Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
OECD 422 - Combined Repeated Dose Toxicity Study and Reproductive/Developmental Toxicity Screening Study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Start: 13 Oct 2020 Experimental Completion: 15 Dec 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
Annex VIII Data Requirement

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

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:
July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
1-Chloro-6-Hydroxyhexane
IUPAC Name:
1-Chloro-6-Hydroxyhexane
Constituent 2
Chemical structure
Reference substance name:
6-chlorohexan-1-ol
EC Number:
217-925-2
EC Name:
6-chlorohexan-1-ol
Cas Number:
2009-83-8
Molecular formula:
C6H13ClO
IUPAC Name:
6-chlorohexan-1-ol
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Han
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: males: 10-11 weeks; females: 13-14 weeks
- Weight at study initiation: 250-289 g for males and approximately 217 to 256 g for females
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Makrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): drinking water
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY:

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

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Oral gavage at a constant dose volume of 5 mL/kg
Vehicle:
propylene glycol
Remarks:
Supplier: Merck Specific gravity: 1.036
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator.
Details of the preparation and dispensing of the test item have been retained in the Study Records.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during
dosing.
Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item. Any residual volumes were discarded.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure (Test Facility Study No. 20257580).
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% of target concentration.
Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was  10%.
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20257580) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20257580.
Duration of treatment / exposure:
Males: Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing,, through the pairing period and thereafter until the day before necropsy, for a minimum of 29 days.
Females that delivered were treated for 51-63 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 40-52 days.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
80 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once daily during the study

BODY WEIGHT: Yes / No / Not specified
- Time schedule for examinations: Males: weekly
Females: weekly from allocation to positive identification of mating and on Days 0, 4, 7, 14 and 20 post coitum. Dams were also weighed on Days 1, 4, 7, 13 post partum and just before to necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was quantitatively 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.

WATER CONSUMPTION AND COMPOUND INTAKE: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples for haematology, coagulation and clinical chemistry were collected, at the end of treatment period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (food deprivation)
- How many animals: 5 males and 5 females (with viable litters) of each group


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples for haematology, coagulation and clinical chemistry were collected, at the end of treatment period
- Animals fasted: Yes (food deprivation)
- How many animals: 5 males and 5 females (with viable litters) of each group


PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: at termination, the timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days.
- Animals fasted: Not specified


URINALYSIS: Yes (only males)
- Time schedule for collection of urine: during the last week of treatment
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: No
- Parameters checked in table [No.13] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during the study, toward the end of treatment
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

Sacrifice and pathology:
METHOD OF SACRIFICE

Parental animals killed for humane reasons were sacrificed by exsanguination under isoflurane anaesthesia.
Parental animals that had completed the scheduled test period, were killed by exsanguination under isoflurane anaesthesia.
offspring:Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital

