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Diss Factsheets

Administrative data

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:
02 Feb - 08 Aug 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 Feb - 08 Aug 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:
July 29, 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD (SD), SPF
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10 weeks (males); 12 weeks (females)
- Weight at study initiation: 355.0 - 439.4 g (males); 242.3 - 295.3 g (females)
- Housing: During acclimation, pre-treatment, treatment, postmating and recovery period: 2 animals per cage; Mating period: 1 male and 1 female; Gestation period: 1 mated female; Lactation period: neonates were kept with the dam; animals were kept in stainless-steel cages (255W×465L×200H mm) and dams with pups in polycarbonate cages (260W x 420D x 180H mm) on aspen animal bedding
- Diet: Lab Diet® #5053 (PMI Nutrition International, USA) irradiated by gamma-ray, ad libitum
- Water: filtered, ultraviolet light-irradiated municipal tap water, ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: Microbial monitoring for diet was performed and a certificate of analysis for the diet was provided by the supplier. The drinking water was analyzed every 6 months for specified contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 - 22.8
- Humidity (%): 40.9 - 53.8
- Air changes (per hr): 10 - 20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was weighed and suspended in corn oil with a magnetic stirrer for about 5 min to prepare the target concentration. The high dose formulation was prepared first and then the lower dose formulations were prepared by diluting the higher dose formulation with corn oil. Dose formulations were prepared at least one time per week and stored at room temperature. Dose formulations were transferred to the animal room at room temperature.

VEHICLE
- Justification for use and choice of vehicle: Corn oil was considered non-toxic with this dose volume (2 mL/kg), and it has been used in previous studies because of the solubility of the test item with this vehicle.
- Amount of vehicle: 2 mL/kg
- Lot/batch no.: MKBZ9899V
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as Day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The verification of the dose level concentration was determined by gaschromatography prior to dosing. Samples were taken from the top, middle and bottom of all dose formulations. Results of dose formulations were 106.84, 102.04, and 101.53% at the dose levels of 7.5, 25, and 75 mg/mL respectively. They were acceptable as the mean concentration was within ±15% of the nominal concentration.
Duration of treatment / exposure:
Main groups:
males: at least 50 days, starting 2 weeks before mating, during mating until day prior to sacrifice
females: for 2 weeks prior to mating and continued until lactation day 13

Recovery groups:
at least 50 days; Animals were not mated and were assigned to 2 weeks of recovery period after the completion of administration.
Frequency of treatment:
once daily, 7 days/week
Details on study schedule:
not applicable
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:
12 (main groups)
6 (recovery groups; for control and high dose groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose level was selected based on the results of a repeated 2-week dose range-finding study in Sprague-Dawley rats in which doses of 15, 60 and 240/150 mg/kg bw/day have been tested. In that study, one found dead female was observed on treatment day 5 at dose level of 240 mg/kg bw/day. The dose level of the high dose group was changed from 240 to 150 mg/kg bw/day on treatment day 6. Increased AST, ALT and yellow discoloration of liver were observed in males at 60 mg/kg bw/day. At 240/150 mg/kg bw/day, general toxicity including soft feces, lacrimation and soiled fur in males, and decreased body weight gain and food consumption in both sexes were observed. In addition, an increased AST and ALT were observed in males. Absolute and relative spleen weights were increased in both sexes. Based on the result of this dose range-finding study, 150 mg/kg bw/day was selected as the high dose, and 50 and 15 mg/kg bw/day were selected as the middle and the low dose, respectively. Animals of vehicle control were administered with the vehicle alone (corn oil).

- Rationale for animal assignment: Animals were selected for use in the study on the basis of adequate body weight (recorded on the day of receipt and of group assignment), estrus cycle and for being free from clinical signs of disease or injuries during the acclimation and pre-treatment periods. They were randomized and assigned to treatment groups to have a similar mean body weight distribution using the Pristima system based on the most recent body weight.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality and morbidity observations were conducted twice daily except for the acclimation and recovery period where animals were observed once daily. In addition, it was conducted once on necropsy day.
- Cage side observations included: mortality/viability, clinical signs and general condition; during the gestation period: signs of abortion or premature delivery

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once per week
- Observations included: evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, pupil size, and unusual respiratory pattern), changes in gait, posture, clonic and tonic movements, stereotypical and bizarre behavior, and difficult and prolonged parturition

BODY WEIGHT: Yes
- Time schedule for examinations: first dosing day and then once per week; additionally on gestation days 0, 7, 14 and on lactation days 0, 4 and 13

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption per animal [g food/animal/day] was calculated by subtracting the amount of residual feed from the amount presented

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OTHER:
For haematology, clinical chemistry, neurobehavioural examination refer to IUCLID section 7.5.1
Oestrous cyclicity (parental animals):
A vaginal smear was taken daily for each female from the beginning of the 14 days prior to mating with continued monitoring into the mating period until there was evidence of mating. Furthermore, regularity and length of the estrus cycle was examined during the treatment period until mating.
In addition, vaginal smear of sacrificed females were taken at termination to examine the stage of the estrus cycle and to allow a correlation with the histopathology of the female reproductive organs.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other: thyroid hormone (T4) and the thyroid stimulating hormone (TSH) in blood on postnatal days 4 and 13

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on treatment day 50
- Maternal/female animals: on lactation day 14; non-parturition females after 27 days from final mating day
- Recovery group: at least 14 days after first scheduled sacrifice of main group males and females

GROSS PATHOLOGY: Yes
The animals were examined carefully for external abnormalities. The abdominal, thoracic and cranial cavities were examined for abnormalities and the organs were removed and examined. Special attention was paid to the organs of the reproductive system. For lactating dams, the number of implantation sites (right and left) was counted. For non-parturition females, uterus implantation sites were examined after staining with ammonium sulfide according to KIT SOPs. Pregnancy was confirmed by implantation sites in the uterus.

