Registration Dossier

Administrative data

Description of key information

NOAEL systemic (OECD 422) = 400 mg/kg bw/day (RTC, 2013)

LOAEL local effects caused by gavage-related reflux of the substance into the respiratory tract (OECD 408) = 50 mg/kg day (BASF, 2020)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

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

OECD 422 (RTC, 2013):

The toxic effects on rats after repeated oral dosing with (Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation of the offspring were investigated. The study was conducted according to OECD 422 guideline and GLP (RTC, 2013).

The vehicle was corn oil. All doses were administered at a constant volume of 4 mL/kg body weight. The dose levels were 0, 40, 125 and 400 mg/kg bw/day.

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 31 or 32 days.

Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum.

The following investigations were performed: body weight, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology and clinical chemistry), litter data, macroscopic observations, organ weights and histopathological examination.

Mortality and fate of females

 

Two cases of premature death occurred during the study. One male of the control group was found dead on Day 2 of treatment but the cause of death could not be determined. One female of the high dose group was found dead on Day 3 of treatment and the cause of death was considered as a consequence of mis-dosing.

Two females were found not pregnant: one in the control and one in the mid-dose group.

In addition, one high dose female was sacrificed for total litter loss on Day 2 post partum.

The number of females with live pups on Day 4 post partum was: 9 in each of the control and mid-dose groups, 10 in the low dose group and 8 in the high dose group.

 

Clinical signsand clinical observations (Functional Observation Battery Tests)

 

Salivation and decreased muscle tone were the main clinical sign observed in all high dose males throughout the study and in all high dose females during post coitum and post partum periods.Decreased muscle tone was considered not to be of toxicological relevance, since no effects were seen in the functional observation battery tests, especially in the grip strength and foot splay tests.

Neurotoxicity assessment (removal of animals from the home cage and open arena)

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.

 

Body weight and body weight gain

 

Males of the high dose showed a slight body weight loss during the first week of the study, and body weights remained slightly decreased compared to the other groups throughout the study. However, these changes were mainly attributable to one male, which was probably mis-dosed. No differences of toxicological significance in body weights were recorded in animals of both sexes compared to the control group, throughout the study.


Food consumption

No effects on food consumption were observed in either males or females.

 

Motor activity and sensory reactivity to stimuli

No relevant differences were noted in all parameters investigated between control and treated groups.

 

Haematology

 

No changes of toxicological significance were observed in haematology parameters and coagulation tests.

 

Clinical chemistry

Due to the absence of related histopathological changes, the recorded alterations were considered of no toxicological relevance.

 

Oestrous cycle, reproductive parameters, pairing combination and mating performance

 

No relevant differences were observed for these parameters between treated and control groups.

All females mated both in the control and treated groups.

Oestrous cycle, copulatory index and fertility index did not show intergroup differences.

 

Implantation, pre-implantation loss data, pre-birth loss data and gestation length of females

 

No relevant differences were observed for these parameters between treated and control groups.

 

Litter data at birth, on Day 1 and on Day 4 post partum of females and sex ratio of pups

 

No significant differences were observed on litter data parameters and sex ratios at birth, on Day 1 and Day 4 post partum.

 

Clinical signs of pups

Clinical signs noted in pups throughout the study were considered unrelated to treatment.

 

Necropsy findings in decedent pups and in pups sacrificed on Day 4 post partum

 

Necropsy findings in decedent pups and in pups sacrificed on Day 4 post partum did not reveal any treatment-related effect.

 

Terminal body weight and organ weights

 

Terminal body weight was slightly decreased in high dose males, while no differences were observed in females.

Differences noted in absolute and relative organ weights between treated and control animals were not accompanied by histological findings. Therefore, they were considered of no toxicological importance.

 

Macroscopic observations

Unscheduled death

Two cases of premature death occurred during the study. One control male for which the cause of death could not be determined and one high dose female for which the cause of death is considered as related to mis-dosing of the test item.

 

Terminal kill

No treatment-related changes were noted.

Microscopic observations

 

No treatment-related findings were noted.

In a single male rat from the high dose, the bronchi and the alveoli in the centriacinar areas contained mucus-like fluid. This change was associated with goblet-cell metaplasia of the epithelium lining the bronchi, peribronchial fibrosis, presence of polymorphonuclear cells and histiocytes within the mucus.

The lung changes are suggested to be related to mis-dosing of the test item.

 

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted.

 

Conclusions

 

On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for both systemic toxicity and reproduction/developmental toxicity was considered 400 mg/kg/day for males and females.

OECD 408 (BASF, 2020):

Methods

(Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 50 (test group 1), 150 (test group 2) and 500 mg/kg body weight/day (mg/kg bw/d;test group 3) over a period of 3 months.Corn oil served as vehicle. During the course of the administration period, food consumption was reduced, and body weight/body weight change values became lower in all animals of test group 3 (500 mg/kg bw/d). Therefore, the dose level was reduced to 300 mg/kg bw/d for male animals (from study day 30 onwards) and for female animals (from study day 56 onwards). The study was conducted according to OECD 408 guideline and GLP.

 

Observation

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 towards the end of the administration period.Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out towards 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.

 

Results

Analytics

The various analyses confirmed

·     the stability of the test-substance preparations for a period of 7 days at room temperature,

·     the homogeneous distribution of the test substance in the vehicle,

·     the correctness of the prepared concentrations.


Findings

Most findings listed below were attributed to local irritation in the respiratory tract/airways after gavage-related reflux:

 

Test group 3: 500/300 mg/kg bw/d

 

Clinical Examinations

·       Male animal No. 36 was sacrificed on study day 35 and all remaining male animals were sacrificed in a moribund condition on study day 36.

