Reaction mass of 3-O-acetyl-1,5-anhydro-4-butyl-2,4-dideoxy-2-methyl-D-xylitol and (2R,3S)-2-methyloctane-1,3-diyl diacetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Combined 28-Day repeated dose toxicity study with the Reproduction/Developmental toxicity screening test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2017-February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

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:

December 2017-February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same 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:

2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl: WI(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. Charles River Den Bosch has general and reproduction/
developmental historical data in this species from the same strain and source. This animal model has
been proven to be susceptible to the effects of reproductive toxicants.

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 were 10-12 weeks old; females were 12-14 weeks old
- Weight at study initiation: males weighed between 265 and 300 g; females weighed between 195 and 237 g
- Fasting period before study: no
- 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 (Macrolon, MIV type). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type). During the lactation phase, females
were housed in Macrolon plastic cages (MIII type). 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) without cage-enrichment, bedding
material, food and water. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles.
For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom) except when interrupted by study procedures/activities.
- Diet: Prepared diets were provided ad libitum throughout the study, except during designated procedures. During the acclimatization period, animals had free access to standard powder rodent diet (SMR/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: 7 days prior to start of the pretest period (females) or 7 days before the commencement of administration (males)

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants.
Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 40-59
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 December 2017 To: 06 February 2018

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
The test item was mixed without the use of a vehicle, directly with the required amount of powder feed. Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used.
Diets were prepared freshly for use at room temperature for a maximum of 4 days. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 4 days (stability was confirmed under Test Facility Study No. 520229 (analytical Method Development and Validation Study)) for supplementing food during the respective food consumption measurement interval.
If not used on the day of preparation, diets remained at room temperature for a maximum of approximately 7.5 hours before they were stored in the freezer. Diets were kept in the freezer (≤ - 15°C) for a maximum of 15 days until first use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diet preparation samples were collected for analysis during Week 1. Concentration was determined for all groups, homogeneity was determined in low and high dose groups. The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results.
Analyses were performed by using a validated analytical procedure. Duplicate sets of samples were used for concentration analysis. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration.
Duplicate sets of samples for the low and high dose groups were used for homogeneity analysis.
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 demonstrated that the test item is stable in diet when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

The test item and control item were administered to the appropriate animals by inclusion in the diet adlibitum from Day 1 onwards for a minimum of 29 days. Males were exposed for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were exposed for 51-63days, i.e. 14 days prior to mating (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 the day of scheduled necropsy. Females which failed to deliver were treated for 43, 44 or 55 days.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, or spilled diet from the food hopper.

Frequency of treatment:

continuously

Dose / conc.:

1 500 other: ppm; 100 mg/kg bw

Dose / conc.:

4 000 other: ppm; 260 mg/kg bw

Dose / conc.:

12 000 other: ppm; 800 mg/kg bw

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
The dose levels were selected based on the results of a 14-day oral dose range finder with dietary administration of Jessemal in rats, and in an attempt to produce graded responses to the test item.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of administration, and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
-Food consumption was quantitatively measured Days 1, 4, 8, 10, 14, 15, 18, 22, 25 and 29, 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, 9, 11 and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflorane)
- Animals fasted: F0-males were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5/sex/dose
- Parameters checked according to Guidelines

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: F0-males were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5/sex/dose
- Parameters checked according to Guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations: Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 6-13).
These tests were performed after completion of clinical observations.
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity

IMMUNOLOGY: No

OTHER: organ weight, reproduction parameters F0, developmental
parameters F1

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight, For the testes of all selected males of Groups 1 and 4 and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Litter observations:

STANDARDISATION OF LITTERS
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
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, plasma thyroid hormone

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
Scheduled necropsies were conducted on the following days:
Males: Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14-16.
Females which failed to deliver: With evidence of mating: Post-coitum Day 25-27.
Without evidence of mating: 26 days after the last day of the mating period.
All males surviving to scheduled necropsy were fasted overnight with a maximum of approximately 24 hours before necropsy. Water was available. F0- females were not fasted overnight.

GROSS PATHOLOGY: Yes, according to Guidelines
HISTOPATHOLOGY: Yes, according to Guidelines

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspringwas sacrificed at PND 14-16.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external examination

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
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, plasma thyroid hormone

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% and or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
non-parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
The motor activity data set was compared using an overall Kruskal-Wallis.
Incidence: 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.

Reproductive indices:

Mating (%): (Number of females mated / Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females / Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1 / Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100

Offspring viability indices:

Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
Viability index (%): (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering) x 100
Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

Slight chromodacryorrhoea observed in a few male and female animals divided over the different groups (including one control female) was considered unrelated to treatment, taking into account its isolated incidence, the minor severity of the effect, its transient occurrence (1-2 days), and the absence of any apparent dose-related trend.
Any other clinical signs noted during the treatment period 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:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A dose-related decrease in body weight gain was observed in males at 4000 and 12000 ppm.
Body weight and body weight gain in males at 12000 ppm were statistically significantly lower than concurrent control values during the entire treatment period (overall weight gain up to 6% lower than control mean). A similar, albeit less severe trend was observed in males at 4000 ppm, achieving statistical significance for body weight gain on Day 15 of the mating period only.
In females at 12000 ppm reduced mean values for body weight and body weight gain were noted during the entire gestation period. Changes compared to the concurrent control group were largest from Days 4-7 post-coitum when almost no body weight gain was recorded. In the subsequent period, body weight gain in the high dose group had almost the same magnitude as that of the concurrent control group. Statistically significantly lower body weights and body weight gains were noted from Days 7-14 post-coitum only. Body weight and body weight gain remained in the same range as controls over the treatment period in the 1500 ppm (both sexes) and 4000 ppm (females) dose groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Lower food consumption (before and after correction for body weight) was observed in males at
12000 ppm from Days 1-4 of both the pre-mating and Days 1-4 of the mating period, followed by no
rmal values in the remainder of the observation period. (Note: There is no explanation for the rela
tively high food consumption recorded for control males in cage 1 from Days 1-4 of the mating period.)
In pregnant females at 12000 ppm, food consumption (before and after correction for body weight)
was lower than the control level over the gestation period (reaching statistical significance only for
absolute food consumption on Days 0-7 and 11-14 post-coitum). On average, absolute and relative
food intake in pregnant females at this high dose level was 21% and 14% lower as compared to the
control group, respectively, during the gestation period, followed by partial recovery during lactation.
During the lactation period, absolute and relative food intake on average was 11% and 5% lower as
compared to the control group.
Food consumption of males and females at the lower dose levels of 1500 and 4000 ppm were
considered to be unaffected by treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes were noted in haematological parameters.
The slight, but statistically significant increase in mean red blood cells observed in males at 12000 ppm at the end of treatment (1.05x of control) occurred in the absence of corroborating changes in other red blood cells parameters, and since only one individual value (one male: 9.50 10E12/L) was slightly above the historical control range, this finding was considered not to be toxicologically relevant.
The statistically significantly lower mean number of platelets noted for females in the 4000 ppm group was considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
There were no treatment-related changes in coagulation parameters [prothrombin time (PT) and activated partial thromboplastin time (APTT)].