ORGAN WEIGHTS: Yes
Paired organs were weighed together unless gross abnormalities were present. However, paired reproductive organs were weighed separately. Animals were fasted overnight prior to necropsy and body weights were recorded on the day of necropsy. Organs were weighed and organ/body weight ratios were calculated.
(for details see 7.5.1)

HISTOPATHOLOGY: Yes
Tissues shown in table 2 from each animal were preserved in 10% neutral buffered formalin, except the eyes (with optic nerve) which were fixed in Davidson’s fixative, and the testes and epididymides which were fixed in Bouin’s fixative. The tissues were placed in the appropriate fixative for approximately 48 hours, and then transferred to 70% ethanol. Formalin was infused into the lung via the trachea and into the urinary bladder.
All tissues except for the reproductive organs collected from the first six animals per sex in the main group were further processed to slides, stained with hematoxylin and eosin (H&E), and examined microscopically. All reproductive organs collected from the main groups were further processed to slides, stained with H&E, and examined microscopically. Target organs from the recovery group animals were additionally evaluated microscopically.
(for details see 7.5.1)
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at postnatal day 13.
- All animals were subjected to external postmortem examinations

GROSS NECROPSY
- Gross necropsy consisted of external examinations
Statistics:
Mean values and standard deviations were calculated. Statistical analyses for comparisons of the various dose groups with the vehicle control group were conducted using the Pristima System (Version 7.X. Xybion Medical Systems Corporation, USA) or Statistical Analysis Systems (SAS/STAT Version 9.2 and 9.4, USA). Data were considered to be significant when p<0.05 or p<0.01.
Multiple comparison tests for different dose groups were conducted. Variance of homogeneity was examined using the Bartlett’s Test. Homogeneous data were analyzed using the Analysis of Variance (ANOVA), and the significance of inter-group differences were analyzed using the Dunnett’s Test. Heterogeneous data were analyzed using the Kruskal-Wallis Test, and the significance of inter-group differences was assessed between the VC and treated groups using the Dunn’s Rank Sum Test.
For comparing the control group with the recovery group, the data were analyzed for homogeneity for variance using the F-test. Homogeneous data were analyzed using the T-test and the significant difference was assessed between control and recovery group using the Dunnett’s Test. Heterogeneous data were analyzed using the Kruskal-Wallis Test and significant difference was assessed between control and recovery group using the Dunn’s Rank Sum Test.
One-way analysis of covariance (ANCOVA) was used to analyze pup body weights. The litter size was used as the covariate. Litter data were statistically evaluated using the statistical unit as a litter.
Data presented as frequencies were analyzed by χ2-test followed by the Fisher's exact test where necessary.
Reproductive indices:
- Precoital Time = No. of days taken to mate
- Mating index (%) = (No. of animals with evidence of mating / No. of animals paired) × 100
- Fertility index (%) = (No. of animals with impregnation or pregnancy / No. of animals paired) × 100
- Fecundity/Pregnancy Index (%) = (No. of animals with impregnation or pregnancy / No. of animals with evidence of mating) x 100
- Unaccounted-for sites (%) = (No. of implantation sites/litter) – (No. of pups born/litter) / No. of implantation sites/litter x 100
- Delivery Index = No. of dams with live pups on Day 0 of lactation / No. of pregnant dams x 100
Offspring viability indices:
- Viability Index = No. of live pups on Day 4 of lactation / No. of live pups at birth x 100
- Weaning Index = No. of live pups on Day 13 of lactation / No. of pups on Day 4 of lactation after culling x 100
- Sex ratio = (No. of male pups on PND 0/litter) / (No. of total pups on PND 0/litter) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related moribund animals were observed in any group throughout the study.
In males at 15, 50 and 150 mg/kg bw/day, salivation was observed in 11, 12, and 18 animals, respectively. In females at 15, 50, and 150 mg/kg bw/day, salivation was observed in 6, 12, and 18 animals, respectively. It was considered test item-related but not toxicologically significant since it was considered to be attributed to the palatability of the test item.
Other clinical signs were observed in this study but were not considered test item-related since these findings were observed with low frequency or occurred sporadically and did not have any dose-response.

In one female of control group found dead on gestation day 21 one early resorption, one late resorption and 7 dead fetuses were observed in the uterine examination. In the result of microscopic findings, moderate hemorrhage, slight alveolar edema and moderate perivascular congestion were observed in the lung. Moderate centrilobular necrosis in liver, right atrium dilatation in heart, slight atrophy in spleen and minimal mononuclear cell infiltration and hemorrhage in uterus were also observed. These were considered to be incidental since it was observed in the control group and there were no correlated microscopic changes in other animals in the same control group.

