Pyridinium toluene-4-sulphonate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

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:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

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

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
- Source: Charles River Laboratories, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes]
- Age at study initiation: approx 12-13 weeks at start of treatment
- Weight at study initiation: males 425-502 g, females 237-306 g
- Fasting period before study: no
- Housing: up to 3 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or indivdually housed
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:
animals received ssniff SM R/M complete diet for rats and mice, Soest, Germany
animals received tap water from municipal supply.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2 - 25.0
- Humidity (%): 29 - 64
- Air changes (per hr): 15 -20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
the test substance is very well water soluble. From the solid test material a stock solution of 741 g/l water was prepared, and further diluted.

- DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

- VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 6, 20, 60 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg body weight
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC with UV detector was used to determine the concentrations in the dosing solutions at 3 occasions during the study. The concentrations determined ranged between 97% and 103% recovery of the nominal concentration.

Duration of treatment / exposure:

males were dosed for 28 days starting 14 days prior to mating. Females were dosed for 14 days prior to mating, during mating, gestation until day 13 post partum.

Frequency of treatment:

daily

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

according to guideline OECD 422 (2016)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: food consumption was determined

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 AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: just before necropsy
- Anaesthetic used for blood collection: Yes pentobarbital
- Animals fasted: Yes
- How many animals: 5 per sex and dose group
- Parameters checked in table [No.?] were examined: see Table on haematology attached as background material

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: just before necropsy
- Animals fasted: Yes
- How many animals: 5 per sex and dose group
- Parameters checked in table [No.?] were examined.: see Table on clinical chemistry and urinalysis attached as background material

URINALYSIS: Yes
- Time schedule for collection of urine: just before necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked in table [No.?] were examined: see Table on clinical chemistry and urinalysis attached as background material

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity.

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table)

see "pathology and histopathology investigations" attached as background material

Statistics:

The statistical evaluation of data (labelled as † in the lists below) was performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS v9.2 (when using Provantis).
In case of the SPSS PC+4.0 program package, the heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of non-normal distribution, the non-parametric method of Kruskal-Wallis One- Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. The Chi-squared test was used for non-continuous data.

Clinical signs:

effects observed, non-treatment-related

Mortality:

mortality observed, non-treatment-related

Description (incidence):

one female of the control group was preterminally euthanised the day after littering for animal welfare reasons (protrusion of the vagina). One female of the high dose group was found dead on the day of littering; the cause of death could not be determined.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males, on the week after initiation of treatment, the body weight gain in the High dose was 74% below control (p<0.01) but the males fully compensated for this in the following week. During the third week the body weight gain was again below the control values (-36%, no statistical significance) and in the fourth week, the animals compensated the difference again. Although there were no statistical differences in the overall body weight gain of the High dose compared to the control, it is noted that the bodyweight gain of the animals was disturbed most probably by the test item. The body weight gain of the Mid dose males were slightly below the Control during the first three weeks, without reaching statistical significances. These minor differences in the Mid dose group were considered to have no toxicological significance. The body weight and body gain weight values of the Low dose males were comparable to the control during the whole observation period.

In the High dose females, during the first week of treatment, the mean body weight gain was near zero (many individuals lost weight) but, as for the males, the weights tended to recover after Day 7. However, the High dose remained below control throughout gestation. After parturition 5/10 dams lost weight or remained at the same weight during the first 4 days of lactation whereas 4/10 dams gained body weight (1/10 dams died on the day of parturition). Statistically significantly lower body weights were recorded from the seventh day of the gestation period until the end of the observed lactation period. Slightly lower bodyweights were recorded also in the female Mid dose group during this period, occasionally reaching statistical significance. These minor differences in the Mid dose group were considered to have no toxicological significance. The body weights of the Low dose females were comparable to the control.

see Figures and Tables on body weight attached as background material

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males, slightly lower food consumption was recorded in the High dose group compared to the controls during the first week, reaching statistical significance. The overall intake over the whole study was similar in all groups.

