Methyl α-D-mannopyranoside

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 September 2021 to 08 December 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 September 2021 to 08 December 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males approx. 15 weeks, females approx. 13-14 weeks
- Weight at study initiation: males: 358 - 405 g (mean: 382.88 g, ± 20% = 306.30 – 459.45 g)
females: 208 - 262 g (mean: 234.00 g, ± 20% = 187.20 – 280.80 g)
- Housing: Animals were housed in groups of 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages during the premating period for both males and females and during post-mating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10 times
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 07 September 2021 To: 29 November 2021

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in aqua ad injectionem.
The application volume for all groups was 5 mL/kg bw/day.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

As the test item was shown to be homogenous according to a previous study (after at least 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and samples were only taken for substance concentration verification in study week 1 (pre-mating period), week 3 (first week of mating), week 5 (gestation) and in the last week of the study (gestation / lactation) from all groups (16 samples).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 3 mL). The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. STUGC21AA0522-6) and until then stored under appropriate conditions based on available stability data. The B-samples were retained at below 15 °C at BSL Munich (test facility) and discarded after completion of the final study report.
Nominal concentrations were within acceptance criterion of ±15%.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to PND 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose and 10 animals per sex for the control were used.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were chosen based on the results of a previous dose-range-finding study (reference 7.5.1-2).

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.


BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4, PND 9 and PND 13 along with pups. Inadvertently, female no. 46 was not weighed on gestation day 0. All animals were weighed directly before termination. Any animals prematurely sacrificed were weighed prior to the sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment prior to or as part of the sacrifice. Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 5 males and females per group
- Parameters checked in table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment prior to or as part of the sacrifice. Blood from the abdominal aorta of the animals was collected in serum separator tubes.
- Animals fasted: No
- How many animals: 5 males and females per group
- Parameters checked in table No.2 were examined.

PLASMA/SERUM HORMONES: Yes
- Time of blood sample collection: at study termination
- Animals fasted: No
- How many animals: Blood samples from the adult males were assessed for serum levels for thyroid hormones (T4).

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to or as part of the sacrifice of the animals.
- Metabolism cages used for collection of urine: No
- Animals fasted: No
- Parameters checked in table No. 3

NEUROBEHAVIOURAL EXAMINATION: Yes
Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the last week of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group outside the home cage using a functional observational battery. Sensory reactivity to different modalities were investigated comprising grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

Oestrous cyclicity (parental animals):

Vaginal smears were examined daily from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals):

The testes from group 1 and 4 males were checked during the histopathological examinations for completeness of cell populations, completeness of stages and degenerative changes.

Litter observations:

PARAMETERS EXAMINED
The duration of gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after the delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring were recorded.
The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD/Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 4 were weighed, Tissues and organs in Table 5 were prepared for microscopic examination.

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry was statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 was considered as statistically significant).

Reproductive indices:

copulation index, fertility index, delivery index

Offspring viability indices:

The number of male and female pups, sex ratio, still births, runts, litter weight data, anogenital distance, nipple retention and external abnormalities were evaluated.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs of toxicological relevance were observed in the control group and in all treatment groups. During the mating period one male of the MD group had abnormal breathing on day 9. During the lactation phase, one female in the control group had severe piloerection on day 0, two females of the LD group had slight piloerection between days 0 and 2 and two females of the HD group had moderate piloerection on day 0 and slight piloerection between days 8 and 11, respectively. One female of the MD group had a hairless area on the neck between days 3 and 12. These signs were only observed in single animals and on a few days during the study and are therefore considered to be incidental and not adverse. One female of the MD group had a scratch/cut with signs of necrosis on the tail due to which the animal was taken out of the study. Regarding detailed clinical observation, no differences to control were noted during the treatment period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two mortalities occurred during the study. One male in the LD (low dose) group was found dead on study day 21, which was the 7th day of the mating phase. One female of the MD (mid dose) group was found in moribund condition on study day 9 of the premating phase. In both above-mentioned animals the cause of death was not evident from the histopathologically evaluated tissues and remained unclear. However, because (i) there were no test item-related findings in any of the organs examined in either of the above animals, (ii) no dose dependency was observed and (iii) no clinical signs of systemic toxicity were observed in the animals of this study, the cause of morbidity in both animals was considered incidental.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The test item had no effect on food consumption in this study. Mean daily food intake of males and females in the dose groups was comparable to the control group in this study except for in week one of the gestation phase females of the MD group consumed statistically significantly less food when compared to control animals (~15% below control). No considerable differences between the groups were observed.

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, the test item showed no relevant effects on clinical biochemical parameters.
A statistically significant increase in glucose (GLUC) was noted only in females in the HD group when compared to the control group (~38% above control). Overall, a slight dose dependent increase in glucose is observed in all dose groups. For the liver parameter AP, a statistically significant increase was noted in females of the MD group when compared to the control group (~107% above control). As the other liver parameters ALAT and ASAT were unaffected and no histopathological changes in the liver were observed in females of the MD group, the mentioned increase is not considered to be related to the treatment with test item. Furthermore, no effects on liver parameters were found for males of all dose groups and no dose dependence was seen in females.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The test item had no toxicologically relevant effect on urinary parameters analysed in selected animals at the end of the treatment period of this study. Findings in single male animals such as high level of glucose when compared to the control group and higher number of leucocytes in a control male are considered to be incidental and without relation to the treatment with test item. No specific changes in urinary parameters were noted in all female dose groups when compared to the control group.

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Under the conditions of this study there were no microscopic changes that could be related to the test item administration. In the lung of some dosed animals, control animals and one decedent animal, minimal mixed cell infiltrates (i.e. minimal interstitial inflammation) was observed. The minor inflammatory changes observed in the lung of the decedent animal and few surviving animals were considered to be caused by an inflammatory process most likely not related to the test item administration and deemed as incidental.
In kidneys from one control animal, one low dose group animal and two high dose group animals dilatation of the renal pelvis and compression of the adjacent renal parenchyma were noted. The pelvic dilatation observed in few animals was caused by an increased hydrostatic pressure in the lower urinary tract and was considered to be most likely incidental.
In addition, the treatment with the test item did not induce any histomorphological effects in the reproductive organs of one animal which was pregnant but did not litter, and in a decedent animal that was euthanized before mating on test day 9.
All other recorded microscopic findings were deemed to be incidental or were within the range of background alterations that may be recorded in Wistar rats.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no morphological findings in the reproductive organs of males which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The litter parameters including the total number of pups, still births and runts born on PND 0, and the number of live pups were not statistically significantly different in test item groups compared to the control group. Observed total mortality in the MD group of 2.10% was achieved by the death of 1/11 pups of a dam and 1/13 pups of another dam and total mortality in the HD group of 0.83% between PNDs 0-4 was achieved by the death of 1/12 pups of one dam, but no mortality of pups occurred in the MD and HD groups between PNDs 4-13. As the mortality was observed for single pups of single dams from the MD and HD groups and only during the first observation days without statistical significance, no effect of the treatment with test item is considered.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No toxicological relevant differences in mean total litter weight, male or female litter weight on PND 0, 4 and 13 were observed in any of the treatment groups. The litter weights were within the normal range of variation in this strain.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Treatment with the test item had no effect on serum T4 levels of pups sacrificed on day 13.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No gross external abnormalities of toxicological relevance were observed for pups of any of the groups on PNDs 0-12 or the day of sacrifice/death. Few isolated external findings in the control group included hairless area on the back of some pups, wound on the snout, PND 0 and PND 6 and in the MD group a dark snout on PND 0. These external findings were considered to be spontaneous and not related to test item treatment.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/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

Conclusions:

No treatment-related adverse effects were observed in the combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with the test substance in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg bw/da. Thus, the NOAEL for general toxicity as well as the NOAEL for reproduction/developmental toxicity is determined to be 1000 mg/kg bw/day.

Executive summary:

The substance was tested in the Combined Repeated Dose Oral Toxicity Study with the Reproduction / Developmental Toxicity Screening Test in Wistar Rats. According to the results of a previous dose range finding study the following doses were tested: 0, 100, 300 and 1000 mg/kg bw/day. 10 animals per sex per day were treated (orally by gavage). The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to PND 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. No clinical signs of toxicological relevance were observed in the control group and in all treatment groups. Detailed clinical observation showed no toxicologically relevant differences in all male and female groups when compared to the control group. No test item-related effects were observed during the functional observation for males and females. The test item had no effect on body weight, body weight change and food consumption in males and females in all dose groups when comparing to control animals. No test item-related effects on the reproduction and developmental parameters analysed in this study were observed, i.e. oestrous cycle, copulation, fertility and delivery indices, number of corpora lutea, implantation sites and live pups, pre- and post-implantation loss, number of male and female pups, sex ratio, still births, runts, litter weight data, anogenital distance, nipple retention and external abnormalities. Treatment with the test item had no effect on serum T4 levels in parental males and offspring. The test item had no toxicologically relevant effect on haematological, coagulation and clinical biochemistry parameters of selected male and female animals analysed at the end of the treatment period of this study. The test item had no toxicologically relevant effects on urinary parameters analysed in selected animals at the end of the treatment period of this study. There were gross lesions observed in male and female treatment groups, which were considered to be not related to the test item. No test item related effects on absolute and relative organ weights were observed in males and females in any of the treatment groups when compared to the control groups. No test item-related microscopic changes were observed in any of the examined tissues and organs either in the decedents or in surviving animals. Based on these results, the NOAEL for general toxicity as well as the NOAEL for reproduction/developmental toxicity is determined to be 1000 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

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:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methyl α-D-mannopyranoside

EC Number:

210-502-3

EC Name:

Methyl α-D-mannopyranoside

Cas Number:

617-04-9

Molecular formula:

C7H14O6

IUPAC Name:

(2R,3S,4S,5S,6S)-2-(hydroxymethyl)-6-methoxyoxane-3,4,5-triol

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males approx. 15 weeks, females approx. 13-14 weeks
- Weight at study initiation: males: 358 - 405 g (mean: 382.88 g, ± 20% = 306.30 – 459.45 g)
females: 208 - 262 g (mean: 234.00 g, ± 20% = 187.20 – 280.80 g)
- Housing: Animals were housed in groups of 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages during the premating period for both males and females and during post-mating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10 times
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 07 September 2021 To: 29 November 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in aqua ad injectionem.
The application volume for all groups was 5 mL/kg bw/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

As the test item was shown to be homogenous according to a previous study (after at least 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and samples were only taken for substance concentration verification in study week 1 (pre-mating period), week 3 (first week of mating), week 5 (gestation) and in the last week of the study (gestation / lactation) from all groups (16 samples).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 3 mL). The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. STUGC21AA0522-6) and until then stored under appropriate conditions based on available stability data. The B-samples were retained at below 15 °C at BSL Munich (test facility) and discarded after completion of the final study report.
Nominal concentrations were within acceptance criterion of ±15%.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to PND 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose and 10 animals per sex for the control were used.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were chosen based on the results of a previous dose-range-finding study (reference 7.5.1-2).

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4, PND 9 and PND 13 along with pups. Inadvertently, female no. 46 was not weighed on gestation day 0. All animals were weighed directly before termination. Any animals prematurely sacrificed were weighed prior to the sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment prior to or as part of the sacrifice. Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 5 males and females per group
- Parameters checked in table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment prior to or as part of the sacrifice. Blood from the abdominal aorta of the animals was collected in serum separator tubes.
- Animals fasted: No
- How many animals: 5 males and females per group
- Parameters checked in table No.2 were examined.

PLASMA/SERUM HORMONES: Yes
- Time of blood sample collection: at study termination
- Animals fasted: No
- How many animals: Blood samples from the adult males were assessed for serum levels for thyroid hormones (T4).

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to or as part of the sacrifice of the animals.
- Metabolism cages used for collection of urine: No
- Animals fasted: No
- Parameters checked in table No. 3

NEUROBEHAVIOURAL EXAMINATION: Yes
Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the last week of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group outside the home cage using a functional observational battery. Sensory reactivity to different modalities were investigated comprising grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 4)

HISTOPATHOLOGY: Yes (see table 5)

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry was statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs of toxicological relevance were observed in the control group and in all treatment groups. During the mating period one male of the MD group had abnormal breathing on day 9. During the lactation phase, one female in the control group had severe piloerection on day 0, two females of the LD group had slight piloerection between days 0 and 2 and two females of the HD group had moderate piloerection on day 0 and slight piloerection between days 8 and 11, respectively. One female of the MD group had a hairless area on the neck between days 3 and 12. These signs were only observed in single animals and on a few days during the study and are therefore considered to be incidental and not adverse. One female of the MD group had a scratch/cut with signs of necrosis on the tail due to which the animal was taken out of the study. Regarding detailed clinical observation, no differences to control were noted during the treatment period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two mortalities occurred during the study. One male in the LD (low dose) group was found dead on study day 21, which was the 7th day of the mating phase. One female of the MD (mid dose) group was found in moribund condition on study day 9 of the premating phase. In both above-mentioned animals the cause of death was not evident from the histopathologically evaluated tissues and remained unclear. However, because (i) there were no test item-related findings in any of the organs examined in either of the above animals, (ii) no dose dependency was observed and (iii) no clinical signs of systemic toxicity were observed in the animals of this study, the cause of morbidity in both animals was considered incidental.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The test item had no effect on food consumption in this study. Mean daily food intake of males and females in the dose groups was comparable to the control group in this study except for in week one of the gestation phase females of the MD group consumed statistically significantly less food when compared to control animals (~15% below control). No considerable differences between the groups were observed.

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, the test item showed no relevant effects on clinical biochemical parameters.
A statistically significant increase in glucose (GLUC) was noted only in females in the HD group when compared to the control group (~38% above control). Overall, a slight dose dependent increase in glucose is observed in all dose groups. For the liver parameter AP, a statistically significant increase was noted in females of the MD group when compared to the control group (~107% above control). As the other liver parameters ALAT and ASAT were unaffected and no histopathological changes in the liver were observed in females of the MD group, the mentioned increase is not considered to be related to the treatment with test item. Furthermore, no effects on liver parameters were found for males of all dose groups and no dose dependence was seen in females.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The test item had no toxicologically relevant effect on urinary parameters analysed in selected animals at the end of the treatment period of this study. Findings in single male animals such as high level of glucose when compared to the control group and higher number of leucocytes in a control male are considered to be incidental and without relation to the treatment with test item. No specific changes in urinary parameters were noted in all female dose groups when compared to the control group.

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Description (incidence and severity):

Treatment with the test item had no effect on serum T4 levels of parental males.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related effects on absolute and relative organ weights were observed in males and females in any of the treatment groups when compared to the control groups.
A statistically significantly higher mean organ to body weight ratio was found for kidneys and heart in males of the HD group when compared to the control group (~15% and ~12% above control, respectively). A slight dose dependence for both organs was observed, however all organ weights are within the range of historical control data and no test item related histopathological changes were found in the kidneys or the heart in any of the animals in all treatment groups. In addition, only one gender was affected. Therefore, these slightly higher mean organ to body weight ratios are considered to be incidental and not related to treatment with the test item.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no gross lesions observed in male and female treatment groups, which were considered to be related to the test item.
Macroscopic examination of organs from males revealed a cyst in the rectum and in the mesenteric lymph nodes of male no. 40 in the HD group, white coloured kidneys in male no. 13 of the LD group, a dilatation of the pelvis of the kidneys in animal nos. 35 and 39 of the HD group, enlarged thyroid glands on both sides in male nos. 1, 2 and 4 of the control group, a spotted thymus in male no. 18 of the LD group, reddish coloured mesenteric lymph nodes in male nos. 2 and 3 of the control group and reddish coloured mandibular lymph nodes in male no. 10 of the control group.
Macroscopic examination of organs from females revealed a pale and spotted lung on the left caudal lobe of female no. 56 of the LD group, an enlarged stomach with a red granular mucosa of female no. 61 of the MD group, a depressed, dark red granular mucosa in the stomach of female nos. 52 and 59 of the LD group, an enlarged stomach, duodenum, jejunum, cecum, colon and ileum, which was in addition dark red in females no. 52 of the LD group and no. 61 of the MD group, a gas filled cecum and colon in female no. 74 of the HD group, a herniation of the liver in female no. 45 of the control group, an abnormally shaped liver in female no. 59 of the LD group, a prolapse of the vagina in female no. 75 of the HD group, a red spotted thymus in female no. 55 of the LD group, a red thymus in female no. 57 of the LD group, a small thymus in females no. 52 of the LD group and no. 61 of the MD group, a brown spotted thymus and red axillary lymph nodes in female no. 76 of the HD group and red mandibular lymph nodes in female no. 61 of the MD group.
All gross lesions recorded at necropsy were considered to be most likely incidental and showed no correlation with histopathology findings.
Decedent male no. 20 was autolytic and the lung failed to collapse when found dead on study day 21. Enlarged adrenal glands, a spotted thymus, dark red axillary lymph nodes and enlarged mesenteric lymph nodes in decedent female no. 63 were found at necropsy on study day 9.
All gross lesions correlating with microscopic findings were unremarkable and could not be related to treatment with the test item. The gross lesions noted at necropsy did not correlate with the histopathologic findings and were considered to be most likely of no toxicological relevance.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Under the conditions of this study there were no microscopic changes that could be related to the test item administration. In the lung of some dosed animals, control animals and one decedent animal, minimal mixed cell infiltrates (i.e. minimal interstitial inflammation) was observed. The minor inflammatory changes observed in the lung of the decedent animal and few surviving animals were considered to be caused by an inflammatory process most likely not related to the test item administration and deemed as incidental.
In kidneys from one control animal, one low dose group animal and two high dose group animals dilatation of the renal pelvis and compression of the adjacent renal parenchyma were noted. The pelvic dilatation observed in few animals was caused by an increased hydrostatic pressure in the lower urinary tract and was considered to be most likely incidental.
In addition, the treatment with the test item did not induce any histomorphological effects in the reproductive organs of one animal which was pregnant but did not litter, and in a decedent animal that was euthanized before mating on test day 9.
All other recorded microscopic findings were deemed to be incidental or were within the range of background alterations that may be recorded in Wistar rats.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

No treatment-related adverse effects were observed in the combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with the test substance in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg bw/da. Thus, the NOAEL for general toxicity is determined to be 1000 mg/kg bw/day.

Executive summary:

The substance was tested in the Combined Repeated Dose Oral Toxicity Study with the Reproduction / Developmental Toxicity Screening Test in Wistar Rats. According to the results of a previous dose range finding study the following doses were tested: 0, 100, 300 and 1000 mg/kg bw/day. 10 animals per sex per day were treated (orally by gavage). The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to PND 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. No clinical signs of toxicological relevance were observed in the control group and in all treatment groups. Detailed clinical observation showed no toxicologically relevant differences in all male and female groups when compared to the control group. No test item-related effects were observed during the functional observation for males and females. The test item had no effect on body weight, body weight change and food consumption in males and females in all dose groups when comparing to control animals. Treatment with the test item had no effect on serum T4 levels in parental males. The test item had no toxicologically relevant effect on haematological, coagulation and clinical biochemistry parameters of selected male and female animals analysed at the end of the treatment period of this study. The test item had no toxicologically relevant effects on urinary parameters analysed in selected animals at the end of the treatment period of this study. There were gross lesions observed in male and female treatment groups, which were considered to be not related to the test item. No test item related effects on absolute and relative organ weights were observed in males and females in any of the treatment groups when compared to the control groups. No test item-related microscopic changes were observed in any of the examined tissues and organs either in the decedents or in surviving animals. Based on these results, the NOAEL for general is determined to be 1000 mg/kg bw/day.