Non-parturition was observed in two females of control group and in one female at 150 mg/kg bw/day. Three females were subjected to unscheduled sacrifice on gestation day 27 and all of them were non-pregnant. In the results of microscopic findings, minimal squamous cell hyperplasia in stomach was observed in one animal at 150 mg/kg bw/day, but there were no other changes observed in the reproductive system in this animal.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item-related deaths occurred in any group throughout the study.
One female of control group was found dead on gestation day (GD) 21. Two females of control group and one female at 150 mg/kg bw/day were subjected to unscheduled sacrifice on gestation day 27 since they were non-pregnant.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item-related changes in body weight and in body weight gain were observed in both sexes during the study. Statistically significant changes in body weight gain during the study were not considered test item-related since they were transient.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related changes in food consumption were observed in both sexes during the study.
A statistically significant change in food consumption during the study was not considered test item-related since there was no correlation with body weight changes and there was no dose-dependency.
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):
In males, total leukocyte count (WBC, 1.40-fold over control), absolute lymphocyte count (LYMA, 1.47-fold over control), absolute monocyte count (MONA, 1.43-fold over control), absolute large unstained cells counts (LUCA, 1.33-fold over control), fibrinogen (FIB, 1.22-fold over control) were increased at 150 mg/kg bw/day and platelet count (PLT, up to 1.21-folds over control) was increased at 50 and 150 mg/kg bw/day. The increases of WBC, LYMA, FIB and PLT were statistically significant. In females, WBC (1.30-fold over control), LYMA (1.37-fold over control), MONA (1.62-fold over control) and LUCA (1.58-fold over control) were increased at 150 mg/kg bw/day. The increases of WBC, MONA and LUCA were statistically significant. These changes recovered or showed a tendency of recovery at the end of the recovery period. Increased leukocyte counts, fibrinogen and platelet count in males and/or females at 150 mg/kg bw/day were correlated with inflammatory changes in the liver.
Other statistically significant changes were not considered test item-related, because these changes were minimal, there were no dose-relationships, and no correlations with microscopic changes and/or observed only in the recovery group.
(for result tables refer to 7.5.1)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males, alanine aminotransferase (ALT, 2.80-fold over control), aspartate aminotransferase (AST, 1.46-fold over control), gamma glutamyl transpeptidase (GGT, 8.47-fold over control) and total bilirubin (TBIL, 1.07-fold over control) were increased at 150 mg/kg bw/day. The increases of ALT, GGT and TBIL were statistically significant. In females, ALT (2.05-fold over control), AST (1.43-fold over control), TBIL (1.21-fold over control) and GGT (8.48-fold over control) were also increased at 150 mg/kg bw/day. The increase of GGT was statistically significant. These changes recovered or showed a tendency to recover at the end of the recovery period. Increases of alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transpeptidase and total bilirubin in males and/or females at 150 mg/kg bw/day were considered to be results of a massive damage of hepatocytes.
Other statistically significant changes were not considered test item-related, because changes were minimal, there were no dose-relationships, no correlated microscopic changes and/or observed only in recovery group.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes in functional behavior examinations were observed in both sexes during the study.
A statistically significant decrease in grip strength of forelimb in males at 50 and 150 mg/kg bw/day was not considered test item-related since there was no dose-dependency and no change in other parameters during the functional behavioral examinations.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Test item-related changes were observed in liver, spleen, hepatic lymph node, and stomach of both sexes at 150 mg/kg bw/day.

Liver:
Slight to marked cholangiofibrosis, minimal to marked hydropic degeneration, minimal to marked necrosis, minimal to marked bile duct hyperplasia, minimal to slight hepatocyte hypertrophy and/or minimal to slight pigmented macrophages in both sexes and slight fibrosis in capsule in one male were observed at 150 mg/kg bw/day. These changes were observed in the periportal region except a fibrosis in capsule. After recovery period, cholangiofibrosis, hydropic degeneration and necrosis were fully recovered and bile duct hyperplasia and hepatocyte hypertrophy showed a tendency of recovery in both sexes at 150 mg/kg bw/day. In the 150 mg/kg bw/day recovery group, minimal to moderate periportal fibrosis in both sexes were also observed and pigmented macrophage was similar to the main groups in both sexes.
Cholangiofibrosis, hydropic degeneration, and necrosis were considered to be an adverse effect of the test-item and are well known and often observed as hepatocellular toxicity caused by various xenobiotics and chemicals. Periportal fibrosis was observed and shown to be a recovery reaction.
Bile duct hyperplasia, hepatocyte hypertrophy, pigmented macrophages and/or fibrosis in the capsule accompanied the above named findings were not considered as an adverse effect at the severity as seen here. These changes were considered to be adaptive changes to a drug-induced increase in a metabolic workload or were considered as secondary findings related to the aforementioned adverse effects in the liver.
Spleen and hepatic lymph node:
Minimal to slight lymphoid hyperplasia in both sexes at 150 mg/kg bw/day and slight chronic inflammation in the capsule in one female at 150 mg/kg bw/day were observed. At the end of the recovery period and lymphoid hyperplasia showed a tendency of recovery in both sexes at 150 mg/kg bw/day and a capsular fibrosis in both sexes and chronic inflammation in capsule were observed in females at 150 mg/kg bw/day. In the hepatic lymph node, moderate lymphoid hyperplasia, slight congestion were observed in both sexes at 150 mg/kg bw/day.
These changes in spleen and hepatic lymph nodes were considered to be associated with a secondary response to the inflammatory changes in the liver.

Stomach:
Minimal to slight squamous cell hyperplasia in the non-glandular region was observed in both sexes at 150 mg/kg bw/day and fully recovered after the recovery period. This change was considered to be non-adverse but an adaptive response following a local irritation caused by the test item since it fully subsided after the recovery period.

Other changes observed in microscopic findings were considered as incidental or spontaneous changes since they were infrequent, generally of low severity, and similarly distributed among the control and the treated groups.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone anylysis:
No test item-related changes in the thyroid hormone (T4) and the thyroid stimulating hormone (TSH) were observed in the adult male animals.
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
A decreased absolute weight of testes and epididymides in males at 150 mg/kg bw/day did not represent a meaningful toxicologic finding as there were no correlated microscopic findings and/or it fully recovered during the recovery period.
Reproductive performance:
no effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to the highest dose tested
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
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
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease in body weight of the F1 pups was observed on PND 13 (male pups: 92% of VC, female pups: 91% of VC) at 150 mg/kg bw/day.
(see table 1)
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:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone anylysis:
No test item-related changes in the thyroid hormone (T4) and the thyroid stimulating hormone (TSH) were observed in pups.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at 50 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Generation:
F1
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
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Table 1. Pups body weight (males)