The food consumption of the females showed similar patterns as the body weight changes; there were consistently significantly lower values in the High dose. After parturition, low food consumption values were recorded for 5/10 dams during the first 4 days of lactation whereas 4/10 dams had similar food consumption as the controls (1/10 dams died on the day of parturition). To a much lesser extent, during the first 3 weeks of treatment slightly lower values were also seen in Mid dose. The food consumption of the Low dose females was comparable to the control at any occasion.

see Tables on food consumption attached as background material

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Significantly lower (p<0.05) activated partial thromboplastin time (APTT) in the male High dose group and significantly lower (p<0.05 or p<0.01) prothrombin time (PTT) in the Mid and High dose male groups were recorded. Considering the individual values of the High dose, 2 of the 5 males had APPT and PPT values below the historical control range (and additionally one animal had 9.9 sec APTT value which is just below the 10.0 sec lower limit of the historical control range). In females all values were normal with clearly no treatment related trend in these parameters. There was no other supporting evidence of any changes in these animals (other clinical pathology parameters, histopathology, etc.) so the relationship between the 2 low APTT and PPT values and treatment was considered to be equivocal. Generally, slightly shorter clotting time parameters is not considered to be an adverse effect. While it is plausible that liver changes may have slightly affected these parameters, these differences are not considered to be an adverse effect of the test item.

Besides this, there were no other statistically significant values recorded in any of the dose groups compared to the control.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant differences were recorded in males: Significantly (p<0.05 or p<0.01) higher cholesterol concentration in the Mid and High dose groups, significantly (p<0.05 or p<0.01) higher phosphorus concentration in all dose groups, significantly (p<0.01) lower sodium concentration in the High dose group, significantly lower (p<0.05 or p<0.01) chloride concentration in all dose groups, significantly (p<0.05) higher albumin level in the Mid dose group, significantly (p<0.05) lower alkaline phosphatase concentration in the Low and Mid dose groups and significantly (p<0.05) higher bile acid concentration in the High dose group. In females all values were normal. As all these values were within the historical ranges, it was considered that there were no adverse effects of treatment at any dose level.

see Table on clinical chemistry parameters attached as background material

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In males, significantly higher absolute and relative kidney weights were recorded in the Mid and High dose groups. The kidney weights in the female dose groups were comparable to the control.

In all male dose groups the absolute and relative liver weights were higher compared to the control. In females, significantly higher relative liver weights were recorded in the Mid and High dose groups.

Weight findings in other organs were considered incidental and not test substance related.

see Table on kidney and liver weights attached as background material

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related findings were seen in the liver and kidneys of the following animals:

Low dose group (30 mg/kg bw/day): Minimal hepatocellular hypertrophy in the centrilobular zone of the liver in 2/12 males.

Mid dose group (100 mg/kg bw/day): Minimal hepatocellular hypertrophy in the centrilobular zone of the liver in 11/12 males and 12/12 females, minimal eosinophilic droplets in the tubular section of the kidneys in 5/12 males.

High dose group (300 mg/kg bw/day): Slight to moderate hepatocellular hypertrophy in the centrilobular zone of the liver in 1/12 males and 7/12 females, slight hepatocellular microvesicular vacuolation in the centrilobular zone of the liver in 2/12 males and 1/12 females, moderate hepatocellular degeneration in the centrilobular and periportal zones of the liver in 5/12 males and 4/12 females, moderate multifocal macrovesicular inflammatory cell infiltration with hepatocellular apoptosis in the liver in 1/12 male, minimal to marked eosinophilic droplets in the tubular section of the kidneys in 9/12 males.

Hepatocellular degeneration was considered as adverse change (seen only in High dose animals), while hypertrophy was considered to be non-adverse adaptive change (seen in Low dose males and Mid and High dose of both sexes). Vacuolation (seen in two High dose males and one High dose female and also in the found dead High dose female) was also regarded as non-adverse.

In the kidneys of 5/12 Mid dose and 9/12 High dose males minimal/marked eosinophilic droplets were seen, correlated with organ weight changes. This was regarded as non-adverse.

