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Administrative data

Description of key information

For general toxicity the no-observed-adverse-effect level (NOAEL) was considered to be 15 mg/kg/day, based on mortality of parents and low bodyweigt/weight gain in offspring.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 March to 1 Novemebr 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: reputable laboratotory animal supplier
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: Males: 343 to 395 g; Females: 237 to 278 g.
- Fasting period before study: no
- Housing: Cages comprised of a polycarbonate body (and floor) with a stainless steel mesh lid; changed at appropriate intervals. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Diet: certified pelleted diet, ad libitum
- Water (e.g. ad libitum): potable water from the public supply, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 ºC
- Humidity (%): 40-70 %
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark.

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

The test substance TCD was prepared for administration as a series of graded concentrations in the vehicle, corn oil.

Starting with the lowest concentration (2 mg/mL), approximately 50 % of the final volume of vehicle was added to the required amount of pre-weighed test substance and magnetically stirred until dissolved. The solution was made up to final volume with vehicle and mixed using a magnetic stirrer until homogeneous. Higher concentrations (6 and 18 mg/mL) were prepared in ascending order of concentration using the same method.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity - before treatment commenced, the suitability of the proposed mixing procedure was determined and specimen formulations were analysed to assess the homogeneity and stability of the test material in the liquid matrix.

Achieved concentration - samples of each formulation prepared for administration in the first and last weeks of treatment were analysed for achieved concentration of the test substance.

Analysis of the concentration of test substance in the fomulation ws undertaken by gas chromatograhy using external standard calibration. The method was validated prior to use.
Duration of treatment / exposure:
At least 4 weeks (28 days)
Frequency of treatment:
Once daily at approximately the same time each day
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
45 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: based on results for 78 day preliminary study
- Rationale for animal assignment: random
- Rationale for selecting satellite groups: not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included:.evidence of ill health or reaction to treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: males/females, daily for week 1-2; twice weekly for week 3 onwards. After mating females were observed on days 0, 6, 13, and 20 of gestation and on days 1 and 6 of lactation.

-BODY WEIGHT: yes
- Time schedule for examinations: males - each week including day of commencement of treatment and necroscopy; females - each week inclusing day of commencm,ent of treatment, the following mating days 0, 6, 13, and 20 after mating and on days 1, 4 and 7 of lactation.


FOOD CONSUMPTION: yes
- Time schedule: males - weekly except during mating (week 3)

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
-Time schedule: week 2 prior to pairing

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 2 prior to pairing

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males during week 5 of treatment, females during Days 4-6 of lactation

- Dose groups that were examined: five lowest numbered males in each group and the five lowest numbered surviving lactating females in each group during Days 4-6 of lactation.

- Battery of functions tested: approach response, touch response, auditory startle reflex, tail pinch response, grip strength, motor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Grip strength, motor activity, body weight, food consumption, clinical pathology, organ weights:

Parametric analysis
Bartlett's test for variance homogeneity (Bartlett 1937)
Williams’ test (Williams 1971, 1972)
Dunnett's test (Dunnett 1955, 1964)
Shirley's test (Shirley 1977)
Steel's test (Steel 1959)
Fisher’s Exact tests (Fisher 1973)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
mortality at the top dose level
Mortality:
mortality observed, treatment-related
Description (incidence):
mortality at the top dose level
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
low bodyweight and weight gain at the top amd mid dose dose level
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
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):
high white blood cell count at top dose level
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
low plasma bile acid in males at top dose level
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Low motor activity in females, during Lactation, at the top dose level
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
males - liver, prostate high; females- adrenal and brain low
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
cortical hypertrophy in females, all dose groups. Involution/atrophy in females at 15 and 45 mg/kg/day
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Two females were killed for welfare reasons on Days 10 or 11 of the pre-mating period.
One female (45mg/kg/day) had a slight convulsion on Day 6, and was killed for welfare reasons on Day 11, after exhibiting a marked convulsion at one to two hours after administration. Macroscopic examination revealed dark kidneys and small thymus. Following microscopic examination, significant findings were seen in the spleen (decreased cellularity of the red pulp) and the adrenals (cortical hypertrophy). The factor contributory to death was poor clinical condition.

One female (45mg/kg/day) had a moderate convulsion on Day 6, showed slight tremor on Day 7, and was killed for welfare reasons on Day 10, after exhibiting a marked convulsion at one to two hours after administration. Macroscopic examination revealed dark kidneys and small thymus. Following microscopic examination, significant findings were seen in the spleen (decreased cellularity of the red pulp) and the adrenals (cortical
hypertrophy). The factor contributory to death was poor clinical condition.

There were no general clinical signs and no signs during the arena observations related to treatment with TCD.

BODY WEIGHT AND WEIGHT GAIN
Overall mean body weight gain (Week 0-5) was slightly high in males treated at 45 mg/kg/day (X 1.3), when compared with Control and was high in females at 15 or 45 mg/kg/day, with relationship to dose (X 1.7 or 1.9, respectively), during the two week pre-mating period.
When compared with Control, body weight gain during gestation was not affected by treatment; however, maternal body weight gain was low in treated females (5, 15 or 45 mg/kg/day) during the first four days of lactation (X 0.36, 0.18 or 0.18, respectively) and overall lactation body weight gain (Days 1 to 7) was X 0.77, 0.50 or 0.70, respectively.