Group

 

Control

15 mg/kg bw/day

50 mg/kg bw/day

150 mg/kg bw/day

PND 0

Mean

7.04

6.75

6.96

7.05

SD

0.44

0.65

0.35

0.50

Covariate adjusted mean

6.97

6.79

7.01

7.01

n

9

12

12

11

PND 4 (preculling)

Mean

11.52

11.11

11.08

10.71

SD

1.25

1.69

0.97

0.85

Covariate adjusted mean

11.35

11.08

11.28

10.65

n

9

12

12

11

PND 4 (postculling)

Mean

11.82

11.38

11.51

11.02

SD

1.32

1.63

0.99

0.83

n

9

12

12

11

PND 13

Mean

35.78

35.13

35.05

32.32+D

SD

1.46

2.93

1.83

2.68

Covariate adjusted mean

35.51

34.98

35.24

32.51*D

n

9

12

12

11

+D = Dunnett LSD Test Significant at the 0.01 level or at 0.05 level (*)   

Table 2. Pups body weight (females)

Group

 

Control

15 mg/kg bw/day

50 mg/kg bw/day

150 mg/kg bw/day

PND 0

Mean

6.58

6.32

6.646.96

6.637.05

SD

0.41

0.63

0.38

0.53

Covariate adjusted mean

6.51

6.36

6.69

6.59

n

9

12

12

11

PND 4 (preculling)

Mean

10.82

10.46

10.66

10.26

SD

1.27

1.67

0.97

0.98

Covariate adjusted mean

10.64

10.44

10.88

10.20

n

9

12

12

11

PND 4 (postculling)

Mean

10.99

10.72

11.02

10.48

SD

1.19

1.67

1.11

1.10

n

9

12

12

11

PND 13

Mean

34.09

33.60

34.19

30.64*R

SD

1.46

3.27

1.71

3.64

Covariate adjusted mean

33.86

33.47

34.36

30.80*D

n

9

12

12

11

* R = Dunn Rank Sum Test Significant at the 0.05 level+D = Dunnett LSD Test Significant at the 0.01 level or at 0.05 level (*)  

Conclusions:
The NOAEL for parental systemic toxicity was considered to be 50 mg/kg bw/day in both sexes, based on cholangiofibrosis, hydropic degeneration and necrosis of liver in both sexes at 150 mg/kg bw/day. The NOAEL for parental fertility was considered to be 150 mg/kg bw/day, since no adverse effects were observed on parental reproductive function.
The NOAEL for developmental toxicity was considered to be 50 mg/kg bw/day based on decreased pup body weights on postnatal day 13 at 150 mg/kg bw/day. This effect was considered to be a secondary effect of parental systemic toxicity. No further effect on development was observed.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

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 29, 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Allyl phenoxyacetate
EC Number:
231-335-2
EC Name:
Allyl phenoxyacetate
Cas Number:
7493-74-5
Molecular formula:
C11H12O3
IUPAC Name:
allyl phenoxyacetate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD (SD), SPF
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10 weeks (males); 12 weeks (females)
- Weight at study initiation: 355.0 - 439.4 g (males); 242.3 - 295.3 g (females)
- Housing: During acclimation, pre-treatment, treatment, postmating and recovery period: 2 animals per cage; Mating period: 1 male and 1 female; Gestation period: 1 mated female; Lactation period: neonates were kept with the dam; animals were kept in stainless-steel cages (255W×465L×200H mm) and dams with pups in polycarbonate cages (260W x 420D x 180H mm) on aspen animal bedding
- Diet: Lab Diet® #5053 (PMI Nutrition International, USA) irradiated by gamma-ray, ad libitum
- Water: filtered, ultraviolet light-irradiated municipal tap water, ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: Microbial monitoring for diet was performed and a certificate of analysis for the diet was provided by the supplier. The drinking water was analyzed every 6 months for specified contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 - 22.8
- Humidity (%): 40.9 - 53.8
- Air changes (per hr): 10 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was weighed and suspended in corn oil with a magnetic stirrer for about 5 min to prepare the target concentration. The high dose formulation was prepared first and then the lower dose formulations were prepared by diluting the higher dose formulation with corn oil. Dose formulations were prepared at least one time per week and stored at room temperature. Dose formulations were transferred to the animal room at room temperature.

VEHICLE
- Justification for use and choice of vehicle: Corn oil was considered non-toxic with this dose volume (2 mL/kg), and it has been used in previous studies because of the solubility of the test item with this vehicle.
- Amount of vehicle: 2 mL/kg
- Lot/batch no.: MKBZ9899V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The verification of the dose level concentration was determined by gaschromatography prior to dosing. Samples were taken from the top, middle and bottom of all dose formulations. Results of dose formulations were 106.84, 102.04, and 101.53% at the dose levels of 7.5, 25, and 75 mg/mL, respectively. They were acceptable as the mean concentration was within ±15% of the nominal concentration.
Duration of treatment / exposure:
Main groups:
males: at least 50 days, starting 2 weeks before mating, during mating until the day prior to sacrifice
females: for 2 weeks prior to mating and continued until lactation day 13

Recovery groups:
at least 50 days; Animals were not mated and were assigned to 2 weeks of recovery period after the completion of administration.
Frequency of treatment:
once daily, 7 days/week
Doses / concentrationsopen allclose all
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:
12 (main groups)
6 (recovery groups; for control and high dose groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose level was selected based on the results of a repeated 2-week dose range-finding study in Sprague-Dawley rats in which doses of 15, 60 and 240/150 mg/kg bw/day have been tested. In that study, one found dead female was observed on treatment day 5 at dose level of 240 mg/kg bw/day. The dose level of the high dose group was changed from 240 to 150 mg/kg bw/day on treatment day 6. Increased AST, ALT and yellow discoloration of liver were observed in males at 60 mg/kg bw/day. At 240/150 mg/kg bw/day, general toxicity including soft feces, lacrimation and soiled fur in males, and decreased body weight gain and food consumption in both sexes were observed. In addition, an increased AST and ALT were observed in males. Absolute and relative spleen weights were increased in both sexes. Based on the result of this dose range-finding study, 150 mg/kg bw/day was selected as the high dose, and 50 and 15 mg/kg bw/day were selected as the middle and the low dose, respectively. Animals of the vehicle control were administered with the vehicle alone (corn oil).