note: Historical control data for F0-males (period 2015-2017): Red blood cells (10E12/L): mean = 8.81; P5-P95 = 8.14-9.45 (n=286).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of treatment, a test-item related increase in mean cholesterol was observed in males at 12000 ppm (1.3 x of control). The mean value was at the upper limit of the historical control data , with 2/5 individual values above the upper limit.
The higher mean plasma activity of alanine aminotransferase (ALAT: 1.7x of control) and aspartate aminotransferase (ASAT: 1.4x of control) recorded in males at 12000 ppm (only statistically significant for ALAT) was attributed to one single male that presented with remarkably increased plasma activity of both hepatic enzymes (ALAT: 117.2 U/L; ASAT: 254.1 U/L) when compared to both concurrent and historical control means (3). The same male was also noted with a relatively high value for bile acids (87.6 μmol/L). At the microscopic level, slight hyperplasia of the bile duct was noted. The second high dose male with increased bile acids (55.2 μmol/L) had the same liver finding. However, hyperplasia of the bile duct (up to a slight degree) was also seen for the remaining three high dose males that had normal values of bile acids.
A relatively high ASAT value (139.8 U/L) was measured in one low dose male as well. In the absence of any comparable change in the mid dose group, no toxicological significance was attached to this isolated finding.
Also in lactating females at 4000 and 12000 ppm, an increase in bile acids (only significant at 4000 ppm) was observed at the end of treatment (4x and 2x of control, respectively). A large intragroup variation was observed at both dose levels. At the individual level, bile acids levels were above the historical control range (3) in 3/5 and 2/5 females at 4000 and 12000 ppm, respectively. One of these two high dose female was noted with minimal hyperplasia of the bile duct. But there were also two other high dose females with the same pathological finding with normal values for bile acids.
In lactating females at 4000 and 12000 ppm, a test item-related increase in total bilirubin was observed at the end of treatment (1.5x and 1.6x of control, respectively). The mean value at 4000 ppm was within but in the upper limit of the historical control data . The mean value at 12000 ppm was outside the historical control range(3).
The significantly higher mean values for urea measured in males at 1500 and 12000 ppm were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Thyroid hormone analyses:
At the end of treatment, serum level of total T4 in F0-males was significantly lower than concurrent control values (0.8x of control). The mean value (3.43 μg/dL) remained within the historical control range (4). At the individual level, 3 out of the 10 males had values at the lower limit or below the historical range (1.74, 2.32 and 3.02 μg/dL for the individual males, respectively).

(3) Historical control data for F0-males (period 2015-2017):
Cholesterol (mmol/L): mean = 1.85; P5-P95 = 1.36-2.42 (n=304).
ALAT (U/L): mean = 46.9; P5-P95 = 30.30-68.00 (n=304).
ASAT (U/L): mean = 81.2; P5-P95 = 65.50-99.50 (n=304).
Bile acids (μmol/L): mean = 23.6; P5-P95 = 9.70-46.0 (n=303).
Urea (mmol/L): mean = 6.8; P5-P95 = 4.00-9.60 (n= 304)
Total T4 (μg/dL): mean 4.65; P5-P95 = 3.01-6.52 (n=724).
Historical control data for non-fasted F0-females (period 2017-2018):
Total bilirubin (μmol/L): mean = 1.9; P5-P95 = 1.25-2.50 (n=60).
Bile acids (μmol/L): mean = 22.1; P5-P95 = 8.45-44.65 (n=60).

(4) Historical control data for F0-males (period 2015-2017):
Total T4 (μg/dL): mean 4.65; P5-P95 = 3.01-6.52 (n=724).

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the treatment period, a statistically significant decrease in mean foreleg grip strength was observed in all treated female groups (mean up to 24% lower than that of the control). This finding was attributed to a relative high mean value in the concurrent control group when compared to available historical control data (1536 vs 953). Given that none of the individual values of treated females was below the historical control range, changes occurred in the absence of a dose-related response and there were no findings during clinical observation or motor activity measurement in this study indicating any abnormalities in the neuromuscular domain, this reduction in foreleg grip strength was regarded as unrelated to treatment. The statistically significant decrease in hindleg grip strength observed in females at 4000 ppm was not considered to be related to treatment as no-dose related trend was observed.
Leg grip strength in males was not affected by treatment.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. (Note: no explanation could be given for the sudden higher activity of control males recorded 30-35 minutes after start of the session with higher counts of both total movements and ambulations).

note: historical values Fore leg grip (gram): mean = 953; P5-P95 = 578-1363 (n=240).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with Jessemal were noted in the liver and the thymus and are summarized in the section 'any other information on results incl. tables'.
Liver, hyperplasia bile duct was present in males starting at 4000 ppm up to slight degree and in females at 12000 ppm at minimal degree.
Infiltration peribiliary was present in males starting at 4000 ppm at minimal degree.
Hepatocellular hypertrophy was present in females treated at 12000 ppm up to slight degree.
Thymus, atrophy was present in females treated at 4000 and 12000 ppm up to slight degree. At 12000 ppm this correlated with the decrease in thymus weight.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were not affected by treatment.
All females had regular cycles, generally of 4 days. Extended di-estrus during pairing occurred in one control female (with normal litter) and one female each at 1500 ppm
(with only one implantation site) and at 4000 ppm. For the latter female, mating could not be confirmed; a finding that was attributed to the massive reduced sperm noted in the epididymides of the male she was paired with.
Given the incidental nature of the extended di-estrus, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, this cycle abnormality did not indicate a relation with treatment.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were noted in spermatogenic profiling and histopathological examination of reproductive organs.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

There were 1/10 control group couples, 1/10 couples treated at 4000 ppm and 3/10 couples treated at 12000 ppm that had no offspring. In addition, there was 1/10 couples treated at 1500 ppm with implantation sites only. For the couple treated at 4000 ppm the lack of offspring could be explained by the marked tubular atrophy in the testes and the massive reduced sperm in the epididymis.This single incidence in the mid-dose group was not considered test item-related. Histopathology did not reveal any changes in the reproductive organs of the other couples that could explain their lack in offspring.

Mating index was not affected by treatment. Except for one female at the mid dose of 4000 ppm (no. 65), all females showed evidence of mating. The mating indices were 90% at 4000 ppm and 100% for the other groups.

Precoital time was not considered to be affected by treatment.
In general, females showed evidence of mating within 5 days, except for one control female which had a precoital time of 13 days. For one female (no. 54) at 1500 ppm mating was overlooked. At necropsy, one implantation site was found in her uterus.

Number of implantation sites was not affected by treatment.
The mean number of implantation sites in females at 12000 ppm was slightly, but statistically significantly lower than that observed in the concurrent controls (11.4 vs 12.6). This finding was considered to be unrelated to treatment with the test item, as all individual values (i.e. 10 to 13 implantation sites) remained well within the historical control data (7).
One control female had 4 implantation sites and one female at 1500 ppm had a single implantation site only. These isolated findings were considered to be unrelated to treatment, as no dose-related trend could be established.

A total of one control female and 3 females at 12000 ppm were not pregnant. The fertility indices were 90, 100, 100 and 70% for the control, 1500 ppm, 4000 ppm and 12000 ppm groups, respectively. The fertility index at the high dose group was outside the range of the historical control data (8).

(7)Historical control data for F0-females (period 2015-2017):
Number of implantation sites: mean = 12; P5-P95 = 5-16 (n=514).
Fertility index (%): mean = 95; P5-P95 = 80-100 (n=53).
Gestation index (%): mean = 99; P5-P95 = 85-100 (n=52).

(8) Historical control data for F0-females (period 2015-2017):
Post-implantation survival index (%): mean = 93; P5-P95 = 84-100 (n=48).
Live birth index (%): mean = 99; P5-P95 = 94-100 (n=48).

Key result

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

111 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: morphological changes in the liver

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

4 000 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
For the pup of the control female that had to be sacrificed in extremis on PND 4, absence of milk in the stomach and lean appearance were noted at last litter check. For the pup of the female at 1500 ppm that had to be sacrificed in extremis on PND 3, blue colour and difficult breathing were observed. No toxicological significance was attached to this isolated finding at the low dose level.
The nature and incidence of the other clinical signs remained within the range considered normal for pups of this age and were, therefore, considered to be unrelated to treatment.

Mortality / viability:

no mortality observed

Description (incidence and severity):

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 99, 98, 100 and 100% for the control, 1500 ppm, 4000 ppm and 12000 ppm groups, respectively.
One pup of the control group and two pups at 1500 ppm had to be sacrificed in extremis or were missing (most likely cannibalised) on PND 3-4. No relation to treatment was attributed to these euthanized/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

At 4000 and 12000 ppm, mean body weights of male and female pups were slightly lower (not statistically significant) than those of control animals on lactation Days 7 and 13. Mean value of combined pup body weight was 6% and 9% lower at 4000 and 12000 ppm, respectively, on lactation Day 13. In general, individual values remained within the range considered normal for pups of this age.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum levels of total T4 in male and female PND14-16 pups were considered not to be affected by treatment. All values remained within the normal range of biological variation.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.