FOOD CONSUMPTION
Overall food consumption of males at 45 mg/kg/day was marginally high in Weeks 4 and 5 (X 1.14), when compared with Control.
Overall food consumption of females treated at 15 or 45 mg/kg/day was slightly low during lactation (X 0.88 or 0.84, respectively).

HEMATOLOGY
Overall white cell and concomitant neutrophil, lymphocyte, eosinophil, basophil, monocyte and large unstained cell counts were high in both sexes treated at 45 mg/kg/day, when compared with Control.

CLINICAL CHEMISTRY

There were no differences in plasma enzyme activity in Week 2, when compared with Control.

In males at 45 mg/kg/day, plasma bile acid concentration was markedly low (X 0.34) and cholesterol concentration was slightly high.

NEUROBEHAVIOUR
Sensory reactivity findings in both sexes were considered to be unaffected by treatment. Motor activity scores for males in Week 5 were considered to be unaffected by treatment. Motor activity in females, during Lactation Days 4-6, treated at 45 mg/kg/day were markedly low.

ORGAN WEIGHTS
Mean adjusted liver weight was low, without dose-relationship, in males receiving 5, 15 or 45 mg/kg/day and mean prostate weight was low at 45 mg/kg/day; mean adrenal weight was high in females at 5, 15 or 45 mg/kg/day (X 1.16, 1.19 or 1.27) and mean brain weight was marginally high, without dose-relationship, in females at 15 or 45 mg/kg/day (both X 1.06), when compared with Control.


GROSS PATHOLOGY
The nature and incidence of all findings were consistent with the commonly seen background of macroscopic changes.

HISTOPATHOLOGY: NON-NEOPLASTIC
Increased cortical hypertrophy was seen in females at all dose groups. Involution/atrophy was seen in females at 15 and 45 mg/kg/day. Increased incidence and severity of cortical tubules with hyaline droplets was seen in males at 45 mg/kg/day.


Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
Key result
Critical effects observed:
no

Two females treated at 45 mg/kg/day had convulsions on Day 6, one of which showed slight tremor on Day 7 and both were killed for welfare reasons after having a marked convulsion on Day 10 or 11. A third female at this dose had a single, moderate convulsion on Day 3 and a fourth female had a convulsive episode on Day 4 of lactation. No other animals were noted to have convulsive episodes, despite extending the post dose observation regimen to the times after dosing that the convulsions occurred. The cause of the convulsions/tremor in these four females is not known. Chin rubbing, with or without salivation was seen in the majority of animals at 45 mg/kg/day and in three females at 15 mg/kg/day during the study, this was considered to be attributable to the palatability of the test substance.

At 45 mg/kg/day, overall body weight gain (Weeks 0-5) was slightly high and food consumption was slightly high during Weeks 4 and 5 in males and body weight gain during the two-week pre-mating period was high in females, which was also seen in females treated at 15 mg/kg/day, but these are considered not to be toxicologically significant. Body weight gain and food consumption were not affected by treatment during gestation. Body weight gain was low during lactation and food consumption was slightly low at 45 or 15 mg/kg/day.

There were some intergroup differences in haematology and blood chemistry parameters but they did not correlate with any microscopic pathology changes and were therefore considered to be of no toxicological significance.

Motor activity scores in females receiving 45 mg/kg/day were markedly low and the forelimb grip strength of both sexes receiving 45 mg/kg/day was marginally low, but both were within the Historical Control Data range. Oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, gestation index, litter size, sex ratio or survival of the offspring were not affected by treatment at 45 mg/kg/day. At 45 mg/kg/day, mean offspring bodyweights on Day 1 of age were low and remained low, to Day 7 of age and one litter had six dead offspring, from 15 born; however mortality in other litters at this dose was within acceptable limits. There were no macroscopic findings related to treatment. Microscopic investigation revealed minimal cortical hypertrophy in the adrenals from females in all treated groups, which correlated with the high mean tissue weight and involution/atrophy of the thymus from the majority of females at 45 mg/kg/day and a single case of thymic atrophy in females at 15 mg/kg/day, which were considered to be related to stress. Among males receiving 45 mg/kg/day, there was an increase in the incidence and severity of renal cortical tubules with hyaline droplets (alpha2 microgloulin nephropathy), which is occasionally seen in males following treatment with a hydrocarbon. Sensory reactivity and grip strength were unaffected by treatment at 45 mg/kg/day.

Conclusions:
For general toxicity the no-observed-adverse-effect level (NOAEL) was considered to be 15 mg/kg/day.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch code 1

Additional information

Treatment at 15 mg/kg/day did not result in any systemic toxicity. At 45 mg/kg/day, there was no effect of treatment on mating performance, fertility or the number of implantations or litter size on Day 1 of age but offspring body weights on Day 1 of age and subsequent weight gains from Day 4 to Day 7 of age were low, but there was no conclusive effect on offspring survival. There was no effect on offspring body weights up to Day 7 of age at 5 or 15 mg/kg/day. For general toxicity the no-observed-adverse-effect level (NOAEL) was considered to be 15 mg/kg/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

One study available, considered to be valid and conducted in accordance with an accepted  test method under GLP.

Repeated dose toxicity: via oral route - systemic effects (target organ) glandular: other

Justification for classification or non-classification

Although mortality occurred in animals at the top dose level this was due to general toxicity and not attributable to the decrease in function of any particular organ. Any effects on organs on surviving animals were not particlularly signifcant (adaptive increase decrease in weight and hypertrophy/atrophy) and therefore the substance is not classifed for STOT-RE.