- Rationale for animal assignment: Animals were selected for use in the study on the basis of adequate body weight (recorded on the day of receipt and of group assignment), estrus cycle and for being free from clinical signs of disease or injuries during the acclimation and pre-treatment periods. They were randomized and assigned to treatment groups to have a similar mean body weight distribution using the Pristima system based on the most recent body weight.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality and morbidity observations were conducted twice daily except for the acclimation and recovery period where animals were observed once daily. In addition, it was conducted once on necropsy day.
- Cage side observations included: mortality/viability, clinical signs and general condition; during the gestation period: signs of abortion or premature delivery

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once per week
- Observations included: evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, pupil size, and unusual respiratory pattern), changes in gait, posture, clonic and tonic movements, stereotypical and bizarre behavior, and difficult and prolonged parturition

BODY WEIGHT: Yes
- Time schedule for examinations: first dosing day and then once per week; additionally on gestation days 0, 7, 14 and on lactation days 0, 4 and 13

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption per animal [g food/animal/day] was calculated by subtracting the amount of residual feed from the amount presented

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HEMATOLOGY: Yes
- Time schedule for collection of blood: day of scheduled sacrifice
- Anaesthetic used for blood collection: not specified
- Animals fasted: 16 h (overnight) prior to scheduled sacrifice
- How many animals: all scheduled sacrifice animals in the main and the recovery groups
- Parameters checked: total leukocyte count (WBC), mean corpuscular hemoglobin (MCH), total red blood cell count (RBC), mean corpuscular hemoglobin concentration (MCHC), hemoglobin (HGB), platelet count (PLT), hematocrit (HCT), reticulocyte count (absolute (RETA) and relative (RET%) counts), mean corpuscular volume (MCV), WBC differential count (absolute and relative (%) differential counts: including neutrophils (NEU), eosinophils (EOS), basophils (BAS), monocytes (MON), lymphocytes (LYM) and other cells as appropriate (i.e.; large unstained cells (LUC)), prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of scheduled sacrifice
- Animals fasted: 16 h (overnight) prior to scheduled sacrifice
- How many animals: all scheduled sacrifice animals in the main and the recovery groups
- Parameters checked: glucose (GLU), alanine aminotransferase (ALT), blood urea nitrogen (BUN), total bilirubin (TBIL), creatinine (CREA), alkaline phosphatase (ALP), total protein (TP), gamma glutamyl transpeptidase (GGT), albumin (ALB), creatine phosphokinase (CK), albumin/globulin ratio (A/G), calcium (Ca), total cholesterol (TCHO), inorganic phosphorus (IP), triglyceride (TG), sodium (Na), phospholipid (PL), potassium (K), aspartate aminotransferase (AST), chloride (Cl)

URINALYSIS:No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: shortly before scheduled sacrifice
- Dose groups that were examined: first six animals per sex in main group (if possible) and in all animals of the recovery group
- Battery of functions tested: sensory function tests (approach and touch response, tail pinch, acoustic startle response and pupillary reflex), grip strength and motor activity

IMMUNOLOGY: No

OTHER:
Thyroid Hormone Analysis:
- Time schedule for examinations: at termination
- Dose groups that were examined: all adult animals in main and recovery group
- Parameters checked: thyroid hormone (T4) and the thyroid stimulating hormone (TSH)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The animals were carefully examined for external abnormalities. The abdominal, thoracic and cranial cavities were examined for abnormalities and the organs were removed and examined. Special attention was paid to the organs of the reproductive system. For lactating dams, the number of implantation sites (right and left) was counted. For non-parturition females, uterus implantation sites were examined after staining with ammonium sulfide according to KIT SOPs. Pregnancy was confirmed by implantation sites in the uterus.

ORGAN WEIGHTS: Yes (see table 1)
Paired organs were weighed together unless gross abnormalities were present. However, paired reproductive organs were weighed separately. Animals were fasted overnight prior to necropsy and body weights were recorded on the day of necropsy. Organs were weighed and organ/body weight ratios were calculated.