Other observations in the kidneys were of low incidence and/or severity and/or distributed across control and dosed animals. They were considered incidental or a common background.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 100 - < 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Cross-reference 2

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

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

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
- Source:
Charles River Laboratories, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes]
- Age at study initiation: approx 12-13 weeks at start of treatment
- Weight at study initiation: males 425-502 g, females 237-306 g
- Fasting period before study: no
- Housing: up to 3 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or indivdually housed
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:
animals received ssniff SM R/M complete diet for rats and mice, Soest, Germany
animals received tap water from municipal supply.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
19.2 - 25.0
- Humidity (%): 29 - 64
- Air changes (per hr): 15 -20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
the test substance is very well water soluble. From the solid test material a stock solution of 741 g/l water was prepared, and further diluted.

- DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

- VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
6, 20, 60 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg body weight
- Lot/batch no. (if required):
- Purity:

Details on mating procedure:

- M/F ratio per cage: 1 : 1
- Length of cohabitation: 5 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC with UV detector was used to determine the concentrations in the dosing solutions at 3 occasions during the study. The concentrations determined ranged between 97% and 103% recovery of the nominal concentration.

Duration of treatment / exposure:

males were dosed for 28 days starting 14 days prior to mating. Females were dosed for 14 days prior to mating, during mating and gestation until day 13 post partum.

Frequency of treatment:

once daily

Details on study schedule:

- Age at mating of the mated animals in the study: 14 - 15 weeks

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a Dose Range Finding study severe toxicity was seen at 1000 mg/kg bw/day causing death of the animals. At 100 and 300 mg/kg bw/day, treatment-related increased kidney and liver weights were recorded, but without any other indication of serious toxic effects in these organs.
- Rationale for animal assignment (if not random):
- Other:

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: food consumption was determined

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 AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
just before necropsy
- Anaesthetic used for blood collection: Yes pentobarbital
- Animals fasted: Yes
- How many animals: 5 per sex and dose group
- Parameters checked in table [No.?] were examined: see Table on haematology attached as background material

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: just before necropsy
- Animals fasted: Yes
- How many animals: 5 per sex and dose group
- Parameters checked in table [No.?] were examined.: see Table on clinical chemistry and urinalysis attached as background material

URINALYSIS: Yes
- Time schedule for collection of urine: just before necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked in table [No.?] were examined: see Table on clinical chemistry and urinalysis attached as background material

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity.

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the treatments starts. Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals):

Parameters examined in P male parental generations: weights and histological examination of testis, epididymis, prostate, seminal vesicles with coagulating glands

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.

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.

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

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after 28 days of dosing
- Maternal animals: All surviving animals on post natal day 14.

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 [#] were prepared for microscopic examination and weighed, respectively: see "pathology and histopathology investigations" attached as background material

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: macroscopic

GROSS NECROPSY
- Gross necropsy consisted of external examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Statistics:

The statistical evaluation of data (labelled as † in the lists below) was performed with
the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS v9.2
(when using Provantis).
In case of the SPSS PC+4.0 program package, the heterogeneity of variance between
groups was checked by Bartlett's test. Where no significant heterogeneity was
detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained
result was significant, then Duncan's Multiple Range test was used to assess the
significance of inter-group differences. Where significant heterogeneity was found,
the normal distribution of data was examined by Kolmogorow-Smirnow test. In the
case of non-normal distribution, the non-parametric method of Kruskal-Wallis One-
Way analysis of variance was applied. If a positive result was detected, the inter-group
comparisons were performed using Mann-Whitney U-test. The Chi-squared test was
used for non-continuous data.
In case of the SAS v9.2 software package (within the validated Provantis system) the
following decision tree was applied automatically for statistical evaluation of
continuous numeric data. The normality and heterogeneity of variance between groups
was checked by Shapiro-Wilk and Levene tests using the most appropriate data format
(log-transformed when justified). Where both tests showed no significant
heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out.
If the obtained result was positive, Dunnett (Multiple Range) test was used to assess
the significance of inter-group differences; identifying differences of <0.05 or <0.01
as appropriate.