NECROPSY
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means
were applied with Bonferroni correction for multiple testing. Mixed modelling techniques,
comparing six different covariance structures, were used in order to select the best fitting
statistical model.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the
overall test is significant.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant clinical signs were noted during daily detailed clinical observations up to 80 mg/kg/day.
At 250 mg/kg/day incidental breathing rales were recorded in 3/10 males and 2/10 females during the first three weeks of treatment. Females at 250 mg/kg/day also showed hunched posture (4/10) and uncoordinated movements (1/10) during this period. Piloerection was recorded in one female during Days 2-10 post-coitum. As clinical signs were transient, they were regarded not toxicologically relevant.
Salivation seen after dosing among animals of the 80 and 250 mg/kg/day dose groups starting at Day 21 of the treatment period was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.
Any other clinical signs noted during the treatment period (alopecia) occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two females were sacrificed for human reasons:
Female No. 66 (80 mg/kg/day) was sacrificed in extremis on Day 23 post-coitum because of delivery difficulties. Bleeding of the genital region was recorded on the day of sacrifice. At necropsy dead/autolytic fetuses were found in the uterus. No microscopic findings of note were recorded; all findings were regarded to be related to the pregnancy and incomplete delivery. These delivery problems in a single female at 80 mg/kg/day were regarded unrelated to treatment with the test item.
Female No. 71 (250 mg/kg/day) was sacrificed in extremis on treatment Day 18 (i.e. the day of mating) due to severe body weight loss (~ 9% in three days, recorded in study raw data) and clinical signs (hunched posture, piloerection and uncoordinated movements). There were no findings of note that could explain the poor condition of this animal.
One female of the 250 mg/kg/day group (No. 80) was euthanized on Lactation Day 2, as she had a total litter loss.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test item-related changes in body weights and body weight gain were observed up to
80 mg/kg/day.
Body weights and body weight gain in males at 250 mg/kg/day were decreased from Day 8 of treatment onwards (up to 10% lower than control for absolute body weight on Day 15 of the
mating period).
In females at 250 mg/kg/day decreased body weight gain was recorded during the lactation period (up to 60% lower than control, statistically significant on Day 13 of lactation only)
resulting in an absolute body weight of 4% lower than control on Lactation Day 13 (not statistically significant).
Any other changes in body weights and body weight gain were considered to be unrelated to treatment regarding the direction of change and absence of a dose-related trend.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded up to 250 mg/kg/day for males and up to 80 mg/kg/day for females.
In females at 250 mg/kg/day food consumption was reduced during the first two weeks of treatment (up to 19% lower than control for absolute food consumption, not statistically significant), which subsequently recovered. In addition, decreased food consumption (before and after correction for body weight) was recorded during Days 4-13 of the lactation period (up to 23% lower than control on Days 7-13 of lactation). This findings was regarded treatment-related.
In males at 250 mg/kg/day food consumption was reduced during the first two weeks of treatment (up to 14% lower than control for absolute food consumption, not statistically
significant), which subsequently recovered. As this finding was transient it was considered of no toxicological relevance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following test item-related changes in hematology parameters were noted in treated males or females. Relative differences in mean values as compared to the control group are
indicated between parentheses.
- Increased red blood cell counts in males at 80 and 250 mg/kg/day (1.05 and 1.08x of control, respectively) with corroborative increased hemoglobin (1.07 and 1.08x of control, respectively) and hematocrit (each 1.08x of control) levels.
- Decreased white blood cell counts in females at 25, 80 and 250 mg/kg/day (0.72, 0.59 and 0.53x of control, respectively) with corroborative decreased neutrophil (0.67, 0.65
and 0.53x of control, respectively), lymphocyte (0.73, 0.56 and 0.53x of control, respectively) and eosinophil (0.72, 0.40 and 0.28x of control, respectively) concentrations (not always statistically significant).
- Decreased reticulocyte levels in females at 250 mg/kg/day (0.62x of control).
- Decreased platelet counts in females at 25, 80 and 250 mg/kg/day (0.90, 0.82 and 0.77x of control, respectively; not always statistically significant).
Any other changes in hematology parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Coagulation
In males and females at 250 mg/kg/day prolonged prothrombin time (PT) was recorded (1.24 and 1.08x of control, respectively), which was considered test item-related.
Prolonged activated partial thromboplastin time (APTT) recorded in males at 25, 80 and 250 mg/kg/day was considered the result of slightly low control values3 and considered not related to treatment with the test item.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following test item-related changes in clinical biochemistry parameters were noted in treated males or females. Relative differences in mean values as compared to the control group are indicated between parentheses:
-Increased liver enzyme activities in males at 25, 80 and 250 mg/kg/day, consisting of increased alanine aminotransferase (ALT, 1.51, 1.70 and 1.52x of control, respectively) and aspartate aminotransferase (AST, 1.14, 1.49 and 1.34x of control,respectively) activities (not statistically significant at the low dose).
-Increased bile acids in males and females at 250 mg/kg/day (BILEAC, 1.99 and 4.16xof control, respectively).
-Increased urea concentrations in males at 250 mg/kg/day (1.77x of control).
-Increased creatinine concentrations in males (1.22x of control) while decreased in females (0.71x of control) at 250 mg/kg/day.
-Decreased cholesterol levels in males at 80 and 250 mg/kg/day (0.66 and 0.55 of control, not statistically significant at the mid dose).
-Decreased total protein and albumin concentrations in females at 250 mg/kg/day (0.87 and 0.81x of control, respectively).
Decreased chloride levels in males at 250 mg/kg/day was considered not toxicologically relevant, considering the magnitude of change and as individual values were within the same range as control.
Increased potassium levels in males and decreased potassium levels in females were considered the result of slightly low or high control values4 and were regarded not related to treatment with the test item.
Any other changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.
Thyroid hormone analyses:
Serum levels of T4 and TSH in F0-males were decreased at 250 mg/kg/day (0.74 and 0.30x of control, respectively). No differences were recorded in females.

