Reaction mass of 2-[4-[(Hexahydro-2,4,6-trioxo-5-pyrimidyl) azo]phenyl]-6-methylbenzothiazole-7-sulphonic acid, compound with 2,2',2''-nitrilotris[ethanol] (1:1) and Lithium 2-[4-[(hexahydro-2,4,6-trioxopyrimidin-5-yl)azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Endpoint summary

Description of key information

The no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and female Wistar rats.

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

Study period:

2016-07-18 to 2018-01-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines; OPPTS 870.3650:

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Batch identification: 0013479406

Species:

rat

Strain:

Wistar

Remarks:

strain Crl:WI(Han)

Details on species / strain selection:

The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH/ Charles River Laboratories, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 13-14 weeks
- Weight at study initiation: Mean weight per group: Male: 391.6 g - 394.7 g; Female: 213.9 g - 219.1 g
- Housing: Individually during the study period; During overnight matings, male and female mating partners were housed together; Pregnant animals and their litters were housed together until PND 13
The bedding and the enrichment is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals) by the producer.
- Diet: Kliba maintenance diet mouse/rat, ad libitum
- Water: Drinking water, ad libitum
- Acclimation period: 3 weeks

DETAILS OF FOOD AND WATER QUALITY:
Food analyses
The food used in the study was assayed for chemical and for microbiological contaminants.
With regard to the analytical findings of chemical and microbiological contaminants and the duration of application, the diet was found to be suitable.

Drinking water analyses
The drinking water is regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms by the municipal authorities.
On the basis of the analytical findings, the drinking water was found to be suitable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15 times
- Photoperiod (hrs dark / hrs light): 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 % suspension in drinking water with 5 mg/100 mL Tween 80

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the preparation of the administration suspensions the test substance was weighed in a
calibrated beaker depending on the dose group, topped up with 0.5 % Carboxymethylcellulose suspension in drinking water with 5 mg/ 100mL Tween 80 and intensely mixed with a magnetic stirrer.
During administration, the preparations were kept homogeneous with a magnetic stirrer

VEHICLE
- Justification for use and choice of vehicle: The test substance is stable in the vehicle.
- Concentration in vehicle: 1 g/100 mL; 3 g/100 mL; 10 g/100 mL
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Method: Quantitative LC/ESI-MS with evaluation by the external standard method.

Analytical verifications of the stability of the test substance in 0.5 % Carboxymethylcellulose suspension in drinking water (with 5 mg/ 100mL Tween 80) for a period of 7 days at room temperature were carried out before the study was initiated.
At the beginning (during premating) and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. These samples were used as a concentration control at the same time. At the above mentioned time points additionally one sample from the mid concentration was taken for concentration control
analysis.
The samples collected at the beginning of the administration period and the first set of samples during lactation were analyzed in the Analytical Laboratory at the Competence Center Analytics.
Due to unexpected high values in the measured analytical samples of the mid and low dose group at the Competence Center Analytics a more sensitive analytical method was used for the remaining samples collected during the study. The additional samples were measured at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology.

The stability of test substance in 0.5 % Carboxymethylcellulose suspension in drinking water + 5 mg/100mL Tween 80 was demonstrated for a period of 7 days at room temperature.
In summary it can be stated that the homogeneous distribution of the test substance in the vehicle was demonstrated and the measured values for the test substance were basically in the expected range of the target concentrations (90 - 110%), demonstrating the correctness of the preparations.

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

once 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

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: By request of the sponsor

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented daily for each animal.
- Cage side observations checked: Signs of morbidity, pertinent behavioral changes and signs of overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: In all animals once before the start of the administration period and thereafter at weekly intervals. (Parameters measured see below table no. 1)

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice.

HAEMATOLOGY: Yes
- Time schedule for collection of blood and no. of animals: From all F0 parental male animals of all test groups shortly before sacrifice and from all F0 parental female animals of all test groups on PND 14
- How many animals: From all F0 parental male animals of all test groups, all F0 parental female animals of all test groups
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- Parameters checked in table no 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In the morning
- Animals fasted: Yes
- How many animals: The first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.
- Parameters checked in table no.3 were examined.