HISTOPATHOLOGY: Yes (see table 2)
Tissues shown in table 2 from each animal were preserved in 10% neutral buffered formalin, except the eyes (with optic nerve) which were fixed in Davidson’s fixative, and the testes and epididymides which were fixed in Bouin’s fixative. The tissues were placed in the appropriate fixative for approximately 48 hours, and then transferred to 70% ethanol. Formalin was infused into the lung via the trachea and into the urinary bladder.
All tissues except reproductive organs collected from first six animals per sex in main group were further processed to slides, stained with hematoxylin and eosin (H&E), and examined microscopically. All reproductive organs collected from the main groups were further processed to slides, stained with H&E, and examined microscopically. Target organs from the recovery group animals were additionally evaluated microscopically.
Statistics:
Mean values and standard deviations were calculated. Statistical analyses for comparisons of the various dose groups with the vehicle control group were conducted using the Pristima System (Version 7.X. Xybion Medical Systems Corporation, USA) or Statistical Analysis Systems (SAS/STAT Version 9.2 and 9.4, USA). Data were considered to be significant when p<0.05 or p<0.01.
Multiple comparison tests for different dose groups were conducted. Variance of homogeneity was examined using the Bartlett’s Test. Homogeneous data were analyzed using the Analysis of Variance (ANOVA), and the significance of inter-group differences were analyzed using the Dunnett’s Test. Heterogeneous data were analyzed using the Kruskal-Wallis Test, and the significance of inter-group differences was assessed between the control and treated groups using the Dunn’s Rank Sum Test.
For comparing the control group with the recovery group, the data were analyzed for homogeneity for variance using the F-test. Homogeneous data were analyzed using the T-test and the significant difference was assessed between control and recovery group using the Dunnett’s Test. Heterogeneous data were analyzed using the Kruskal-Wallis Test and significant difference was assessed between control and recovery group using the Dunn’s Rank Sum Test.
Data presented as frequencies were analyzed by χ2-test followed by the Fisher's exact test where necessary.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related moribund animals were observed in any group throughout the study.
In males at 15, 50 and 150 mg/kg bw/day, salivation was observed in 11, 12, and 18 animals, respectively. In females at 15, 50, and 150 mg/kg bw/day, salivation was observed in 6, 12, and 18 animals, respectively. It was considered test item-related but not toxicologically significant since it was considered to be attributed to the palatability of the test item.
Other clinical signs were observed in this study but were not considered test item-related since these findings were observed with low frequency or occurred sporadically and did not have any dose-response.

In one female of control group found dead on gestation day 21 one early resorption, one late resorption and 7 dead fetuses were observed in the uterine examination. In the result of microscopic findings, moderate hemorrhage, slight alveolar edema and moderate perivascular congestion were observed in the lung. Moderate centrilobular necrosis in liver, right atrium dilatation in heart, slight atrophy in spleen and minimal mononuclear cell infiltration and hemorrhage in uterus were also observed. These were considered to be incidental since it was observed in the control group and there were no correlated microscopic changes in other animals in the same control group.

Non-parturition was observed in two females of control group and in one female at 150 mg/kg bw/day. Three females were subjected to unscheduled sacrifice on gestation day 27 and all of them were non-pregnant. In the results of microscopic findings, minimal squamous cell hyperplasia in stomach was observed in one animal at 150 mg/kg bw/day, but there were no changes observed in the reproductive system in this animal.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item-related deaths occurred in any group throughout the study.
One female of control group was found dead on gestation day (GD) 21. Two females of control group and one female at 150 mg/kg bw/day were subjected to unscheduled sacrifice on gestation day 27 since they were non-pregnant.


Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item-related changes in body weight and in body weight gain were observed in both sexes during the study. Statistically significant changes in body weight gain during the study were not considered test item-related since they were transient.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related changes in food consumption were observed in both sexes during the study.
A statistically significant change in food consumption during the study was not considered test item-related since there was no correlation with body weight changes and there was no dose-dependency.
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):
In males, total leukocyte count (WBC, 1.40-fold over control), absolute lymphocyte count (LYMA, 1.47-fold over control), absolute monocyte count (MONA, 1.43-fold over control), absolute large unstained cells counts (LUCA, 1.33-fold over control), fibrinogen (FIB, 1.22-fold over control) were increased at 150 mg/kg bw/day and platelet count (PLT, up to 1.21-folds over control) was increased at 50 and 150 mg/kg bw/day. The increases of WBC, LYMA, FIB and PLT were statistically significant. In females, WBC (1.30-fold over control), LYMA (1.37-fold over control), MONA (1.62-fold over control) and LUCA (1.58-fold over control) were increased at 150 mg/kg bw/day. The increases of WBC, MONA and LUCA were statistically significant. These changes recovered or showed a tendency of recovery at the end of the recovery period. Increased leukocyte counts, fibrinogen and platelet count in males and/or females at 150 mg/kg bw/day were correlated with inflammatory changes in the liver.
Other statistically significant changes were not considered test item-related, because these changes were minimal, there were no dose-relationships, and no correlations with microscopic changes and/or observed only in the recovery group.
(see table 3)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males, alanine aminotransferase (ALT, 2.80-fold over control), aspartate aminotransferase (AST, 1.46-fold over control), gamma glutamyl transpeptidase (GGT, 8.47-fold over control) and total bilirubin (TBIL, 1.07-fold over control) were increased at 150 mg/kg bw/day. The increases of ALT, GGT and TBIL were statistically significant. In females, ALT (2.05-fold over control), AST (1.43-fold over control), TBIL (1.21-fold over control) and GGT (8.48-fold over control) were also increased at 150 mg/kg bw/day. The increase of GGT was statistically significant. These changes recovered or showed a tendency to recover at the end of the recovery period. Increases of alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transpeptidase and total bilirubin in males and/or females at 150 mg/kg bw/day were considered to be results due to a massive damage of hepatocytes.
Other statistically significant changes were not considered test item-related, because changes were minimal, there were no dose-relationships, no correlated microscopic changes and/or observed only in recovery group.
(see table 4)
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes in functional behavior examinations were observed in both sexes during the study.
A statistically significant decrease in grip strength of forelimb in males at 50 and 150 mg/kg bw/day was not considered test item-related since there was no dose-dependency and no change in other parameters during the functional behavioral examinations.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increased absolute and relative weights of liver (absolute weights: up to 1.27-folds over control, relative weights: up to 1.24-folds over control) and spleen (absolute weights: up to 1.42-folds over control, relative weights: up to 1.37-folds over control) were noted in both sexes at 150 mg/kg bw/day. (see table 4) Also, decreased absolute weights of the left and right testis (88% of control, respectively), and the left and right epididymides (91% of control, respectively) were observed. These changes did not represent a meaningful toxicologic finding as there were no correlated microscopic findings and/or it fully recovered during the recovery period.
Other statistically significant changes were not considered test item-related, because changes were minimal, there were no dose-relationships, no correlated microscopic changes and/or observed only in recovery group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related changes were observed in liver, stomach, spleen and hepatic lymph node of males and/or females at 150 mg/kg bw/day.
In the liver, yellow or dark-red discoloration, yellow focus/foci, enlarged, small, abnormal shape and/or irregular surface of all lobe or specific lobes (caudate, left or median lobe) were observed in both sexes at 150 mg/kg bw/day. After recovery period, a yellow discoloration and/or irregular liver surface were still observed in both sexes at 150 mg/kg bw/day.
In the stomach, adhesion of the stomach (stomach to liver) was observed in one male at the recovery at 150 mg/kg bw/day.
In the spleen, enlargement was observed in one male at 150 mg/kg bw/day. After the recovery period, enlargement was also observed in one female at 150 mg/kg bw/day and white discoloration was observed in one male and female at 150 mg/kg bw/day.
Enlarged hepatic lymph nodes were observed in both sexes at 150 mg/kg bw/day, but they were not observed any more at the end of the recovery period.
Other macroscopic findings noted in the main and the recovery groups were considered to be incidental or spontaneous changes that are commonly seen in SD rats with a given low incidence or severity.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related changes were observed in liver, spleen, hepatic lymph node, and stomach of both sexes at 150 mg/kg bw/day.