Endocrine findings:
no effects observed
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The increase of urinary protein recorded in animals receiving 60 mg/kg/day was not dose related, therefore it was considered to be incidental.The statistically significant decrease of urinary volume recorded in animals dosed at 180mg/lg/day was within the range of expected biological variation, therefore it was considered to be incidental.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were considered unaffected by treatment with the test item. Motor activity was considered not to be affected by treatment with the test item. Lower mean total movements and ambulations in males and females at 250 mg/kg/day (not statistically significant) were considered not toxicologically relevant as in general individual values were within the same range as controls and mean values remained within the historical control range. All groups showed a similar motor activity habituation profile with a decreasing trend in
activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were test item-related macroscopic findings consisting of:An accentuated lobular liver pattern was recorded in 4/10 males at 25 mg/kg/day and 10/10 males at 80 and 250 mg/kg/day. Pale discoloration was recorded in 3/10 males at 80 mg/kg/day and in 9/10 males at 250 mg/kg/day. The microscopic correlate for these findings was hepatocellular vacuolation of the periportal area. A reduced size of the prostate gland and seminal vesicles was recorded in 1/10 males at 250 mg/kg/day (no microscopic correlate). A reduced size of the thymus was recorded in 1/10 females at 250 mg/kg/day. The microscopic correlate for this finding was decreased lymphoid cellularity. The remainder of the recorded macroscopic findings were within the range of background
gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher liver weights and lower spleen, prostate gland, seminal vesicles and thymus (females) weights were noted. For the higher thyroid gland (females) weights a test
item-relationship could not be excluded. Statistically significant, test item-related higher liver weights were recorded at 80 and 250 mg/kg/day in males (relative to body weight only) and in females (absolute and relative to body weight). The microscopic correlate for the higher liver weight at 250 mg/kg/day was diffuse hepatocellular hypertrophy in males and cytoplasmic rarefaction (glycogen storage) in females.
Statistically significant, test item-related lower spleen weights were noted in males at 250 mg/kg/day (absolute only) and in females at 80 and 250 mg/kg/day (absolute and/or
relative to body weight). The microscopic correlate in females was decreased lymphoid cellularity and reduced extramedullary hematopoiesis. For males there was no microscopic correlate.
Statistically significant, test item-related lower prostate gland weights (absolute and relative to body weight) and seminal vesicles weights (absolute only) were noted in males at 250 mg/kg/day. There was no microscopic correlate for these lower weights.
Statistically significant, test item-related lower thymus weights were recorded in females at 80 and 250 mg/kg/day (absolute and relative to body weight). The microscopic correlate was
decreased lymphoid cellularity.
Statistically significant higher thyroid gland weights were noted in females at 80 and 250 mg/kg/day (relative to body weight only), both at comparable magnitude and without microscopic correlate. This weight change was regarded possibly test item-related.
There were no other test item-related organ weight changes. Some organ weight differences in males at 250 mg/kg/day were statistically significant when compared to the control group
but were considered to be the result of a test item related effect on final body weight. These included lower absolute weights for heart, testes and epididymides and higher brain relative to body weights.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 80 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
mortality
Remarks on result:
other:
Remarks:
based on a premature decedent, and adverse effects on body weight and food consumption

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based in the results of the present Study (OECD 422), the following No Observed Adverse Effects levels (NOAELS) of the test item were established:

Parental NOAEL: 80 mg/kg/day (based on a premature decedent, and adverse effects on body weight and food consumption).