Other effects:

no effects observed

Description (incidence and severity):

The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment.The post-implantation survival indices were 83, 88, 90 and 83% for the control, 1500 ppm, 4000 ppm and 12000 ppm groups, respectively.
The lower post-implantation survival index noted in the control and 12000 ppm groups when compared to the historical control mean data (83 vs 93%) was attributed to one control female that delivered 6 pups out of 12 implantations sites and one high dose female that delivered 4 pups out of 11 implantations sites. As a dose-related response was absent, this findings was not considered to be related to treatment.
For one female at 4000 ppm, the number of pups was slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 16 days of lactation. No toxicological relevance was attached to this finding in the current study.

The mean number of living pups at first litter check (live litter size) was considered to be unaffected by treatment.
The slightly lower mean number of living pups at first litter check in the 12000 ppm group (9.0 versus 10.1 in the concurrent control group) was caused mainly by one female which had one live pup only (next to 3 dead pups). Such small litter sizes are observed occasionally in this type of study. Moreover, in the current study there were also two relatively small litters in the control group (4 live pups each), and one low dose female with one implantation site only. All remaining litters in the high dose group had 9-12 living pups at first litter check, which is within the normal range. As such, the observation of one litter with one live pup only in the high dose group was considered to be unrelated to the test item.
Lactation index (number of live offspring on PND 13 after littering as percentage of number of live offspring on PND4 after culling) was not affected by treatment. The lactation indices were 99% at 4000 ppm and 100% for the other groups.
One pup at 4000 ppm was missing (most likely cannibalised) on PND 7. No relation to treatment was attributed to this isolated finding since it did not show a dose-related trend and remained within the range considered normal for pups of this age.
Sex ratio was considered unaffected by treatment.
The shift in the sex ratio towards higher numbers of living female pups in the 12000 ppm group was caused by one litter that had only one living, female pup at first litter check (i.e. 100% female).
Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment.
The statistically significantly higher mean normalized anogenital distance of female pups at 4000 ppm occurred without a dose related-trend and, therefore, this observation was not considered to be related to treatment.
Treatment up to and including 12000 ppm had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

862 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 354 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Accuracy: Mean accuracy values were 97, 99 and 96% in the low, mid and high dose groups, respectively. No test item was detected in the control diets.

Homogeneity: coefficient of variation was 2.3 and 3.0% in the low and high dose, respectively.

Mean test article intake over the study period was as follows:

		Mean over means intake [mg test item/kg body weight] (mean range indicated within brackets)
	Group no.	2	3	4
	Nominal dietary inclusion level (ppm)	1500	4000	12000
Sex	Study period
Males	Pre-mating	114 (107-122)	307 (282-322)	878 (805-934)
	Post-mating	107 (97-126)	275 (253-321)	842 (813-867)
	Mean of means^a	111	293	862
Females	Pre-mating	107 (100-110)	282 (246-299)	840 (754-934)
	Post-coitum	149 (133-160)	454 (410-499)	1077 (1056-1096)
	Lactation	315 (228-365)	882 (652-1030)	2200 (1617-2470)
	Mean of means^a	188	534	1354
^aMean of means of all periods, weighed for number of measurement intervals per period: Males: ((5 x mean premating) + (4 x mean mating)) / 9 Females: ((5 x mean premating) + (6 x mean post-coitum) + (5 x mean lactation)) / 16

Mean Percent Organ Weight Differences from Control Groups

	Males					Females
Dose level (ppm):	1500		4000		12000	1500	4000	12000
LIVER
Absolute	1		1		17**	11	11	14
Relative to body weight	4		6		24**	10	13*	20**
THYMUS
Absolute	-7		-10		-9	-12	-21	-32*
Relative to body weight	-5		-5		-3	-14	-20	-28
*:P<0.05, **:P<0.01

Summary Test Item-Related Microscopic Findings – Liver

	Males				Females
Dose level (ppm):	0	1500	4000	12000	0	1500	4000	12000
LIVER^a	5	5	5	5	5	6	5	5
Hyperplasia bile duct
Minimal	-	-	3	1	-	-	-	3
Slight	-	-	-	4	-	-	-	-
Infiltration peribiliary
Minimal	-	-	3	2	-	-	-	-
Hypertrophy hepatocellular
Minimal	-	-	-	-	-	-	-	4
Slight	-	-	-	-	-	-	-	1

^a = Number of tissues examined from each group

Summary Test Item-Related Microscopic Findings – Thymus

	Females
Dose level (ppm):	0	1500	4000	12000
THYMUS^a	5	6	5	5
Atrophy
Minimal	-	-	-	1
Slight	-	-	1	1

^a = Number of tissues examined from each group.

Conclusions:

Overall conclusion on Repeated dose and reproductive toxicity: The parental NOAEL for systemic toxicityis 1500 ppm, corresponding to 111 mg/kg bw/day in males and 188 mg/kg bw/day in females (based on minimal to slight bile duct hyperplasia accompanied by intrahepatic peribiliary infiltration in the males at and from 4000 ppm onwards. In females minimal bile duct hyperplasia was seen in the high dose at 12000 ppm).
Fertility NOAELis at least 12000 ppm, corresponding to 862 mg/kg bw/day in males and 1354 mg/kg bw/day in females. The slightly decreased fertility (3/10 not pregnant) is not considered adverse in absence of any other fertility or reproductive related effects.
Developmental NOAEL: At least 12000 ppm, corresponding to 1354 mg/kg bw/day in females.

Executive summary:

The substance is tested in an OECD TG 422 repeated dose/ reproscreen study. The substance was administered via the diet to Wistar Han rats at dietary concentrations of 1500, 4000 and 12000 ppm (10 rats/sex/dose level anticipated dose levels are 800, 260 and 100 mg/kg bw). Concurrent controls (10 rats/sex) received basal powder diet without the test item. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 51-63 days). Females that failed to deliver pups were treated for 43, 44 or 55 days.Chemical analyses of dietary preparations 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 (for 5 selected animals/sex/group),body weight and food consumption, estrous cycle determination, clinical pathology (for 5 selected animals/sex/group), measurement of thyroid hormone T4 (F0-males), 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). Analysis of diet preparations showed that the powder diets were prepared accurately and homogeneously.

Parental results

Clinical signs, body weight and food consumption: Reduced mean body weight gain was observed in males at 4000 and 12000 ppm (dose-dependently) during the entire treatment period (not always statistically significant). In females at 12000 ppm reduced mean body weight gain was noted during the entire gestation period (not always statistically significant). Changes compared to the concurrent control group were largest from Days 4-7 post-coitum when almost no body weight gain was recorded. In the subsequent period, body weight gain in the high dose group had almost the same magnitude as that of the concurrent control group. The resulting reductions in mean body weights of males at 4000 and 12000 ppm and females at 12000 ppm at end of treatment were modest (i.e. < 10%) and therefore considered not to be adverse.

The reduced body weight gain was accompanied by lower food consumption in males at 12000 ppm from Days 1-4 of both the pre-mating (absolute and relative food consumption were 17% and 29% lower as compared to controls) and Days 1-4 of the mating period (absolute and relative food consumption were 17% and 26% lower as compared to controls). However, normal values were measured in the remainder of the observation period. In pregnant females at 12000 ppm, food consumption was lower than the control level over the gestation period (reaching statistical significance only for absolute food consumption on Days 0-7 and 11-14 post-coitum). On average, absolute and relative food intake in pregnant females at this high dose level was 21% and 14% lower as compared to the control group, respectively, during the gestation period, followed by partial recovery during lactation. At the end of lactation, absolute and relative food intake on average was 11% and 5% lower as compared to the control group.

Haematology:No heamatology effects were seen.

Biochemical parameters: In lactating females at 4000 and 12000 ppm, a test item-related increase in total bilirubin was observed at the end of treatment (1.5x and 1.6x of control, respectively). The mean value at 4000 ppm was at the upper limit of the historical control data. The mean value at 12000 ppm was outside the historical control range. This increase was / was not accompanied with effects on haematology. Cholesterol was increased but in males only (1.3x control) which can be related to the increased liver weight. Bile acids were slightly increased at the high dose in both sexes (ca 50%) but not dose related and not statistically significant. Only the mid dose in females reached statistical significance but this can also be attributed to the fairly low control value.

Organ related effects: There were not treatment related organ weights effects, macroscopic or histopathological effects, except for the liver and thymus.