Reproductive indices:

mating, fertility, gestation indices were determined for parental animals

Offspring viability indices:

survival, pre-implantation mortality, intrauterine mortality, total mortality, sex ratio indices were determined for the F1 generation

Clinical signs:

effects observed, non-treatment-related

Mortality:

mortality observed, non-treatment-related

Description (incidence):

one female of the control group was preterminally euthanised the day after littering for animal welfare reasons (protrusion of the vagina). One female of the high dose group was found dead on the day of littering; the cause of death could not be determined.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males, on the week after initiation of treatment, the body weight gain in the High dose was 74% below control (p<0.01) but the males fully compensated for this in the following week. During the third week the body weight gain was again below the control values (-36%, no statistical significance) and in the fourth week, the animals compensated the difference again. Although there were no statistical differences in the overall body weight gain of the High dose compared to the control, it is noted that the bodyweight gain of the animals was disturbed most probably by the test item. The body weight gain of the Mid dose males were slightly below the Control during the first three weeks, without reaching statistical significances. These minor differences in the Mid dose group were considered to have no toxicological significance. The body weight and body gain weight values of the Low dose males were comparable to the control during the whole observation period.

In the High dose females, during the first week of treatment, the mean body weight gain was near zero (many individuals lost weight) but, as for the males, the weights tended to recover after Day 7. However, the High dose remained below control throughout gestation. After parturition 5/10 dams lost weight or remained at the same weight during the first 4 days of lactation whereas 4/10 dams gained body weight (1/10 dams died on the day of parturition). Statistically significantly lower body weights were recorded from the seventh day of the gestation period until the end of the observed lactation period. Slightly lower bodyweights were recorded also in the female Mid dose group during this period, occasionally reaching statistical significance. These minor differences in the Mid dose group were considered to have no toxicological significance. The body weights of the Low dose females were comparable to the control.

see Figures and Tables on body weight attached as background material

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males, slightly lower food consumption was recorded in the High dose group compared to the controls during the first week, reaching statistical significance. The overall intake over the whole study was similar in all groups.

The food consumption of the females showed similar patterns as the body weight changes; there were consistently significantly lower values in the High dose. After parturition, low food consumption values were recorded for 5/10 dams during the first 4 days of lactation whereas 4/10 dams had similar food consumption as the controls (1/10 dams died on the day of parturition). To a much lesser extent, during the first 3 weeks of treatment slightly lower values were also seen in Mid dose. The food consumption of the Low dose females was comparable to the control at any occasion.

see Tables on food consumption attached as background material

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Significantly lower (p<0.05) activated partial thromboplastin time (APTT) in the male High dose group and significantly lower (p<0.05 or p<0.01) prothrombin time (PTT) in the Mid and High dose male groups were recorded. Considering the individual values of the High dose, 2 of the 5 males had APPT and PPT values below the historical control range (and additionally one animal had 9.9 sec APTT value which is just below the 10.0 sec lower limit of the historical control range). In females all values were normal with clearly no treatment related trend in these parameters. There was no other supporting evidence of any changes in these animals (other clinical pathology parameters, histopathology, etc.) so the relationship between the 2 low APTT and PPT values and treatment was considered to be equivocal. Generally, slightly shorter clotting time parameters is not considered to be an adverse effect. While it is plausible that liver changes may have slightly affected these parameters, these differences are not considered to be an adverse effect of the test item.

Besides this, there were no other statistically significant values recorded in any of the dose groups compared to the control.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant differences were recorded in males: Significantly (p<0.05 or p<0.01) higher cholesterol concentration in the Mid and High dose groups, significantly (p<0.05 or p<0.01) higher phosphorus concentration in all dose groups, significantly (p<0.01) lower sodium concentration in the High dose group, significantly lower (p<0.05 or p<0.01) chloride concentration in all dose groups, significantly (p<0.05) higher albumin level in the Mid dose group, significantly (p<0.05) lower alkaline phosphatase concentration in the Low and Mid dose groups and significantly (p<0.05) higher bile acid concentration in the High dose group. In females all values were normal. As all these values were within the historical ranges, it was considered that there were no adverse effects of treatment at any dose level.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

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 findings were seen in the liver and kidneys of the following animals:

Low dose group (30 mg/kg bw/day): Minimal hepatocellular hypertrophy in the centrilobular zone of the liver in 2/12 males.