In this study, a marked reduction of total T4 and TSH was observed in high dose groups (in males) which was considered to be test item-related. However, under the conditions of this screening study no adverse effect was observed that could be linked to the reduction of total T4 and therefore this reduction was not taken into account when determining the parental NOAEL.
Executive summary:

The purpose of this study was to determine the potential toxic effects of the test substance when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The dose levels in this study were selected to be 0, 25, 80 and 250 mg/kg/day, based on the results of the Dose Range Finder.

The study design was as follows:

Group

No.

Treatment

Dose Level

(mg/kg/day)

Dose Volume

(mL/kg)

Dose

Concentration

(mg/mL)

Number of Animals

Males

Females

1

Vehicle

0

5

0

10

10

2

Test Item

25

5

5

10

10

3

80

5

15

10

10

4

250

5

50

10

10

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle, clinical pathology, measurement of thyroid hormone T4 and TSH (F0-animals), gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).

toxicity was observed up to 80 mg/kg/day.

One female at 250 mg/kg/day was sacrificed on Day 0 post-coitum with severe body weight loss, hunched posture, piloerection and uncoordinated movements. There were no findings of note that could explain the poor condition of this animal. Premature mortality was recorded in one animal only, but a relation to treatment with the test item could not be excluded. This premature decedent was regarded adverse.

One female at 80 mg/kg/day was sacrificed on Day 23 post-coitum because of delivery difficulties. Bleeding of the genital region was recorded on the day of sacrifice. At necropsy dead/autolytic fetuses were found in the uterus. No microscopic findings of note were recorded; all findings were regarded to be related to the pregnancy and incomplete delivery.

This was regarded unrelated to treatment with the test item.

Decreased body weights and/or body weight gain were recorded at 250 mg/kg/day. In males body weights were lower from Day 8 of treatment onwards while in females reduced body weight gain was recorded during the lactation period, correlating with a decreased food consumption. Considering the magnitude of change these effects were regarded adverse.

Non-adverse test item-related morphologic alterations were present in liver of males starting at 25 mg/kg/day (periportal hepatocellular vacuolation, peribiliary infiltrates and diffuse hepatocellular hypertrophy; higher weights and macroscopic accentuated lobular pattern and/or pale discoloration; higher ALT, AST and bile acid values) and liver of females starting at 80 mg/kg/day (cytoplasmic rarefaction; higher weights), thymus of females starting at 80 mg/kg/day (decreased lymphoid cellularity; lower weights and reduced size; lower lymphocyte counts) and at 250 mg/kg/day in prostate gland of males (increased severity of inflammatory cell infiltrates), bone marrow (femur and sternum) of both sexes (decreased cellularity), spleen females (decreased lymphoid cellularity, decreased extramedullary hematopoiesis; lower weights; lower white blood cell, lymphocyte, neutrophil, reticulocyte and eosinophil counts) and mesenteric lymph node females (decreased lymphoid cellularity).

In addition, lower spleen, prostate gland and seminal vesicles weights in males at 250 mg/kg/day, lower spleen weight in females at 80 mg/kg/day and higher thyroid gland weights in females at 80 and 250 mg/kg/day were noted, all without microscopic correlates and non-adverse.

Additional non-adverse changes in hematology and clinical biochemistry parameters comprised of: increased red blood cell, hemoglobin and hematocrit counts, increased urea and creatinine concentrations, decreased cholesterol levels (males) and decreased platelet counts, decreased creatinine, potassium, total protein and albumin concentrations (females).

Non-adverse changes in coagulation parameters were recorded: increased prothrombin time at 250 mg/kg/day (both sexes).

Serum levels of T4 and TSH in F0-males were decreased at 250 mg/kg/day, which was considered to be test item-related.

There were no test item-related effects on the reproductive performance.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex).