2-Butenedioic acid (Z)-, ester with 1,2-propanediol, compd. with 2-(dibutylamino)ethanol

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2016 to May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1998

Deviations:

yes

Remarks:

but without any influence on the 90 day study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BYK Chemie LS-15-109A, LS-15-109B, LS-15-109C
- Expiration date of the lot/batch: 1-Nov-2018
- Purity test date: UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable

Species:

rat

Strain:

other: Han Wistar (RccHan; WIST)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Ltd
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 41 - 47 days
- Weight at study initiation: males 128-178 g, females 107-153 g
- Fasting period before study: no
- Housing: 5 animals per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days

DETAILS OF FOOD AND WATER QUALITY: Teklad 2014C diet; potable water from public supply

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amounts of test material were weighed. Approx. 50% of the final volume of vehicle was added and magnetically stirred until the test item was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 60, 100, 140 mg/ml
- Amount of vehicle (if gavage): 5 ml / kg bw
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of dose formulations were analysed by GC with flame ionisation detector. Mean concentrations were within +/- 6% of the nominal concentrations.

Duration of treatment / exposure:

90 days plus 28 days without treatment for recovery animals

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 animals per sex per dose, plus 5 animals per sex per dose in the control and high dose groups for the recovery period

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels of 0, 300, 500 and 700 mg/kg/day were selected with reference to an acute study, a 7-day dose range finder and an OECD 422 study. In the acute study, mortality was observed at 2000 mg/kg (2/3 animals), there was no mortality at 300 mg/kg. It was therefore considered that the LD50, in female rats was between 300 and 2000 mg/kg. In the 7-day repeat dose range finding study at 800 mg/kg/day, decreased activity, hunched posture, tremors, crouching, recumbency and tonic convulsions were observed in both sexes. Body weight loss was noted (Day 0-3) in both sexes (approximately 9%), with a decrease in food consumption. There were minor changes in haematological parameters; decreased reticulocyte counts, lower red blood cell distribution width and decreased mean platelet volume. Minor changes were seen in clinical chemistry parameters: high glucose concentration, low creatinine concentration and high plasma calcium. In males there were high thyroid and kidney weights. There were no macroscopic findings. In the OECD 422 study, dose levels of 0, 30, 100 and 300 mg/kg/day were assessed. In that study, there were no significant clinical signs or changes in body weight gain, food consumption or clinical pathology investigations. In addition, there was no significant organ weight, macroscopic or histopathological changes. The NOAEL was considered to be 300 mg/kg/day.

It was, therefore, considered that 800 mg/kg/day exceeded the maximum tolerated dose (MTD), so 700 mg/kg/day was considered appropriate for the high dose group in this study. To examine the possible dose response and no-adverse-effect a low dose of 300 mg/kg/day and intermediate dose (approximate geometric mean) of 500 mg/kg/day was selected. Additional animals were included in the control and high dose groups for a recovery period without treatment.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: once per week starting 1 week before first adminstration

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and in week 12
- Dose groups that were examined: all animals (pre-treatment), animals of high dose group and control group (week 12)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13 and at the end of the recovery period
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: all animals
- Parameters listed in table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13 and at the end of the recovery period
- Animals fasted: Yes
- How many animals: all animals
- Parameters listed in table No.2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: week 13 and at the end of the recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters listed in table No.3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly arena observations
- Dose groups that were examined: all animals of all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other: week 12, all animals

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

Estrous cycles by vaginal smear: for 14 days during weeks 10 and 11 of treatment, all groups

Sperm analysis: after scheduled sacrifice of each male, sperm motility (all groups), morphology and homogenisation-resistant spermatid count (groups 1 and 4)

Statistics:

The following sequence of statistical tests was used for grip strength, motor activity, body weight, sperm analysis, organ weight and clinical pathology data: A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972). The test statistic compares the mean square, NMS, for the deviations of the observed means from the maximum likelihood means, calculated under a constraint of monotonicity with the usual error mean square, EMS. The null hypothesis is that the true means are monotonically ordered. The test statistic is F1 = NMS/EMS which can be compared with standard tables of the F distribution with 1 and error degrees of freedom. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead. Where there were only two groups, comparisons were made using t-tests.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. This test is designed to be used when the main test for comparison of the means is a non-parametric monotonic trend test, such as Shirley's test (Shirley 1977).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Signs associated with dosing including swaying, unsteady gait and tremor in up to two males receiving 700 mg/kg/day. These were predominately seen within 5 minutes of dosing, and were occasionally present 30 minutes to 1 hour after dosing. In addition, chin rubbing and salivation has been seen in all animals receiving 700 mg/kg/day from Day 7 and 5, respectively, and in occasional animals receiving 300 or 500 mg/kg/day.