Mid dose group (100 mg/kg bw/day): Minimal hepatocellular hypertrophy in the centrilobular zone of the liver in 11/12 males and 12/12 females, minimal eosinophilic droplets in the tubular section of the kidneys in 5/12 males.

High dose group (300 mg/kg bw/day): Slight to moderate hepatocellular hypertrophy in the centrilobular zone of the liver in 1/12 males and 7/12 females, slight hepatocellular microvesicular vacuolation in the centrilobular zone of the liver in 2/12 males and 1/12 females, moderate hepatocellular degeneration in the centrilobular and periportal zones of the liver in 5/12 males and 4/12 females, moderate multifocal macrovesicular inflammatory cell infiltration with hepatocellular apoptosis in the liver in 1/12 male, minimal to marked eosinophilic droplets in the tubular section of the kidneys in 9/12 males.

Hepatocellular degeneration was considered as adverse change (seen only in High dose animals), while hypertrophy was considered to be non-adverse adaptive change (seen in Low dose males and Mid and High dose of both sexes). Vacuolation (seen in two High dose males and one High dose female and also in the found dead High dose female) was also regarded as non-adverse.

In the kidneys of 5/12 Mid dose and 9/12 High dose males minimal/marked eosinophilic droplets were seen, correlated with organ weight changes. This was regarded as non-adverse.

Other observations in the kidneys were of low incidence and/or severity and/or distributed across control and dosed animals. They were considered incidental or a common background.

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

see Table on oestrous cycles attached in background material

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

based on organ weights

see Table on organ weights, males attached in background material

Reproductive performance:

no effects observed

Description (incidence and severity):

see Table on reproductive indices attached in background material

Key result

Dose descriptor:

NOAEL

Effect level:

> 100 - < 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: general parental toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL = highest dose tested

Remarks on result:

other: NOAEL for reproductive function

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The number of implantations in pregnant females was similar in all groups. In the Low and Mid dose groups the number of viable pups on PND0, 4 and 13 as well as pup survival indices on PND0, 4 and 13 were comparable to control values.

In the High dose group the number of live born was slightly lower (p<0.05), indicating a slightly increased prenatal mortality.

From the 10 litters in the High dose group all pups of 4 litters died during the first 4 postnatal days. From 1 litter half of the pups died during this period. Survival and growth of pups in the remaining 4 litters was similar to control. In this group one dam died on the day of littering. The animal gave birth to one dead and 5 liveborns. After the dam was found dead, the 5 living pups were euthanized because of animal welfare reasons.

The dams (#4501, 4506, 4507, 4509, 4511) of the 4 litters in which high pup mortality was present, showed body weight loss during these 4 postnatal days (PND). This body weight loss was linked with reduced food consumption during this period (see Tables 4 and 5).

The differences between the Control and the High dose group are considered to be test item-related and correlated with reduced feed intake and body weight loss of the dams during these 4 postnatal days (it is noted that significant body weight loss in dams is likely to be associated with reduced milk production). In the High dose group, the number of litters with 100% mortality of pups during the period to PND4 was 4 of 9 litters, whereas in the other groups, 1 in 12 was considered normal.

From PND4 the High dose dams gained body weight at a rate similar to controls, and during this period there was no further pup mortality in the High dose group. The data appears to confirm that the principal effect was on the dam, including body weight loss from PND0 to PND4, with mortality of pups as a consequence, rather than a direct toxic effect on the pups.

see Table on offspring, summary of mortality and viability attached in background material
see Table on delivery, litter viability, sex ratio attached in background material
see Table 4 and Table 5 individual data of high dose females attached in background material

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The Low and Mid dose group values showed no significant differences compared to controls in the F1 generation.

