Pyridinium toluene-4-sulphonate

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the NOAEL of 100 mg/kg body weight/d and the liver effects observed at the dose level of 300 mg/kg body weight/day in the sub-acute repeated dose oral toxicity study WS400304 has to be classified STOT RE 2.