·       Female animal No. 78 was found dead on study day 77.

·        Hunched posture shortly after application was observed in 9 of 10 male as well as 8 of 10 female animals, i.e. starting in the first male and female animals on study day 15. Addtionally, semi-closed eyelids and respiration sounds were observed in all male and female animals at the same time. Poor general condition and labored respiration were observed occasionally in male and female animals.

·        Reduced food consumption and impaired body weight development were observed in all male animals from the second week of application onwards. The same was true for female animals towards the end of the application period.

 

Clinical Pathology (females only)

·       Increased total white blood cell (WBC), absolute and relative neutrophil as well as absolute lymphocyte and monocyte counts.

·       Decreased absolute reticulocyte and relative lymphocyte counts.

·       Increased inorganic phosphate, urea potassium and LDL-cholesterol values.

·       Decreased albumin levels.

 

Pathology (females only)

·        Decreased mean final body weights in female animals (not statistically significant).

·        Several findings occurred in the nasal cavity, i.e. erosion/ulceration in all, neutrophilic inflammation in 9, multifocal squamous cell metaplasia in 1 and multifocal regeneration of olfactory epithelium in 4 female animals.

·        Multifocal squamous cell metaplasia was observed in larynx of all females.

·        In the lungs, atelectasis was observed in one female and active chronic inflammation in two female animals.

 

 

Test group 2: 150 mg/kg bw/d

 

Clinical Examinations

·        Two male animals were found dead each on study days 15 and 34.

·        Reduced food consumption and impaired body weight development were observed in all male animals from the fourth week of application onwards.

 

Clinical Pathology

·       Increased inorganic phosphate levels in male animals.

·       Decreased glucose levels in male animals.

 

Pathology

·        Decreased mean final body weights in male (statistically significant) and female animals (not statistically significant).

·        In the nasal cavity, several findings occurred, i.e. erosion/ulceration in all male and female animals, neutrophilic inflammation in 7 male and 2 female animals, multifocal squamous cell metaplasia in 4 male animals and multifocal regeneration of olfactory epithelium in each 7 male and female animals.

·        In the larynx, multifocal squamous cell metaplasia was observed of 9 male and 8 female animals. In addition, erosion/ulceration occurred in a single male animal.

·        In the trachea, multifocal epithelial degeneration/regeneration was observed in 5 male animals.

·        In the lungs, atelectasis was observed in one female and active chronic inflammation in 5 males and 1 female animal.

 

 

Test group 1: 50 mg/kg bw/d

 

Clinical Examinations

·        Three male animals were found dead or sacrificed moribund each on study days 57, 68 and 83. One female animal was found dead on study day 78 and one female animal was sacrificed moribund on study day 63.

·        Impaired body weight development was observed in all male animals during the last 2 weeks of application.

 

Clinical Pathology

·        No treatment-related, adverse effects were observed.

 

Pathology

·        Decreased mean final body weights in male animals (statistically significant).

·        In the nasal cavity, several findings occurred, i.e. erosion/ulceration in 8 male and 9 female animals, neutrophilic inflammation in 2 male and 5 female animals, multifocal squamous cell metaplasia in 1 male animal and multifocal regeneration of olfactory epithelium in each 8 male and female animals.

·        In the larynx, multifocal squamous cell metaplasia was observed in 3 male and 2 female animals. In addition, erosion/ulceration occurred in 3 male animals and an individual female.

·        In the lungs, active chronic inflammation occurred in 4 male and 3 female animals. Multifocal neutrophilic inflammation was observed in one male and two female animals.

 


 

Conclusion

Under the conditions of this studythe oral administration of (Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid by gavage to male and female Wistar rats over a period of 3 months revealed adverse findings at a dose level of 50 mg/kg bw/d and higher. These adverse signs of toxicity were considered to be the consequence of severe local irritation after gavage-related reflux into the respiratory tract/airways. The irritation was considered to be a consequence of reflux of gastric contents (with low pH values) and the test substance itself, which is classified as skin and eye irritating (H315 and H319).

In the male and female animals, the same locations in the respiratory system were affected, i.e.nasal cavity, larynx, and lungs. In nearly all male and female decedents, yellow translucent material (interpreted as test substance) was seen in alveoli of the lungs.

Thus, the lowest observed adverse effect level (LOAEL) was set to 50mg/kg bw/d for male and female Wistar rats. A no observed adverse effect level (NOAEL) could neither be established for male nor for female Wistar rats and a meaningful assessment of potential target organ toxicity was not possible.

Justification for classification or non-classification

In the OECD 422 the NOAEL (No Observed Adverse Effect Level) for both systemic toxicity and reproduction/developmental toxicity was considered 400 mg/kg/day for males and females, the highest dose level tested. In the OECD 408 adverse signs of toxicity were observed even at the lowest dose level of 50 mg/kg bw/d. These adverse signs of toxicity were considered to be the consequence of severe local irritation related to gavage-related reflux of the test substance (classified as skin and eye irritating; H315 and H319) into the respiratory tract/airways.

A no observed adverse effect level (NOAEL) could neither be established for male nor for female Wistar rats and a meaningful assessment of potential target organ toxicity was not possible due to the severe local irritation and the mortalities caused by the substance reflux at all dose levels. These effects are not relevant for STOT RE classification as they are related to the gavaging procedure of the test material-vehicle formulation and are not expected to occure under real life exposure conditions in man. In addition no specific target organ toxicity was observed in the OECD 422. Thus, the test substance is not classified with regard to repeated dose toxicity according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP), respectively.