Relative liver weights were increased accompanied by minimal hyperplasia only in females and only in the high dose only. Minimal to slight hyperplasia of the bile duct was seen in males at the mid dose (3 animals) and high dose (1 animal), respectively, accompanied by intrahepatic peribiliary. infiltration. These bile effects are not very common effects and considered for deriving the NOAEL.

Non-adverse test item-related atrophy was present in the thymus of females at 4000 and 12000 ppm. This morphologic alteration occurred in the absence of any other findings in that organ and, at the low severity observed in this study, was not considered adverse. No thyroid effects were seen but at 12000 ppm, serum level of total T4 in F0-males was reduced by approximately 23% compared to control values. The mean value (3.43 µg/dL) remained within the historical control range (see note), but at the individual level, 3 out of the 10 males had values at the lower limit or below the historical range (1.74, 2.32 and 3.02 µg/dL). A possible relation to treatment could not be excluded. This reduction of total T4 occurred in the absence of corroborating changes in either organ weight or morphology of the thyroid gland.  Due to the limited data available from this type of screening study, possible adversity of this effect could not be assessed. Therefore, this reduction in total T4 in males sat 12000 ppm was not taken into account when determining the parental NOAEL.

In conclusion, Parental NOAEL is 1500 ppm, corresponding to 111 mg/kg bw/day in males and 188 mg/kg bw/day in females (based on minimal to slight bile duct hyperplasia accompanied by intrahepatic peribiliary infiltration in the males at and from 4000 ppm onwards. In females minimal bile duct hyperplasia was seen in the high dose at 12000 ppm).

Note: [1]Historical control data for F0-males (period 2015-2017): Total T4 (µg/dL): mean 4.65; P5-P95 = 3.01-6.52 (n=724).

 

Fertility results

At 12000 ppm, three out of the ten high dose females were not pregnant, which could not be related to any other reproductive parameters measured. Histopathology did not reveal any changes in the reproductive organs of these females or the males they were mated with that could explain their lack in offspring e.g. oestrus cycle effects or implantation loss or effects in spermatogenesis and are therefore not considered as adverse.

Developmental results

At 4000 and 12000 ppm, a trend towards slightly lower body weight gain of male and female pups was noted on lactation Day 7 and Day 13, resulting in 6% and 9% lower mean combined body weights on lactation Day 13 (not statistically significant). At birth, mean pup body weights in all treated groups were comparable to those of the concurrent control group, andno lack of maternal care was observed. Given the fact that the changes in body weight were only slight (< 10%) with most individual values remaining within the normal range of biological variation, this finding was not considered to be adverse.

Overall conclusion on Repeated dose and reproductive toxicity

The parental NOAEL for systemic toxicityis 1500 ppm, corresponding to 111 mg/kg bw/day in males and 188 mg/kg bw/day in females (based on minimal to slight bile duct hyperplasia accompanied by intrahepatic peribiliary infiltration in the males at and from 4000 ppm onwards. In females minimal bile duct hyperplasia was seen in the high dose at 12000 ppm).

Fertility NOAELis at least 12000 ppm, corresponding to 862 mg/kg bw/day in males and 1354 mg/kg bw/day in females. The slightly decreased fertility (3/10 not pregnant) is not considered adverse in absence of any other fertility or reproductive related effects.

Developmental NOAEL: At least 12000 ppm, corresponding to 1354 mg/kg bw/day in females.

 

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2017-February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developme ntal Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/De velopmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 421, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2016

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)

Version / remarks:

2008

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: other: Crl: WI(Han)

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 were 10-12 weeks old; females were 12-14 weeks old
- Weight at study initiation: males weighed between 265 and 300 g; females weighed between 195 and 237 g
- Fasting period before study: no
- 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 (Macrolon, MIV type). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type). During the lactation phase, females were housed in Macrolon plastic cages (MIII type). 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) without cage-enrichment, bedding material, food and water. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J .Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles.
For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom) except when interrupted by study procedures/activities.
- Diet: Prepared diets were provided ad libitum throughout the study, except during designated procedures. During the acclimatization period, animals had free access to standard powder rodent diet (SMR/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: 7 days prior to start of the pretest period (females) or 7 days before the commencement of administration (males)

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants.
Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 40-59
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 December 2017 To: 06 February 2018

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
The test item was mixed without the use of a vehicle, directly with the required amount of powder feed. Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used.
Diets were prepared freshly for use at room temperature for a maximum of 4 days. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 4 days (stability was confirmed under Test Facility Study No. 520229 (analytical Method Development and Validation Study)) for supplementing food during the respective food consumption measurement interval.
If not used on the day of preparation, diets remained at room temperature for a maximum of approximately 7.5 hours before they were stored in the freezer. Diets were kept in the freezer (≤ - 15°C) for a maximum of 15 days until first use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diet preparation samples were collected for analysis during Week 1. Concentration was determined for all groups, homogeneity was determined in low and high dose groups. The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results.
Analyses were performed by using a validated analytical procedure. Duplicate sets of samples were used for concentration analysis. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration.
Duplicate sets of samples for the low and high dose groups were used for homogeneity analysis.
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 demonstrated that the test item is stable in diet when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 post-coitum

Duration of treatment / exposure:

The test item and control item were administered to the appropriate animals by inclusion in the diet
adlibitum from Day 1 onwards for a minimum of 29 days. Males were exposed for 29 days, up to and
including the day before scheduled necropsy. This included a minimum of 14 days prior to mating
and during the mating period. Females that delivered were exposed for 51-63days, i.e. 14 days prior
to mating (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 the day of scheduled necropsy. Females which
failed to deliver were treated for 43, 44 or 55 days.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk,
from exposure to maternal urine/feces, or spilled diet from the food hopper.

Frequency of treatment:

continuously

Dose / conc.:

1 500 ppm

Dose / conc.:

4 000 ppm

Dose / conc.:

12 000 ppm

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

Details on study design
- Dose selection rationale:
The dose levels were selected based on the results of a 14-day oral dose range finder with dietary administration of Jessemal in rats, and in an attempt to produce graded responses to the test item.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of administration, and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
-Food consumption was quantitatively measured Days 1, 4, 8, 10, 14, 15, 18, 22, 25 and 29, 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 postcoitum and during lactation on PND 1, 4, 7, 9, 11 and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflorane)
- Animals fasted: F0-males were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5/sex/dose
- Parameters checked according to Guidelines

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: F0-males were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5/sex/dose
- Parameters checked according to Guidelines

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations: Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 6-13).
These tests were performed after completion of clinical observations.
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity

Estrous cyclicity (parental animals)
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed.
Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible
from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
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, plasma thyroid hormone level

GROSS EXAMINATION OF DEAD PUPS:
yes.
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally).
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

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% and or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
non-parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
The motor activity data set was compared using an overall Kruskal-Wallis.
Incidence: 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.

Indices:

Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
Viability index (%): (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering) x 100
Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

Slight chromodacryorrhoea observed in a few male and female animals divided over the different groups (including one control female) was considered unrelated to treatment, taking into account its
isolated incidence, the minor severity of the effect, its transient occurrence (1-2 days), and the absence of any apparent dose-related trend.
Any other clinical signs noted during the treatment period 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:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A dose-related decrease in body weight gain was observed in males at 4000 and 12000 ppm.
Body weight and body weight gain in males at 12000 ppm were statistically significantly lower than concurrent control values during the entire treatment period (overall weight gain up to 6% lower than control mean). A similar, albeit less severe trend was observed in males at 4000 ppm, achieving statistical significance for body weight gain on Day 15 of the mating period only.
In females at 12000 ppm reduced mean values for body weight and body weight gain were noted during the entire gestation period. Changes compared to the concurrent control group were largest from Days 4-7 post-coitum when almost no body weight gain was recorded. In the subsequent period, body weight gain in the high dose group had almost the same magnitude as that of the concurrent control group. Statistically significantly lower body weights and body weight gains were noted from Days 7-14 post-coitum only. Body weight and body weight gain remained in the same range as controls over the treatment period in the 1500 ppm (both sexes) and 4000 ppm (females) dose groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

12000 ppm from Days 1-4 of both the pre-mating and Days 1-4 of the mating period, followed by no rmal values in the remainder of the observation period. (Note: There is no explanation for the relatively high food consumption recorded for control males in cage 1 from Days 1-4 of the mating period.)
In pregnant females at 12000 ppm, food consumption (before and after correction for body weight) was lower than the control level over the gestation period (reaching statistical significance only for absolute food consumption on Days 0-7 and 11-14 post-coitum). On average, absolute and relative food intake in pregnant females at this high dose level was 21% and 14% lower as compared to the control group, respectively, during the gestation period, followed by partial recovery during lactation.
During the lactation period, absolute and relative food intake on average was 11% and 5% lower as compared to the control group.
Food consumption of males and females at the lower dose levels of 1500 and 4000 ppm were considered to be unaffected by treatment.

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):

No toxicologically relevant changes were noted in haematological parameters.
The slight, but statistically significant increase in mean red blood cells observed in males at 12000 ppm at the end of treatment (1.05x of control) occurred in the absence of corroborating changes in other
red blood cells parameters, and since only one individual value (one male: 9.50 10E12/L) was slightly above the historical control range, this finding was considered not to be toxicologically relevant.
The statistically significantly lower mean number of platelets noted for females in the 4000 ppm group was considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
There were no treatment-related changes in coagulation parameters [prothrombin time (PT) and activated partial thromboplastin time (APTT)].

note: Historical control data for F0-males (period 2015-2017): Red blood cells (10E12/L): mean = 8.81; P5-P95 = 8.14-9.45 (n=286).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of treatment, a test-item related increase in mean cholesterol was observed in males at 12000 ppm (1.3 x of control). The mean value was at the upper limit of the historical control data , with 2/5 individual values above the upper limit.
The higher mean plasma activity of alanine aminotransferase (ALAT: 1.7x of control) and aspartate aminotransferase (ASAT: 1.4x of control) recorded in males at 12000 ppm (only statistically significant for ALAT) was attributed to one single male that presented with remarkably increased plasma activity of both hepatic enzymes (ALAT: 117.2 U/L; ASAT: 254.1 U/L) when compared to both concurrent and historical control means (3). The same male was also noted with a relatively high value for bile acids (87.6 μmol/L). At the microscopic level, slight hyperplasia of the bile duct was noted. The second high dose male with increased bile acids (55.2 μmol/L) had the same liver finding.
However, hyperplasia of the bile duct (up to a slight degree) was also seen for the remaining three high dose males that had normal values of bile acids.
A relatively high ASAT value (139.8 U/L) was measured in one low dose male as well. In the absence of any comparable change in the mid dose group, no toxicological significance was attached to this isolated finding.
Also in lactating females at 4000 and 12000 ppm, an increase in bile acids (only significant at 4000 ppm) was observed at the end of treatment (4x and 2x of control, respectively). A large intragroup variation was observed at both dose levels. At the individual level, bile acids levels were above the historical control range (3) in 3/5 and 2/5 females at 4000 and 12000 ppm, respectively. One of these two high dose female was noted with minimal hyperplasia of the bile duct. But there were also two other high dose females with the same pathological finding with normal values for bile acids.
In lactating females at 4000 and 12000 ppm, a test item-related increase in total bilirubin was observed at the end of treatment (1.5x and 1.6x of control, respectively). The mean value at 4000 ppm was within but in the upper limit of the historical control data. The mean value at 12000 ppm was outside the historical control range(3).
The significantly higher mean values for urea measured in males at 1500 and 12000 ppm were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Thyroid hormone analyses:
At the end of treatment, serum level of total T4 in F0-males was significantly lower than concurrent control values (0.8x of control). The mean value (3.43 μg/dL) remained within the historical control range (4). At the individual level, 3 out of the 10 males had values at the lower limit or below the historical range (1.74, 2.32 and 3.02 μg/dL for the individual males, respectively).

(3) Historical control data for F0-males (period 2015-2017):
Cholesterol (mmol/L): mean = 1.85; P5-P95 = 1.36-2.42 (n=304).
ALAT (U/L): mean = 46.9; P5-P95 = 30.30-68.00 (n=304).
ASAT (U/L): mean = 81.2; P5-P95 = 65.50-99.50 (n=304).
Bile acids (μmol/L): mean = 23.6; P5-P95 = 9.70-46.0 (n=303).
Urea (mmol/L): mean = 6.8; P5-P95 = 4.00-9.60 (n= 304)
Total T4 (μg/dL): mean 4.65; P5-P95 = 3.01-6.52 (n=724).
Historical control data for non-fasted F0-females (period 2017-2018):
Total bilirubin (μmol/L): mean = 1.9; P5-P95 = 1.25-2.50 (n=60).
Bile acids (μmol/L): mean = 22.1; P5-P95 = 8.45-44.65 (n=60).

(4) Historical control data for F0-males (period 2015-2017):
Total T4 (μg/dL): mean 4.65; P5-P95 = 3.01-6.52 (n=724).

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the treatment period, a statistically significant decrease in mean foreleg grip strength was observed in all treated female groups (mean up to 24% lower than that of the control). This finding was attributed to a relative high mean value in the concurrent control group when compared to available historical control data (1536 vs 953). Given that none of the individual values of treated females was below the historical control range, changes occurred in the absence of a dose-related response and there were no findings during clinical observation or motor activity measurement in this study indicating any abnormalities in the neuromuscular domain, this reduction in foreleg grip strength was regarded as unrelated to treatment. The statistically significant decrease in hindleg grip strength observed in females at 4000 ppm was not considered to be related to treatment as no-dose related trend was observed.
Leg grip strength in males was not affected by treatment.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. (Note: no explanation could be given for the sudden higher activity of control males recorded 30-35 minutes after start of the session with higher counts of both total movements and ambulations).

note: historical values Fore leg grip (gram): mean = 953; P5-P95 = 578-1363 (n=240).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item-related higher liver weights (absolute and/or relative to body weights) were noted in the 4000 ppm-treated females and the 12000 ppm-treated males and females, and test item-related lower thymus weights (absolute and relative to body weight) were present in the 12000 ppm-treated females (see 'any other information on results incl. tables').
There were no other test item-related organ weight changes. Some other organ weight differences, including the lower absolute adrenal weight in males at 12000 ppm, 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 and/or lacked a dose-response.
The significantly lower uterus weights (absolute and relative to body weight) noted in mid and high dose groups were not related to the test item, but caused by relative high individual values in the control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.
All 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:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with Jessemal were noted in the liver and the thymus and are summarized in the section 'any other information on results incl. tables'.
Liver, hyperplasia bile duct was present in males starting at 4000 ppm up to slight degree and in females at 12000 ppm at minimal degree.
Infiltration peribiliary was present in males starting at 4000 ppm at minimal degree.
Hepatocellular hypertrophy was present in females treated at 12000 ppm up to slight degree.
Thymus, atrophy was present in females treated at 4000 and 12000 ppm up to slight degree. At 12000 ppm this correlated with the decrease in thymus weight.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Description (incidence and severity):

No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment. The post-implantation survival indices were 83, 88, 90 and 83% for the control, 1500 ppm, 4000 ppm and 12000 ppm groups, respectively.
The lower post-implantation survival index noted in the control and 12000 ppm groups when compared to the historical control mean data (8) (83 vs 93%) was attributed to one control female that delivered 6 pups out of 12 implantations sites and one high dose female that delivered 4 pups out of 11 implantations sites. As a dose-related response was absent, this findings was not considered to be related to treatment.
For one female at 4000 ppm, the number of pups was slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 16 days of lactation. No toxicological relevance was attached to this finding in the current study.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The mean number of living pups at first litter check (live litter size) was considered to be unaffected by treatment.
The slightly lower mean number of living pups at first litter check in the 12000 ppm group (9.0 versus 10.1 in the concurrent control group) was caused mainly by one female which had one live pup only (next to 3 dead pups). Such small litter sizes are observed occasionally in this type of study. Moreover, in the current study there were also two relatively small litters in the control group (4 live pups each), and one low dose female with one implantation site only. All remaining litters in the high dose group had 9-12 living pups at first litter check, which is within the normal range (9). As such, the observation of one litter with one live pup only in the high dose group was considered to be unrelated to the test item.

(9) Historical control data for F0-females (period 2015-2017):
Living pups at first litter check: mean = 11.4; P5-P95 = 6.00-15.00 (n=794).
Live birth index (%): mean = 99; P5-P95 = 94-100 (n=48).

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Description (incidence and severity):

Live birth index (number of live offspring on PND 1 after littering as percentage of total number of offspring born) was not affected by treatment. The live birth indices were 97, 99, 100 and 95% for the control, 1500 ppm, 4000 ppm and 12000 ppm groups, respectively.
At first litter check, 3 pups of the control group, 1 pup at 1500 ppm and 3 pups at 12000 ppm were found dead. No relation to treatment was attributed to these dead pups since the mortality incidence did not show a doserelated trend and live birth indices remained within the range considered normal for pups of this age (9).

(9) Historical control data for F0-females (period 2015-2017):
Living pups at first litter check: mean = 11.4; P5-P95 = 6.00-15.00 (n=794).
Live birth index (%): mean = 99; P5-P95 = 94-100 (n=48).

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Gestation index and duration of gestation were not considered to be affected by treatment.
The gestation indices were 90% at 1500 ppm and 100% for the other groups. One female at 1500 ppm (with a single implantation site only) delivered no offspring.
As this occurred in absence of a dose-response relationship and mean remained well within normal values for rats of this age and strain (7), this finding in one low dose female wasconsidered to be unrelated to treatment.
One control female and one female at 4000 ppm delivered after 23 days. In the absence of a dose-related trend, this finding was considered to be unrelated to treatment with the test item.

(7) Historical control data for F0-females (period 2015-2017):
Number of implantation sites: mean = 12; P5-P95 = 5-16 (n=514).
Fertility index (%): mean = 95; P5-P95 = 80-100 (n=53).
Gestation index (%): mean = 99; P5-P95 = 85-100 (n=52).

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

A total of one control female and 3 females at 12000 ppm were not pregnant. The fertility indices were 90, 100, 100 and 70% for the control, 1500 ppm, 4000 ppm and 12000 ppm groups, respectively. The fertility index at the high dose group was outside the range of the historical control data.

Historical control data for F0-females (period 2015-2017):
Fertility index (%): mean = 95; P5-P95 = 80-100 (n=53).

Other effects:

no effects observed

Description (incidence and severity):

Lactation index (number of live offspring on PND 13 after littering as percentage of number of live offspring on PND4 after culling) was not affected by treatment. The lactation indices were 99% at 4000 ppm and 100% for the other groups.
One pup at 4000 ppm was missing (most likely cannibalised) on PND 7. No relation to treatment was attributed to this isolated finding since it did not show a dose-related trend and remained within the range considered normal for pups of this age.

Key result

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

111 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: morphological changes in the liver

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

At 4000 and 12000 ppm, mean body weights of male and female pups were slightly lower (not statistically significant) than those of control animals on lactation Days 7 and 13. Mean value of combined pup body weight was 6% and 9% lower at 4000 and 12000 ppm, respectively, on lactation Day 13. In general, individual values remained within the range considered normal for pups of this age.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Live birth index (number of live offspring on PND 1 after littering as percentage of total number of offspring born) was not affected by treatment. The live birth indices were 97, 99, 100 and 95% for the control, 1500 ppm, 4000 ppm and 12000 ppm groups, respectively.
At first litter check, 3 pups of the control group, 1 pup at 1500 ppm and 3 pups at 12000 ppm were found dead. No relation to treatment was attributed to these dead pups since the mortality incidence did not show a doserelated trend and live birth indices remained within the range considered normal for pups of this age.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was considered unaffected by treatment.
The shift in the sex ratio towards higher numbers of living female pups in the 12000 ppm group was caused by litter 71 that had only one living, female pup at first litter check (i.e. 100% female).

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean number of living pups at first litter check (live litter size) was considered to be unaffected by treatment.
The slightly lower mean number of living pups at first litter check in the 12000 ppm group (9.0 versus 10.1 in the concurrent control group) was caused mainly by one female which had one live pup only (next to 3 dead pups). Such small litter sizes are observed occasionally in this type of study. Moreover, in the current study there were also two relatively small litters in the control group (4 live pups each), and one low dose female with one implantation site only. All remaining litters in the high dose group had 9-12 living pups at first litter check, which is within the normal range9. As such, the observation of one litter with one live pup only in the high dose group was considered to be unrelated to the test item.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 99, 98, 100 and 100% for the control, 1500 ppm, 4000 ppm and 12000 ppm groups, respectively.
One pup of the control group and two pups at 1500 ppm had to be sacrificed in extremis or were missing (most likely cannibalised) on PND 3-4. No relation to treatment was attributed to these euthanized/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age

External malformations:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment.
The statistically significantly higher mean normalized anogenital distance of female pups at 4000 ppm occurred without a dose related-trend and, therefore, this observation was not considered to be related to treatment.
Treatment up to and including 12000 ppm had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 862 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 354 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

The conclusion on developmental toxicity is presented in the fertility section.

Executive summary:

The developmental toxicity is presented in the fertility section.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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:

2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Type:

Constituent

Test material form:

liquid

Test animals

Species:

rat

Strain:

other: Crl: WI(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. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

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 were 10-12 weeks old; females were 12-14 weeks old
- Weight at study initiation: males weighed between 265 and 300 g; females weighed between 195 and 237 g
- Fasting period before study: no
- 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 (Macrolon, MIV type). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type). During the lactation phase, females were housed in Macrolon plastic cages (MIII type). 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) without cage-enrichment, bedding material, food and water. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom) except when interrupted by study procedures/activities.
- Diet: Prepared diets were provided ad libitum throughout the study, except during designated procedures. During the acclimatization period, animals had free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: 7 days prior to start of the pretest period (females) or 7 days before the commencement of administration (males)

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility.
It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 40-59
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 December 2017 To: 06 February 2018

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The oral route of administration via dietary inclusion was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

- DIET PREPARATION
The test item was mixed without the use of a vehicle, directly with the required amount of powder feed. Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used.
Diets were prepared freshly for use at room temperature for a maximum of 4 days. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 4 days (stability was confirmed under Test Facility Study No. 520229 (analytical Method Development and Validation Study)) for supplementing food during the respective food consumption measurement interval.
If not used on the day of preparation, diets remained at room temperature for a maximum of approximately 7.5 hours before they were stored in the freezer. Diets were kept in the freezer (≤ - 15°C) for a maximum of 15 days until first use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diet preparation samples were collected for analysis during Week 1. Concentration was determined for all groups, homogeneity was determined in low and high dose groups. The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results.
Analyses were performed by using a validated analytical procedure.
Duplicate sets of samples were used for concentration analysis. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration.
Duplicate sets of samples for the low and high dose groups were used for homogeneity analysis. 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 demonstrated that the test item is stable in diet when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

The test item and control item were administered to the appropriate animals by inclusion in the diet ad libitum from Day 1 onwards for a minimum of 29 days. Males were exposed for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were exposed for 51-63 days, i.e. 14 days prior to mating (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 the day of scheduled necropsy. Females which failed to deliver were treated for 43, 44 or 55 days.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, or spilled diet from the food hopper.

Frequency of treatment:

continuously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 500 other: ppm; 100 mg/kg/bw

Doses / concentrations 2

Dose / conc.:

4 000 other: ppm; 260 mg/kg bw

Doses / concentrations 3

Dose / conc.:

12 000 other: ppm; 800 mg/kg bw

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
The dose levels were selected based on the results of a 14-day oral dose range finder with dietary administration of Jessemal in rats, and in an attempt to produce graded responses to the test item.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of administration, and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
-Food consumption was quantitatively measured Days 1, 4, 8, 10, 14, 15, 18, 22, 25 and 29, 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, 9, 11 and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflorane)
- Animals fasted: F0-males were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5/sex/dose
- Parameters checked according to Guidelines

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: F0-males were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5/sex/dose
- Parameters checked according to Guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations: Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 6-13). These tests were performed after completion of clinical observations.
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity

IMMUNOLOGY: No

OTHER: organ weight, estrous cycle determination, reproduction parameters F0, developmental parameters F1

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, according to Guidelines

HISTOPATHOLOGY: Yes, according to Guidelines

Other examinations:

reproduction parameters and devlopmental parameters are included in sections 7.8.1. and 7.8.2, respectively

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% and or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
non-parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
The motor activity data set was compared using an overall Kruskal-Wallis.
Incidence: 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:

no effects observed

Description (incidence and severity):

Slight chromodacryorrhoea observed in a few male and female animals divided over the different groups (including one control female) was considered unrelated to treatment, taking into account its isolated incidence, the minor severity of the effect, its transient occurrence (1-2 days), and the absence of any apparent dose-related trend.
Any other clinical signs noted during the treatment period 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:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A dose-related decrease in body weight gain was observed in males at 4000 and 12000 ppm.
Body weight and body weight gain in males at 12000 ppm were statistically significantly lower than concurrent control values during the entire treatment period (overall weight gain up to 6% lower than control mean). A similar, albeit less severe trend was observed in males at 4000 ppm, achieving statistical significance for body weight gain on Day 15 of the mating period only.
In females at 12000 ppm reduced mean values for body weight and body weight gain were noted during the entire gestation period. Changes compared to the concurrent control group were largest from Days 4-7 post-coitum when almost no body weight gain was recorded. In the subsequent period, body weight gain in the high dose group had almost the same magnitude as that of the concurrent control group. Statistically significantly lower body weights and body weight gains were noted from Days 7-14 post-coitum only. Body weight and body weight gain remained in the same range as controls over the treatment period in the 1500 ppm (both sexes) and 4000 ppm (females) dose groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Lower food consumption (before and after correction for body weight) was observed in males at 12000 ppm from Days 1-4 of both the pre-mating and Days 1-4 of the mating period, followed by normal values in the remainder of the observation period. (Note: There is no explanation for the relatively high food consumption recorded for control males in cage 1 from Days 1-4 of the mating period.)
In pregnant females at 12000 ppm, food consumption (before and after correction for body weight) was lower than the control level over the gestation period (reaching statistical significance only for absolute food consumption on Days 0-7 and 11-14 post-coitum). On average, absolute and relative food intake in pregnant females at this high dose level was 21% and 14% lower as compared to the control group, respectively, during the gestation period, followed by partial recovery during lactation. During the lactation period, absolute and relative food intake on average was 11% and 5% lower as compared to the control group.
Food consumption of males and females at the lower dose levels of 1500 and 4000 ppm were considered to be unaffected by treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in haematological parameters.
The slight, but statistically significant increase in mean red blood cells observed in males at 12000 ppm at the end of treatment (1.05x of control) occurred in the absence of corroborating changes in other red blood cells parameters, and since only one individual value (one male: 9.50 10E12/L) was slightly above the historical control range, this finding was considered not to be toxicologically relevant.
The statistically significantly lower mean number of platelets noted for females in the 4000 ppm group was considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
There were no treatment-related changes in coagulation parameters [prothrombin time (PT) and activated partial thromboplastin time (APTT)].

note: Historical control data for F0-males (period 2015-2017): Red blood cells (10E12/L): mean = 8.81; P5-P95 = 8.14-9.45 (n=286).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of treatment, a test-item related increase in mean cholesterol was observed in males at 12000 ppm (1.3 x of control). The mean value was at the upper limit of the historical control data , with 2/5 individual values above the upper limit.
The higher mean plasma activity of alanine aminotransferase (ALAT: 1.7x of control) and aspartate aminotransferase (ASAT: 1.4x of control) recorded in males at 12000 ppm (only statistically significant for ALAT) was attributed to one single male that presented with remarkably increased plasma activity of both hepatic enzymes (ALAT: 117.2 U/L; ASAT: 254.1 U/L) when compared to both concurrent and historical control means (3). The same male was also noted with a relatively high value for bile acids (87.6 µmol/L). At the microscopic level, slight hyperplasia of the bile duct was noted. The second high dose male with increased bile acids (55.2 µmol/L) had the same liver finding. However, hyperplasia of the bile duct (up to a slight degree) was also seen for the remaining three high dose males that had normal values of bile acids.
A relatively high ASAT value (139.8 U/L) was measured in one low dose male as well. In the absence of any comparable change in the mid dose group, no toxicological significance was attached to this isolated finding.
Also in lactating females at 4000 and 12000 ppm, an increase in bile acids (only significant at 4000 ppm) was observed at the end of treatment (4x and 2x of control, respectively). A large intragroup variation was observed at both dose levels. At the individual level, bile acids levels were above the historical control range (3) in 3/5 and 2/5 females at 4000 and 12000 ppm, respectively. One of these two high dose female was noted with minimal hyperplasia of the bile duct. But there were also two other high dose females with the same pathological finding with normal values for bile acids.
In lactating females at 4000 and 12000 ppm, a test item-related increase in total bilirubin was observed at the end of treatment (1.5x and 1.6x of control, respectively). The mean value at 4000 ppm was within but in the upper limit of the historical control data . The mean value at 12000 ppm was outside the historical control range(3).
The significantly higher mean values for urea measured in males at 1500 and 12000 ppm were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Thyroid hormone analyses:
At the end of treatment, serum level of total T4 in F0-males was significantly lower than concurrent control values (0.8x of control). The mean value (3.43 µg/dL) remained within the historical control range (4). At the individual level, 3 out of the 10 males had values at the lower limit or below the historical range (1.74, 2.32 and 3.02 µg/dL for the individual males, respectively).

(3) Historical control data for F0-males (period 2015-2017):
Cholesterol (mmol/L): mean = 1.85; P5-P95 = 1.36-2.42 (n=304).
ALAT (U/L): mean = 46.9; P5-P95 = 30.30-68.00 (n=304).
ASAT (U/L): mean = 81.2; P5-P95 = 65.50-99.50 (n=304).
Bile acids (µmol/L): mean = 23.6; P5-P95 = 9.70-46.0 (n=303).
Urea (mmol/L): mean = 6.8; P5-P95 = 4.00-9.60 (n= 304)
Total T4 (µg/dL): mean 4.65; P5-P95 = 3.01-6.52 (n=724).
Historical control data for non-fasted F0-females (period 2017-2018):
Total bilirubin (µmol/L): mean = 1.9; P5-P95 = 1.25-2.50 (n=60).
Bile acids (µmol/L): mean = 22.1; P5-P95 = 8.45-44.65 (n=60).

(4) Historical control data for F0-males (period 2015-2017):
Total T4 (µg/dL): mean 4.65; P5-P95 = 3.01-6.52 (n=724).

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the treatment period, a statistically significant decrease in mean foreleg grip strength was observed in all treated female groups (mean up to 24% lower than that of the control). This finding was attributed to a relative high mean value in the concurrent control group when compared to available historical control data (1536 vs 953). Given that none of the individual values of treated females was below the historical control range, changes occurred in the absence of a dose-related response and there were no findings during clinical observation or motor activity measurement in this study indicating any abnormalities in the neuromuscular domain, this reduction in foreleg grip strength was regarded as unrelated to treatment. The statistically significant decrease in hindleg grip strength observed in females at 4000 ppm was not considered to be related to treatment as no-dose related trend was observed.
Leg grip strength in males was not affected by treatment.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. (Note: no explanation could be given for the sudden higher activity of control males recorded 30-35 minutes after start of the session with higher counts of both total movements and ambulations).

note: historical values Fore leg grip (gram): mean = 953; P5-P95 = 578-1363 (n=240).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item-related higher liver weights (absolute and/or relative to body weights) were noted in the 4000 ppm-treated females and the 12000 ppm-treated males and females, and test item-related lower thymus weights (absolute and relative to body weight) were present in the 12000 ppm-treated females (see 'any other information on results incl. tables').
There were no other test item-related organ weight changes. Some other organ weight differences, including the lower absolute adrenal weight in males at 12000 ppm, 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 and/or lacked a dose-response.
The significantly lower uterus weights (absolute and relative to body weight) noted in mid and high dose groups were not related to the test item, but caused by relative high individual values in the control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.
All 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:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with Jessemal were noted in the liver and the thymus and are summarized in the section 'any other information on results incl. tables'.
Liver, hyperplasia bile duct was present in males starting at 4000 ppm up to slight degree and in females at 12000 ppm at minimal degree.
Infiltration peribiliary was present in males starting at 4000 ppm at minimal degree.
Hepatocellular hypertrophy was present in females treated at 12000 ppm up to slight degree.
Thymus, atrophy was present in females treated at 4000 and 12000 ppm up to slight degree. At 12000 ppm this correlated with the decrease in thymus weight.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

effects on reproduction and developmental parameters are described in sections 7.8.1 and 7.8.2

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

111 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: morphological changes in the liver

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

4 000 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Accuracy: Mean accuracy values were 97, 99 and 96% in the low, mid and high dose groups, respectively. No test item was detected in the control diets.

Homogeneity: coefficient of variation was 2.3 and 3.0% in the low and high dose, respectively.

Mean test article intake over the study period was as follows:

		Mean over means intake [mg test item/kg body weight] (mean range indicated within brackets)
	Group no.	2	3	4
	Nominal dietary inclusion level (ppm)	1500	4000	12000
Sex	Study period
Males	Pre-mating	114 (107-122)	307 (282-322)	878 (805-934)
	Post-mating	107 (97-126)	275 (253-321)	842 (813-867)
	Mean of means^a	111	293	862
Females	Pre-mating	107 (100-110)	282 (246-299)	840 (754-934)
	Post-coitum	149 (133-160)	454 (410-499)	1077 (1056-1096)
	Lactation	315 (228-365)	882 (652-1030)	2200 (1617-2470)
	Mean of means^a	188	534	1354
^aMean of means of all periods, weighed for number of measurement intervals per period: Males: ((5 x mean premating) + (4 x mean mating)) / 9 Females: ((5 x mean premating) + (6 x mean post-coitum) + (5 x mean lactation)) / 16

Mean Percent Organ Weight Differences from Control Groups

	Males					Females
Dose level (ppm):	1500		4000		12000	1500	4000	12000
LIVER
Absolute	1		1		17**	11	11	14
Relative to body weight	4		6		24**	10	13*	20**
THYMUS
Absolute	-7		-10		-9	-12	-21	-32*
Relative to body weight	-5		-5		-3	-14	-20	-28
*:P<0.05, **:P<0.01

Summary Test Item-Related Microscopic Findings – Liver

	Males				Females
Dose level (ppm):	0	1500	4000	12000	0	1500	4000	12000
LIVER^a	5	5	5	5	5	6	5	5
Hyperplasia bile duct
Minimal	-	-	3	1	-	-	-	3
Slight	-	-	-	4	-	-	-	-
Infiltration peribiliary
Minimal	-	-	3	2	-	-	-	-
Hypertrophy hepatocellular
Minimal	-	-	-	-	-	-	-	4
Slight	-	-	-	-	-	-	-	1

^a = Number of tissues examined from each group

Summary Test Item-Related Microscopic Findings – Thymus

	Females
Dose level (ppm):	0	1500	4000	12000
THYMUS^a	5	6	5	5
Atrophy
Minimal	-	-	-	1
Slight	-	-	1	1

^a = Number of tissues examined from each group.

Applicant's summary and conclusion

Conclusions:

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, a parental no-observed-adverse-effect level (NOAEL) of the substance was established to be 1500 ppm, corresponding to 111 mg/kg bw/day in males and 188 mg/kg bw/day in females (based on minimal to slight bile duct hyperplasia accompanied by intrahepatic peribiliary infiltration in the males at and from 4000 ppm onwards. In females minimal bile duct hyperplasia was seen in the high dose at 12000 ppm).
Note: In this study, a reduction of total T4 was observed in F0-males at 12000 ppm which was considered to be compound-related. However, possible adversity of this effect could not be assessed within this type of screening study and was, therefore, not taken into account when determining the parental NOAEL.

Executive summary:

The substance was administered via the diet to Wistar Han rats at dietary concentrations of 1500, 4000 and 12000 ppm (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received basal powder diet without the test item. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 51-63 days). Females that failed to deliver pups were treated for 43, 44 or 55 days.Chemical analyses of dietary preparations 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 (for 5 selected animals/sex/group),body weight and food consumption, estrous cycle determination,clinical pathology (for 5 selected animals/sex/group),measurement of thyroid hormone T4 (F₀-males), 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)).

Analysis of diet preparations showed that the powder diets were prepared accurately and homogeneously.

Parental results

Reduced mean body weight gain was observed in males at 4000 and 12000 ppm (dose-dependently) during the entire treatment period (not always statistically significant). In females at 12000 ppm reduced mean body weight gain was noted during the entire gestation period (not always statistically significant). Changes compared to the concurrent control group were largest from Days 4-7 post-coitum when almost no body weight gain was recorded. In the subsequent period, body weight gain in the high dose group had almost the same magnitude as that of the concurrent control group. The resulting reductions in mean body weights of males at 4000 and 12000 ppm and females at 12000 ppm at end of treatment were modest (i.e. < 10%) and therefore considered not to be adverse.

The reduced body weight gain was accompanied by lower food consumption in males at 12000 ppm from Days 1-4 of both the pre-mating (absolute and relative food consumption were 17% and 29% lower as compared to controls) and Days 1-4 of the mating period (absolute and relative food consumption were 17% and 26% lower as compared to controls). However, normal values were measured in the remainder of the observation period. In pregnant females at 12000 ppm, food consumption was lower than the control level over the gestation period (reaching statistical significance only for absolute food consumption on Days 0-7 and 11-14 post-coitum). On average, absolute and relative food intake in pregnant females at this high dose level was 21% and 14% lower as compared to the control group, respectively, during the gestation period, followed by partial recovery during lactation. At the end of lactation, absolute and relative food intake on average was 11% and 5% lower as compared to the control group.

No heamatology effects were seen.

Biochemical parameters: In lactating females at 4000 and 12000 ppm, a test item-related increase in total bilirubin was observed at the end of treatment (1.5x and 1.6x of control, respectively). The mean value at 4000 ppm was at the upper limit of the historical control data. The mean value at 12000 ppm was outside the historical control range. This increase was / was not accompanied with effects on haematology.

Cholesterol was increased but in males only (1.3x control) which can be related to the increased liver weight.

Bile acids were slightly increased at the high dose in both sexes (ca 50%) but not dose related and not statistically significant. Only the mid dose in females reached statistical significance but this can also be attributed to the fairly low control value.

Organ related effects: There were not treatment related organ weights effects, macroscopic or histopathological effects, except for the liver and thymus. .

Relative liver weights were increased accompanied by minimal hyperplasia only in females and only in the high dose only. Minimal to slight hyperplasia of the bile duct was seen in males at the mid dose (3 animals) and high dose (1 animal), respectively, accompanied by intrahepatic peribiliary infiltration. These bile effects are not very common effects and considered for deriving the NOAEL

Non-adverse test item-related atrophy was present in the thymus of females at 4000 and 12000 ppm. This morphologic alteration occurred in the absence of any other findings in that organ and, at the low severity observed in this study, was not considered adverse.

No thyroid effects were seen but at 12000 ppm, serum level of total T4 in F₀-males was reduced by approximately 23% compared to control values.The mean value (3.43 µg/dL) remained within the historical control range[1], but at the individual level, 3 out of the 10 males had values at the lower limit or below the historical range (1.74, 2.32 and 3.02 µg/dL). A possible relation to treatment could not be excluded. This reduction of total T4 occurred in the absence of corroborating changes in either organ weight or morphology of the thyroid gland.  Due to the limited data available from this type of screening study, possible adversity of this effect could not be assessed. Therefore, this reduction in total T4 in males sat 12000 ppm was not taken into account when determining the parental NOAEL.

[1]Historical control data for F₀-males (period 2015-2017):

                             Total T4 (µg/dL): mean 4.65; P5-P95 = 3.01-6.52 (n=724).

 

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following parental No Observed Adverse Effect Level (NOAEL) for the substance was established:

Parental NOAEL: 1500 ppm, corresponding to 111 mg/kg bw/day in males and 188 mg/kg bw/day in females (based on minimal to slight bile duct hyperplasia accompanied by intrahepatic peribiliary infiltration in the males at and from 4000 ppm onwards. In females minimal bile duct hyperplasia was seen in the high dose at 12000 ppm).

 Note: In this study, a reduction of total T4 was observed in F₀-males at 12000 ppm which was considered to be compound-related. However, possible adversity of this effect could not be assessed within this type of screening study and was, therefore, not taken into account when determining the parental NOAEL.