Thyroid Hormones
Blood samples from all dams at PND 14 and all males at termination.
Blood samples from the adult males were assessed for serum levels for thyroid hormones (T4).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 1. On study day 30; 2. On study day 56
- Dose groups that were examined: 1. Five male animals per group; 2. Five female animals (with litter) per group
- Battery of functions tested: followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests (see table no. 8 A)- C)).
Motor activity measurement : The Measurement of motor activity (MA) was measured at the end of the administration period in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was determined once a week as representative value over a period of 3 days for the male and female parental animals, with the following exceptions:
- Water consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals)
- Water consumption of the females with evidence of sperm was determined on gestation days (GD) 0-1, 7-8, 14-15 and 20-21.
- Water consumption of the F0 females, which gave birth to a litter was determined for PND 1-2, 4-5, 7-8, 10-11 and 13-14.

FOOD CONSUMPTION: Yes
Food consumption was determined once a week for male and female parental animals, with the following exceptions:
Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0 -7, 7 - 14 and 14 - 20.
- Food consumption of F0 females which gave birth to a litter was determined on PND 1 -4, 4 - 7, 7 - 10 and 10 - 13.
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The male and female animals were sacrificed 34 and 58/62 days, respectively, after the beginning of the administration, and examined.

ORGAN WEIGHTS
See table no. 4 and 5

Organ/tissue fixation (all parental animals) see table no.6

HISTOPATHOLOGY: Yes (see table no.7)

Statistics:

Blood parameters (clinical pathology)
Statistical test: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians.

Weight parameters (pathology)
Statistical test: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.

Water consumption, food consumption (parental animals), body weight and body weight change (parental animals)
Statistical test: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (twosided) for the hypothesis of equal means.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All high- and mid-dose male and female F0 animals showed discolored feces (orange) from premating day 1 (test group 3) and 3 (test group 2) till the end of the study.
All high-dose male and female F0 animals showed discolored urine (yellow) from premating day 10 till the end of the study.
Several male animals of test groups 3 showed salivation after treatment (grade: slight to severe) during several parts of the mating period and during the entire post-mating period.
During a part of the gestation period one female of test group 3 and during several parts of the lactation period several females of test group 3 showed salivation after treatment (grade: slight to moderate).
One mid-dose female (No.122) showed a swelling at the right forelimb from the end of premating till the end of the study because of an injury.
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the low-dose male and female F0 animals during the entire study period.

Detailed clinical observations
All male and all female animals of all dose groups (100, 300 and 1000 mg/kg bw/d) did not show any abnormalities.
One mid-dose female (No.122) showed a swelling on the right forelimb because of an injury.

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any of the groups.
One animal of dose group 1 (No. 117 - 300 mg/kg bw/d) was sacrificed premature on premating day 6 because it escaped from the cage during the first premating week [one week before start of the mating period] and paired with male animal No. 18.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights of all male and female parental animals in all test substance-treated groups were comparable to the concurrent control values during the entire study period.
This includes the statistically significantly decreased values during premating days 7 - 13 in test groups 1-3 and the statistically significantly increased values in the high-dose females during premating days 0 - 7.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of all male and female F0 animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

Water consumption of all male and female F0 animals of all test substance-treated groups (100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control values throughout the entire study.
The supposedly decreased water consumption of all test substance-treated F0 females during one single timepoint at the end of the lactation period (PND 13 – 14) was assessed as incidental.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed.
In males of test group 3 (1000 mg/kg bw/d) absolute and relative lymphocyte counts were increased (absolute counts not statistically significant). However, both parameter values were within historical control range (males, absolute lymphocytes 3.16-5.96 Giga/L; relative lymphocytes 70.0-82.0 %). In males of test group 2 (300 mg/kg bw/d) relative monocyte counts were significantly lower compared to controls, but the change was not dose-dependent.
Therefore, the mentioned changes were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.