The body weights of all freshly delivered pups of the 10 High dose litters including those dying until postnatal day 4 were not significantly different to controls. The surviving pups in the High dose group had body weights and body weight gains that were generally not significantly different to controls.

see Table on litter body weight development attached in background material

Gross pathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Compared to the control, there were no statistically significant differences in thyroid hormone concentration levels recorded in any of the PND13 pup dose groups. The thyroid gland weights of the PND13 pups were also statistically not different from the Control group. In summary, there were no effects on the thyroid hormone levels or on the thyroid gland weights in the PND13 pups that were ascribed to the test item.

There were no statistically significant differences in the anogenital distance between the test item treated groups (males/females) and the control on PND0. There was no nipples/areolae presence seen in any of the male pups on PND13.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 100 - < 300 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

dams were dosed

Sex:

male/female

Basis for effect level:

mortality

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

other: very likely malnutrition due to maternal toxicity

Treatment related:

yes

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 422 reproduction / developmental toxicity screening study

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Charles River Laboratories, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes]
- Age at study initiation: approx 12-13 weeks at start of treatment
- Weight at study initiation: males 425-502 g, females 237-306 g
- Fasting period before study: no
- Housing: up to 3 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or indivdually housed
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:
animals received ssniff SM R/M complete diet for rats and mice, Soest, Germany
animals received tap water from municipal supply.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
19.2 - 25.0
- Humidity (%): 29 - 64
- Air changes (per hr): 15 -20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
the test substance is very well water soluble. From the solid test material a stock solution of 741 g/l water was prepared, and further diluted.

- DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

- VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
6, 20, 60 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg body weight
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC with UV detector was used to determine the concentrations in the dosing solutions at 3 occasions during the study. The concentrations determined ranged between 97% and 103% recovery of the nominal concentration.

Details on mating procedure:

- If cohoused:
- M/F ratio per cage: 1 : 1
- Length of cohabitation: up to 5 days
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smea] referred to as day 0 of pregnancy
- Any other deviations from standard protocol:

Duration of treatment / exposure:

males were dosed for 28 days starting 14 days prior to mating. Females were dosed for 14 days prior to mating, during mating and gestation until day 13 post partum.

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: in a dose range finding study severe toxicity was seen at 1000 mg/kg bw/d causing death of animals. At 100 and 300 mg/kg/d treatment related increased kidney and liver weights were recorded, but without any other indication of serious toxic effects in these organs.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations:on gestation days 0, 3, 7, 10, 14, 17, and 20 and on post-partal days 0, 4, 7, 10, 13

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on post partum day # 14
- Organs examined: see Table on pathology and histopathology investigations attached as background material

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
- Other: number of pre-implantation mortality

Fetal examinations:

Females were allowed to litter and rear their offspring until post natal day 13. The delivery process was observed as carefully as possible. All observations were recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy until the completion of parturition.

Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups) and to detect the presence of gross abnormalities. Observations were reported individually for each adult animal. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.

Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND0) and on PND4 and PND13, with accuracy of 0.01g. All litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded.

The anogenital distance (AGD) of each pup was measured at the time of the first weighing (PND0). Presence of nipples/areolae in male pups were recorded on PND13.

All pups were examined externally at weighing on PND4. One male and one female pup (where possible) was allocated randomly for culling for blood sampling on PND4.

