mean T4 and TSH levels.

At pathology, in animals treated at 1000 mg/kg/day, dose-related hepatocellular hypertrophy in the liver, hyaline droplets in males, tubular dilation and basophilia in the kidneys, and hypertrophy of cortical cells in the adrenals (males only) were observed and correlated with increased mean organ weights.

In animals treated at 300 mg/kg/day, dose-related hepatocellular hypertrophy in the liver, hyaline droplets, tubular dilation and basophilia in the kidneys from males were observed, and correlated with increased mean organ weights in males.

In animals treated at 100 mg/kg/day, dose-related hepatocellular hypertrophy in the liver from males only, hyaline droplets in males and tubular dilation in females.

There were no test item-related effects on estrous cycles during the first 2 weeks of treatment and on mean fertility, mating and delivery data.

 

These effects on clinical signs, mean body weight change and mean food consumption, and at laboratory investigations and pathology, were considered as test item-related and non-adverse.

 

Conclusion

The test item was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, gestation and until Day 14p.p., at dose-levels of 100, 300 and 1000 mg/kg/day.

 

Based on the experimental conditions and results of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity (systemic and local) was considered to be 1000 mg/kg/day based on the absence of adverse effects at this dose and the non-adverse findings in clinical signs, body weight change, food consumption and hematology, blood biochemistry, urinalysis and pathology,

.