In females of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d), calcium levels were significantly higher compared to controls. The calcium medians and means of test groups 1 and 3 were within, those of test group 2 above, and mean and median of the controls below the historical control range (females, calcium 2.56-2.66 mmol/L). Calcium alterations in the test groups were not dose-dependently changed. Alanine aminotransferase (ALT) activities in males of test group 2 (300 mg/kg bw/d) were significant lower compared to controls, but the change was also not dose-dependent. Therefore, the mentioned alterations among clinical chemistry parameters were regarded as incidental and not treatment-related.

Thyroid hormones
In parental males of test group 2 (300 mg/kg bw/d) significant higher T4 levels compared to controls were observed.
However, this change was not dose-dependent and therefore it was regarded as incidental and not treatment-related.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Home cage observations:
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.

Open field observations:
The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.

Sensorimotor tests/reflexes:
There were no test substance-related findings in male and female animals of all test groups.

Quantitative Parameters:
No test substance-related impaired parameters were observed in male and female animals of all test groups.

Motor activity measurement (MA)
No statistically significant changes on motor activity data (summation of all intervals) was observed in all male and female animals of all dose groups in comparison to the concurrent control group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Minor changes in thymus, adrenal gland, kidney and thyroid gland weight are considered to be of no significant toxicologic relevance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

An orange yellow discoloration of the content of the gastrointestinal tract (particularly of the stomach, jejunum, cecum or colon) was observed in 2/19 animals of the 100 mg/kg/d, 5/20 animals of the 300 mg/kg/d and 16/20 animals of the 1000 mg/kg/d treated group. The highest incidence of this finding was found in the glandular stomach.
The discoloration of the gastrointestinal content, supposed to be related to the chemical properties of the test item could not be related to any histopathologic change of the mucosa or underlying structures.
Except for the discolored gastrointestinal content, all gross findings in this study are considered spontaneous, incidental or related to technical procedures and consistent with the usual pattern of findings in animals of this strain and age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The discoloration of the gastrointestinal content, supposed to be related to the chemical properties of the test item could not be related to any histopathologic change of the mucosa or underlying structures.
Except for the discolored gastrointestinal content, all histopathologic findings in this study are considered spontaneous, incidental or related to technical procedures and consistent with the usual pattern of findings in animals of this strain and age.

Histopathological findings: neoplastic:

not examined

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 test substance related adverse effects were observed.

Key result

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and guideline compliant study

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

Additional information

Repeated dose toxicity

The test item was assessed for repeated dose toxicity and reproduction/developmental toxicity according to OECD 422 and GLP.

The test item was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (0.5 % Carboxymethylcellulose suspension in drinking water + 5 mg/100mL Tween 80). The duration of treatment covered a 2-week pre-mating and a mating period, approximately 7 days post-mating in males, and the entire gestation period as well as approximately 23 days of the lactation period and 31 days post-mating in females (for sperm negative female).

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females.

A detailed clinical observation (DCO) was performed in all animals before the start of the administration period and, as a rule, thereafter at weekly intervals.

Water consumption of the F0 parents was determined for premating days 0 - 3 and 7–10. In dams water consumption was determined for gestation days (GD) 0 - 1, 7 - 8, 14 - 15 and 20 - 21 and lactation days (PND) 1 - 2, 4 - 5, 7 - 8, 10 - 11 and 13 - 14.

Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for GD 0 - 7, 7 - 14, 14 - 20 and PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13.

Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on GD 0, 7, 14 and 20, on the day of parturition (PND 0) and on PND 4, 7, 10 and 13.

Estrous cycle data were evaluated for F0 generation females over a two week period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.

Clinico-chemical and hematological examinations were performed in 5 animals per sex and group towards the end of the administration period.

Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement.

At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group.

All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

No test substance-related clinical, reproductive or clinical pathology adverse findings were noted at 100, 300, 1000 mg/kg bw/d for the F0 parental animals.

In conclusion, under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the oral administration of the test item by gavage to male and female Wistar rats did not result in signs of systemic toxicity up to a dose level of 1000 mg/kg bw/d. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and female Wistar rats.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes. In an OECD 422 study in rats no effects on general systemic toxicity were observed, therefore the NOAEL for male and female rats was determined to be 1000 mg/kg bw. As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.