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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-08 until 2018-11-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
12 Sep 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Kaiser-Friedrich-Stra ße 7, 55116 Mainz, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
CAS nr used in the past for this type of chemical was 85204-21-3, after analytical charaterisation renamed and new CAS 1471311-93-9 was applied.
Batch identification: 14671736W0
Purity: 100% UVCB substance
Content: 100 g/100 g (UVCB)
Content of Triethanolamine: 74.2 g/100 g (Analytical Report, study code: 17L00233)
Specific details on test material used for the study:
Please note: This kind of substance was in the past handle under the CAS nr 85204-21-3
After analytical characterisation the substance was renamed and received a new CAS nr. Now the substance is characterised by the CAS 1471311-93-9 EC Number: 939-488-3

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: BASF and 14671736W0
- Expiration date of the lot/batch: 01 May 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the vehicle: stability in drinking water with 5 mg/100 mL Cremophor EL over a period of 7 days at room temperature verified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: prepared once weekly, specific amount of test substance was weighed and filled up with deionized water containing Cremophor EL
- Final dilution of a dissolved liquid: suspension in deionized water with 5 mg/100 mL Cremophor EL, respectively

FORM AS APPLIED IN THE TEST: suspension; preparations were kept homogeneous with a magnetic stirrer

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Weight at study initiation (mean): 174 g male, 123 g female; weight variation of the animals used did not exceed 20 percent of the mean weight of each sex
- Housing: group, 5 animals per cage, polysulfonate cages (TECNIPLAST, Hohenpeißenberg, Germany)
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat “GLP”, meal (Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum, drinking water
- Acclimation period: up to 6 days

DETAILS OF FOOD AND WATER QUALITY:
- diet: assayed for chemical and microbiological contaminants by the supplier
- drinking water: regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring Department of BASF SE as well as for the presence of microorganisms by a contract laboratory

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (06.00-18.00 h / 18.00-06.00 h)

IN-LIFE DATES: From: 2018-01-16 To: 2018-04-24 / 2018-04-25

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
deionized water containing 5 mg/100 mL Cremophor EL
Analytical verification of doses or concentrations:
yes
Remarks:
performed in all concentrations at the beginning and towards the end of the administration period
Details on analytical verification of doses or concentrations:
- samples stored in a freezer
- HPLC, with reversed-phase (RP-18e) stationary phase and mobile phase based on Acetonitrile/(NH4) H2PO4 Buffer
- approx. 1.3 min retention time
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
1 g/ 100 mL
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
3 g/ 100 mL
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
10 g/ 100 mL
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on available study information e.g. 85R0586/11S125
- Rationale for animal assignment: list of randomization instructions was compiled with a computer
- Fasting period before blood sampling for clinical biochemistry: at least 16 hours

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, before the administration as well as within 2 hours and within 5 hours after the administration
- Parameters: any abnormal clinically signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
- Parameters: Abnormal behavior when handled, Fur, Skin, Posture, Salivation, Respiration, Activity/arousal level, Tremors, Convulsions, Abnormal movements, Impairment of gait, Lacrimation, Palpebral closure, Exophthalmus, Feces (appearance/consistency), Urine, Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period (randomization), during the administration period on day 0 and thereafter at weekly intervals

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly
- Parameter: mean food consumption in grams per animal and day

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily by visual inspection
- Parameter: any overt changes in volume

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the start of the administration period on day 0 and on study day 91
- Dose groups that were examined: control and high-dose animals
- Parameters: eyes examined for any changes using an ophthalmoscope after administration of a mydriatic agent