Liver:
Slight to marked cholangiofibrosis, minimal to marked hydropic degeneration, minimal to marked necrosis, minimal to marked bile duct hyperplasia, minimal to slight hepatocyte hypertrophy and/or minimal to slight pigmented macrophages in both sexes and slight fibrosis in capsule in one male were observed at 150 mg/kg bw/day. These changes were observed in the periportal region except a fibrosis in capsule. After recovery period, cholangiofibrosis, hydropic degeneration and necrosis were fully recovered and bile duct hyperplasia and hepatocyte hypertrophy showed a tendency of recovery in both sexes at 150 mg/kg bw/day. In the 150 mg/kg bw/day recovery group, minimal to moderate periportal fibrosis in both sexes were also observed and pigmented macrophage was similar to the main groups in both sexes.
Cholangiofibrosis, hydropic degeneration, and necrosis were considered to be an adverse effect of the test-item and are well known and often observed as hepatocellular toxicity caused by various xenobiotics and chemicals. Periportal fibrosis was observed and shown to be a recovery reaction.
Bile duct hyperplasia, hepatocyte hypertrophy, pigmented macrophages and/or fibrosis in the capsule accompanied the above named findings were not considered as an adverse effect at the severity as seen here. These changes were considered to be adaptive changes to a drug-induced increase in a metabolic workload or were considered as secondary findings related to the aforementioned adverse effects in the liver.

Spleen and hepatic lymph node:
Minimal to slight lymphoid hyperplasia in both sexes at 150 mg/kg bw/day and slight chronic inflammation in the capsule in one female at 150 mg/kg bw/day were observed. At the end of the recovery period, lymphoid hyperplasia showed a tendency of recovery in both sexes at 150 mg/kg bw/day and a capsular fibrosis in both sexes and chronic inflammation in capsule were observed in females at 150 mg/kg bw/day. In the hepatic lymph node, moderate lymphoid hyperplasia and slight congestion were observed in both sexes at 150 mg/kg bw/day.
These changes in spleen and hepatic lymph nodes were considered to be associated with a secondary response to the inflammatory changes in the liver.

Stomach:
Minimal to slight squamous cell hyperplasia in the non-glandular region was observed in both sexes at 150 mg/kg bw/day and fully recovered after the recovery period. This change was considered to be non-adverse but an adaptive response following a local irritation caused by the test item since it fully subsided after the recovery period.

Other changes observed in microscopic findings were considered as incidental or spontaneous changes since they were infrequent, generally of low severity, and similarly distributed among the control and the treated groups.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone anylysis:
No test item-related changes in the thyroid hormone (T4) and the thyroid stimulating hormone (TSH) were observed in the adult male animals.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at 50 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
gross pathology
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios

Target system / organ toxicity

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

Any other information on results incl. tables

Table 3. Haematological findings

Group

 

WBC (x10³/µL)

 

LYMA (x10³/µL)

 

MONA

(x10³/µL)

LUCA (x10³/µL)

 

FIB (mg/dL)

 

PLT (10³/µL)

 