Salivation was also observed during the weekly physical examination in all treated groups. In addition, there was a higher incidence of brown staining on the upper dorsal surface in females receiving 700 mg/kg/day.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain (Week 0-13) was statistically significantly low, compared to the Controls, in females receiving 500 or 700 mg/kg/day (83 or 90% of control, respectively) and in all treated male groups (86, 93 and 91% of control for 300, 500 or 700 mg/kg/day, respectively) during treatment. However, in both sexes, this lacked dose-relationship.

Body weight of previously treated females indicated minor weight gain during the 4-week recovery period, with Control females at stasis during this period. In males, compared with Controls, there was significant weight gain indicating full recovery.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was slightly low in females receiving 500 or 700 mg/kg/day (88% of control), reflecting the trend seen with body weight.
Food consumption was considered unaffected by treatment in males and females given 300 mg/kg/day.
Food consumption during recovery was essentially similar to treatment.

Food efficiency:

not specified

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The haematological investigations in Week 13 indicated, when compared with the Controls, high red cell distribution width and slightly high reticulocyte counts in males receiving 700 mg/kg/day with the latter still apparent after 4 weeks of recovery, shorter activated partial thromboplastin time in males and females, in a dose-related manner, attaining statistical significant in both sexes at 700 mg/kg/day, although individual values for females were within the concurrent control range. The shorter activated partial thromboplastin time was still apparent after 4 weeks of recovery, attaining statistical significance in males. In treated females, when compared with controls, lymphocyte and monocytes counts were generally high, with an associated increase in the overall white blood cell count.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The biochemical examination of the blood plasma indicated, when compared to the controls, slightly high alanine amino-transferase activities in males receiving 500 or 700 mg/kg/day. In females, alanine amino-transferase activity was statistically significantly, slightly high, but there was no dose-relationship. Urea concentration was also slightly low and cholesterol concentration was high in treated females attaining statistical significance at 500 or 700 mg/kg/day, although the majority of individual cholesterol values were within the concurrent control range. Potassium concentration was high in females receiving 700 mg/kg/day.
Following a 4 week recovery period animals previously treated with BYK-LP X 20954 values were similar to those of the Control animals, indicating recovery for previously affected parameters.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The urinalysis investigation in Week 13 indicated, when compared with the controls, an increase of urinary volume in males receiving 500 or 700 mg/kg/day, and all treated females (although this lacked dose-relationship) and a marginal increase in total protein and increased total glucose, attaining statistical significance, in males receiving 700 mg/kg/day and treated females, however, this also lacked a dose-relationship in the females.
All other differences from controls were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation.
Following a 4 week recovery period animals previously treated with BYK-LP X 20954 values were similar to those of the Control animals.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Sensory reactivity responses and grip strength were unaffected by treatment.
Motor activity scores (low and high beam scores) showed considerable variability at 500 or 700 mg/kg/day in both sexes, and to a lesser extent in females at 300 mg/kg/day, this finding was considered an unclear relationship to treatment although it was not considered adverse.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The analysis of organ weights after 13 weeks of treatment indicated, when compared to the Controls, a slight increase in body weight adjusted liver and an increase in body weight adjusted kidney weights in males and females given 500 or 700 mg/kg/day, attaining statistical significance in the majority of these groups. In females, uterus and cervix weights were slightly low, when compared with controls, in a dose-related manner, although this did not attain statistical significance.
The analysis of organ weights after 4 weeks of recovery indicated in females, ovary and uterus and cervix weights were slightly low when compared with Controls, however, in the absence of any macroscopic pathology it was not considered toxicologically significant.
Full recovery, was apparent of all other previously affected organs, including body weight adjusted liver and body weight adjusted kidney weights in males and females.

see "overall remarks, attachments" : Table 13 Organ weights

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment with BYK-LP X 20954 were seen in the spleen and kidneys of males at all dose levels.
see "overall remarks, attachments" : Histopathology

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

All other findings were considered incidental and unrelated to treatment, attributed to normal biological variation.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 700 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: NOAEL refers to the highest dose level administered to the animals.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Sperm analysis

No adverse effects on sperm motility or concentration were observed following 13 weeks treatment with BYK-LP X 20954 at doses up to 700 mg/kg/day. Testicular spermatid numbers at 700 mg/kg/day appeared slightly higher than the Control values; this was statistically significant but was considered not to be toxicologically significant.

A lower percentage normal sperm was apparent following BYK-LP X 20954 treatment at 700 mg/kg/day when compared with Controls. A concomitant increase in abnormal sperm was observed, the majority of which were decapitate. These changes were statistically significant.

Following a 4 week recovery period, males previously treated with BYK-LP X 20954 showed sperm motility, concentration and morphology values similar to those of the Control animals.

Estrous cycles

There was no effect of treatment on estrous cycles.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

700 mg/kg bw/day

Study duration:

subchronic

Species:

rat

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

The NOAEL of the sub-chronic oral toxicity study was derived at the highest dose level, i.e. 700 mg/kg bw/day. This result does not necessitate any classification regarding repeated exposure according to REGULATION (EC) 1272/2008.