Statistics:

The statistical evaluation of data (labelled as † in the lists below) was performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS v9.2 (when using Provantis).
In case of the SPSS PC+4.0 program package, the heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of non-normal distribution, the non-parametric method of Kruskal-Wallis One- Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. The Chi-squared test was used for non-continuous data.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

one female of the control group was preterminally euthanised the day after littering for animal welfare reasons (protrusion of the vagina). One female of the high dose group was found dead on the day of littering; the cause of death could not be determined.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the High dose females, during the first week of treatment, the mean body weight gain was near zero (many individuals lost weight) but, as for the males, the weights tended to recover after Day 7. However, the High dose remained below control throughout gestation. After parturition 5/10 dams lost weight or remained at the same weight during the first 4 days of lactation whereas 4/10 dams gained body weight (1/10 dams died on the day of parturition). Statistically significantly lower body weights were recorded from the seventh day of the gestation period until the end of the observed lactation period. Slightly lower bodyweights were recorded also in the female Mid dose group during this period, occasionally reaching statistical significance. These minor differences in the Mid dose group were considered to have no toxicological significance. The body weights of the Low dose females were comparable to the control.

see Figures and Tables on body weight and Table 4 and Table 5 individual data of high dose females attached as background material

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption of the females showed similar patterns as the body weight changes; there were consistently significantly lower values in the High dose. After parturition, low food consumption values were recorded for 5/10 dams during the first 4 days of lactation whereas 4/10 dams had similar food consumption as the controls (1/10 dams died on the day of parturition). To a much lesser extent, during the first 3 weeks of treatment slightly lower values were also seen in Mid dose. The food consumption of the Low dose females was comparable to the control at any occasion.

see Tables on food consumption and Table 4 and Table 5 individual data of high dose females attached as background material

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In females, significantly higher relative liver weights were recorded in the Mid and High dose groups.

Weight findings in other organs were considered incidental and not test substance related.

see Table on kidney and liver weights attached as background material

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related findings were seen in the liver of the following animals:

Mid dose group (100 mg/kg bw/day): Minimal hepatocellular hypertrophy in the centrilobular zone of the liver in 12/12 females.

High dose group (300 mg/kg bw/day): Slight to moderate hepatocellular hypertrophy in the centrilobular zone of the liver in 7/12 females, slight hepatocellular microvesicular vacuolation in the centrilobular zone of the liver in 1/12 females, moderate hepatocellular degeneration in the centrilobular and periportal zones of the liver in 4/12 females (# 4506, 4507, 4511, 4512).

Hepatocellular degeneration was considered as adverse change (seen only in High dose animals), while hypertrophy was considered to be non-adverse adaptive change (seen in Mid and High dose of both sexes). Vacuolation (seen in one High dose female and also in the found dead High dose female) was also regarded as non-adverse.

see Tables on individual histopathology data of high dose females attached as background material

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

two of the 12 high dose females (# 4504, 4512) were not pregnant (for unknown reasons)

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

not examined

Total litter losses by resorption:

no effects observed

Early or late resorptions:

not examined

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

The number of implantations in pregnant females was similar in all groups, but the number of live born was slightly lower (p<0.05) in the High dose, indicating a slightly increased prenatal mortality. In the High dose group, the number of pups born alive was significantly lower than in the control group.

see Table on pre-natal and post-natal survival indices attached as background material
see Table on "Offspring summary of mortality and viability" attached as background material
see Table Summary of dead pups attached as background material

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

two (# 4504, 4512) of the 12 females were not pregnant after mating.

Other effects:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

> 100 - < 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

dead fetuses

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

see Table on litter body weight development attached as background material

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

The number of live born was slightly lower (p<0.05) in the High dose, indicating a slightly increased prenatal mortality. In the High dose group, the number of pups born alive was significantly lower than in the control group.

see Table on pre-natal and post-natal survival indices attached as background material

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

effects observed, treatment-related

Description (incidence and severity):

all pups of the following four females # 4506, 4507, 4509, 4511 and half of the pups of female # 4501 died during the first four days after birth (PPD 0 - 4). These five females had significantly low food consumption during this period and lost weight. In addition, in three of the females (# 4506, 4507, 4511) hepatocellular degeneration was observed during histology after necropsy.

see Tables on food consumption and body weight "Table 4 and Table 5 individual data of high dose females" attached as background material
see Tables on individual histopathology data of high dose females attached as background material
see Table on pre-natal and post-natal survival indices attached as background material

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

> 100 - < 300 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

dams were dosed

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in postnatal survival

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects