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

Endpoint summary

Description of key information

Repeated dose / Reproscreen study: NOAEL is 111 mg/kg bw based on minimal to slight hyperplasion in the liver bile ducts.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

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 (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

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

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.

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.

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

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

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

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:

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

111 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

good

System:

hepatobiliary

Organ:

liver

Additional information

In an repeated dose/reproscreen study according to OECD TG 422, 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).

Justification for classification or non-classification

Based on the observed effects and NOAEL value in the repeated dose toxicity study with the reproduction/developmental toxicity screening test the substance does not need to be classified for repeated dose toxicity when considering the criteria in the EU CLP (EC No. 1272/2008 and its amendments).