males

Control

n

12

12

12

12

12

12

mean

10.45

8.01

0.35

0.12

182.6

994.3

SD

2.165

1.414

0.134

0.047

9.06

86.03

15 mg/kg bw/day

n

12

12

12

12

12

12

mean

12.33

9.75

0.44

0.14

186.0

974.8

SD

2.937

2.488

0.221

0.052

7.16

111.10

50 mg/kg bw/day

n

12

12

12

12

12

12

mean

11.52

9.07

0.4

0.14

191.8

1118.4*R

SD

3.175

2.476

0.148

0.075

11.87

82.91

150 mg/kg bw/day

n

12

12

12

12

12

12

mean

14.64+D

11.76+D

0.50

0.16

223.2+R

1200.6+R

SD

3.732

3.182

0.265

0.079

30.48

227.41

Recovery group - Control

n

6

6

6

6

6

6

mean

10.17

7.42

0.39

0.10

199.9

1095.7

SD

2.447

1.609

0.127

0.041

10.97

88.91

Recovery group 150 mg/kg bw/day

n

6

6

6

6

6

6

mean

10.93

8.90

0.29

0.12

202.4

1105.0

SD

1.803

1.311

0.092

0.038

7.12

166.78

females

Control

n

9

9

9

9

9

9

mean

9.37

6.22

0.47

0.12

216.3

1190.2

SD

1.673

1.994

0.12

0.049

22.14

236.35

15 mg/kg bw/day

n

12

12

12

12

12

12

mean

8.66

5.81

0.42

0.11

14.7

1194.0

SD

2.041

1.726

0.152

0.035

0.46

221.87

50 mg/kg bw/day

n

12

12

12

12

12

12

mean

9.42

6.08

0.38

0.12

15.2*R

1237.7

SD

2.808

2.307

0.123

0.050

0.60

139.20

150 mg/kg bw/day

n

11

11

11

11

11

11

mean

12.14*D

8.55

0.76+D

0.19*R

15.1

1289.4

SD

2.691

2.644

0.186

0.086

1.03

329.31

Recovery group - Control

n

6

6

6

6

6

6

mean

6.86

5.42

0.17

0.08

172.1

1093.2

SD

0.903

0.646

0.071

0.025

6.48

118.97

Recovery group 150 mg/kg bw/day

n

6

6

6

6

6

6

mean

6.23

4.92

0.20

0.08

181.1*D

1219.7

SD

1.172

1.042

0.106

0.033

3.84

270.14

R = Dunn Rank Sum Test Significant at the 0.01 level (+) or at 0.05 level (*)

D = Dunnett LSD Test Significant at the 0.01 level (+) or at 0.05 level (*)

WBC = total leukocyte count

LYMA = absolute lymphocyte count

MONA = absolute monocyte count

LUCA = absolute large unstained cells counts

FIB = fibrinogen

PLT = platelet count

Table 3. Clinical biochemistry findings

Group

 

ALT (IU/L)

AST (IU/L)

GGT (IU/L)

TBIL (mg/dL)

males

Control

n

12

12

12

12

mean

44.3

172.6

0.49

0.183

SD

42.60

83.42

0.497

0.1209

15 mg/kg bw/day

n

12

12

12

12

mean

34.4

149.4

0.41

0.152

SD

11.40

35.09

0.293

0.0270

50 mg/kg bw/day

n

12

12

12

12

mean

34.9

152.0

0.73

0.150

SD

6.52

16.69

0.308

0.0234

150 mg/kg bw/day

n

12

12

12

12

mean

124.0*R

251.3

4.15+R

0.195*R

SD

181.65

216.19

6.111

0.0341

Recovery group - Control

n

6

6

6

6

mean

47.4

168.5

0.44

0.131

SD

26.76

40.47

0.148

0.0105

Recovery group 150 mg/kg bw/day

n

6

6

6

6

mean

53.8

162.5

0.80+D

0.140

SD

12.46

36.32

0.207

0.0290

females

Control

n

9

9

9

9

mean

65.5

181.8

1.18

0.118

SD

22.29

46.30

0.747

0.0227

15 mg/kg bw/day

n

12

12

12

12

mean

59.8

164.5

0.78

0.122

SD

15.82

32.37

0.355

0.0178

50 mg/kg bw/day

n

12

12

12

12

mean

70.1

162.1

1.45

0.135

SD

16.19

27.38

0.564

0.0237

150 mg/kg bw/day

n

11

11

11

11

mean

134.0

259.5

10.01+R

0.143

SD

176.91

210.57

8.246

0.0336

Recovery group - Control

n

6

6

6

6

mean

44.1

142.8

1.08

0.197

SD

10.60

25.76

0.252

0.0289

Recovery group 150 mg/kg bw/day

n

6

6

6

6

mean

43.2

190.2+D

1.15

0.175

SD

13.61

25.34

0.425

0.0341

R = Dunn Rank Sum Test Significant at the 0.01 level (+) or at 0.05 level (*)

D = Dunnett LSD Test Significant at the 0.01 level (+) or at 0.05 level (*)

ALT = alanine aminotransferase

AST = aspartate aminotransferase

GGT = gamma glutamyl transpeptidase

TBIL = total bilirubin

Table 4. Findings in organ weight

Group

 

Liver absolut (g)

Liver relative (g/kg bw)

males

Control

n

12

12

mean

13.623

2.7296

SD

1.8269

0.17755

15 mg/kg bw/day

n

12

12

mean

13.787

2.7626

SD

1.2970

0.21793

50 mg/kg bw/day

n

12

12

mean

14.437

2.8353

SD

1.7329

0.19210

150 mg/kg bw/day

n

12

12

mean

16.385+D

3.3854+D

SD

1.5829

0.23912

Recovery group - Control

n

6

6

mean

14.781

2.7965

SD

1.1645

0.19102

Recovery group 150 mg/kg bw/day

n

6

6

mean

15.223

2.8543

SD

1.2307

0.20696

females

Control

n

9

9

mean

12.366

3.7479

SD

1.3052

0.33675

15 mg/kg bw/day

n

12

12

mean

12.924

3.8280

SD

0.8599

0.21893

50 mg/kg bw/day

n

12

12

mean

12.632

3.7100

SD

1.0564

0.18965

150 mg/kg bw/day

n

11

11

mean

15.757+R

4.5949+R

SD

2.2078

0.52216

Recovery group - Control

n

6

6

mean

8.684

2.8012

SD

0.5044

0.06238

Recovery group 150 mg/kg bw/day

n

6

6

mean

9.687*D

3.1183+R

SD

0.7912

0.18695

R = Dunn Rank Sum Test Significant at the 0.01 level (+) or at 0.05 level (*)

D = Dunnett LSD Test Significant at the 0.01 level (+) or at 0.05 level (*)    

                 

Applicant's summary and conclusion

Conclusions:
The NOAEL for systemic toxicity was considered to be 50 mg/kg bw/day in both sexes, based on cholangiofibrosis, hydropic degeneration and necrosis of liver in both sexes at 150 mg/kg bw/day.