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning of day of study day 92 (male) and 93 (female)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Anticoagulant used: EDTA-K3
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- all details given for heamatology apply
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: in the afternoon of the study day 81
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (no food or drinking water provided during the unrinalysis)
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional observational battery (FOB)
- Time schedule for examinations: at the end of the administration period at about 10:00 h
- Dose groups that were examined: all dose groups, all animals
- Battery of functions tested: passive observations, open field observations, sensory motor activity, reflex test
- Parameters:
- Home cage observations: Posture, Tremors, Convulsions, Abnormal movements, Gait, Other findings
- Open field observations: Behavior on removal from the cage, Fur, Skin, Salivation, Nasal discharge, Lacrimation, Eyes/ pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/ stereotypes, Gait, Activity/ arousal level, Feces excreted within 2 minutes (appearance/ consistency), Urine excreted within 2 minutes (amount/ color), Rearing within 2 minutes
- Sensory motor tests/ reflexes: Reaction to an object being moved towards the face (approach response), Touch sensitivity (touch response), Vision (visual placing response), Pupillary reflex, Pinna reflex, Audition (auditory startle response), Coordination of movements (righting response), Behavior during handling, Vocalization, Pain perception (tail pinch), Grip strength of forelimbs, Grip strength of hindlimbs, Landing foot-splay test

Motor activity:
- Time schedule for examinations: the same day as the FOB from 14:00 h, each measurement lasted exactly 1 hour
- Dose groups that were examined: all dose groups, all animals
- Parameters: animals individually house in cages, 18 beams were allocated per cage, number of beam interrupts was counted over 12 intervals for 5 minutes per interval, rats were fasted during the measurement

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Parameters: Organ weights and incidence of gross lesions for the following organs:
- Organs examined: Anesthetized animals (final body weight), Adrenal glands (fixed), Brain, Epididymides, Heart, Kidneys, Liver, Ovaries (fixed), Spleen, Testes, Thymus (fixed), Thyroid glands (with parathyroid glands) (fixed), Uterus with cervix

HISTOPATHOLOGY: Yes (see table)
Statistics:
CLINICAL EXAMINATIONS
- Body weight, body weight change: DUNNETT's test (two-sided) for the hypothesis of equal means
- Rearing, grip strength forelimbs, grip strength hindlimbs, footsplay test, motor activity: Non-parametric one-way analysis using KRUSKALWALLIS test (two-sided), if p<=0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians

CLINICAL PATHOLOGY
- blood paramters:
- parameters with bidirectional changes, Non-parametric one-way analysis using KRUSKAL-WALLIS test; if p<=0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
- parameters with unidirectional changes, Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
- Urinalysis, (apart from urine volume, specific gravity, color and turbidity): Pairwise comparison with the WILCOXON-test (one-sided) for the hypothesis of equal medians (if exactly the same numbers of the dose group and the control, no statistical test)
- Urine volume and specific gravity: Non-parametric one-way analysis using KRUSKAL-WALLIS test, if p<=0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
- Urine color and turbidity: not evaluated statistically

PATHOLOGY
- Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided), if p<=0.05, a pairwise comparison of each test group with the control group was performed using WILCOXON-test (two-sided) for the equal medians

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related, adverse effects were obtained in any test group (100, 300 and 1000 mg/kg bw/d).
- salivation shortly after treatment was observed in all animals of the highest test group on several days
From the temporary, short appearance immediately after dosing it was concluded that the finding was induced by a bad taste of the test substance or local affection of the upper digestive tract.
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- no test substance-related, adverse effects on body weight development were obtained
- mean body weights and body weight change values of male and female animals did not show any significant deviations to the control
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- no test substance-related, adverse changes observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
- no test substance-related, adverse changes observed with regard to water consumption
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- no treatment-related findings observed
- other apparent findings were assessed as being incidental in nature since they occurred in control as well as in treated animals without a dose-response relationship
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- no treatment-related changes among hematological parameters observed
- observed effects were within historical control range or statistically significant but not dose-dependent and therefore 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
- observed effects were within historical control range or statistically significant but not dose-dependent and therefore regarded as incidental and not treatment-related
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
- in males of the highest test group there were significant increased ketone bodies found, considered an isolated finding, which was not accompanied by any changed metabolism parameter
- this alteration was regarded as potentially treatment-related, but non-adverse
- no treatment-related, adverse changes among urinalysis parameters were observed
- potential in kidneys observed for concentrating the urine, regarded as an adaptive but not as an adverse effect
- observed effects were within historical control range or statistically significant but not dose-dependent and therefore regarded as incidental and not treatment-related
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
- deviations from "zero values" obtained
- findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only
- observations were considered to have been incidental
- no test substance-related deviations to the control animals were noted for male and female animals
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- there were significantly increases in absolute and relative organ weights in some test groups, results given in table 5 and table 6, respectively
- significant weight increases of the kidneys in males (relative: 0.70%) and females (absolute/ relative: 1.77 g/ 0.78%) of the highest test group, above the historical control range (males, relative: 0.544-0.654%; females, absolute: 1.388-1.628 g; relative: 0.652-0.746%)
- no histopathological correlate was noted, these changes were considered treatment-related but not adverse
- weak significant absolute weight increase of the kidneys in females of test group 2 above the historical range (1.69 g)
- no significant relative weight deviations were noted, the absolute weight increase was regarded as possibly treatment-related and not adverse

- significant mean absolute liver weight increase in females of test group 3 (5.87 g) was beyond the historical control range (4.883-5.577 g)
- relative weight was not statistically significant changed and no histopathological correlate was observed, this change was considered to be possibly treatment-related but not adverse

- significant decrease of the heart in males of the highest test group (1.01 g) within the historical control values (0.905-1.134 g)
- no histopathological correlate observed, regarded as incidental and not treatment related

- incidentally significant spleen weight increase in males, not related to treatment
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- findings occurred individually and were considered to be incidental or spontaneous in origin and without any relation to treatment
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- findings occurred either individually or were biologically equally distributed over control and treatment groups
- findings considered to be incidental or spontaneous in origin and without any relation to treatment

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: there were no adverse signs of toxicity even at a dose level of 1000 mg/kg bw/d

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 5: Absolute organ weights

 

Male animals

Female animals

Test group (mg/kg bw/d)

1

(100)

2

(300)

3

(1000)

1

(100)

2

(300)

3

(1000)

Kidneys

 

 

 

+5.04%

+11.80%*

+17.43%**

Liver

 

 

 

+5.26%

+1.84%

+12.16%**

Heart

-0.93%

-2.60%

-5.86%*

 

 

 

Spleen

+14.38%*

+11.64%

+5.99%

 

 

 

* : p ≤ 0.05, **: p ≤ 0.01

Table 6: Relative organ weights

 

Male animals

Female animals

Test group (mg/kg bw/d)

1

(100)

2

(300)

3

(1000)

1

(100)

2

(300)

3

(1000)

Kidneys

+3.54%

-0.06%

+12.61%**

+5.54%

+9.87%

+13.68%*

* : p ≤ 0.05, **: p ≤ 0.01

Applicant's summary and conclusion

Conclusions:
The test substance did not reveal adverse signs of toxicity even at a dose level of 1000 mg/kg bw/d by gavage to male and female Wistar rats over a period of 3 months .
Thus, the no observed adverse effect level (NOAEL) was set to 1000 mg/kg bw/d for male and female Wistar rats.
Executive summary:

The test substance was administered orally by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 100 (test group 1), 300 (test group 2) and 1000 mg/kg body weight/day (mg/kg bw/d; test group 3) over a period of 3 months according to OECD 408.

Deionized water containing 5mg/100mL Cremophor EL served as vehicle, control animals were dosed daily with the vehicle only.

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Ophthalmological examinations were performed before the beginning and at the end of the administration period. A functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out at the end of the administration period. Clinico-chemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period, all animals were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations.

The various analyses confirmed the stability of the test-substance preparations for a period of 7 days at room temperature and the correctness of the prepared concentrations.

There were no treatment-related, adverse effects observed after the examination of clinical parameters, clinical pathology and pathology in any of the three dose groups.

The findings let to the conclusion that the oral administration of the test item by gavage to male and female Wistar rats over a period of 3 months did not reveal adverse signs of toxicity even at a dose level of 1000 mg/kg bw/d.

Thus, the no observed adverse effect level (NOAEL) was set to 1000 mg/kg bw/d for male and female Wistar rats.