4-oxo-4-(p-tolyl)butyric acid adduct with 4-ethylmorpholine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12.07.2016 - 28.04.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

419-240-6

EC Name:

-

Cas Number:

171054-89-0

Molecular formula:

Hill formula: C28H37NO7 CAS formula: (C11H12O3)2.C6H13NO

IUPAC Name:

bis(4-(4-methylphenyl)-4-oxobutanoic acid); 4-ethylmorpholine

Test material form:

solid: particulate/powder

Details on test material:

light beige
- Density: 1,24 g/cm3
Batch no. 41505042

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Han (IGS)

Details on test animals or test system and environmental conditions:

test animals:
Source: Charles River, Sandhofer Weg 7, 97633 Sulzfeld, Germany
Age at acclimatisation Males: 11-12 weeks; females: 11-12 weeks
Weight at acclimatisation: 277 g – 324 g (males); 177 g – 210 g (females)
Health status: Specific pathogen free (SPF)
Pregnancy status females: Nulliparous, non-pregnant
Acclimatisation: 6 to 9 days (according to start of experimental cohort)

animal husbandry:
Room: SPF barrier (males & females in OP2)
Housing conditions: Clean conventional housing: airing with approx. 10 air changes per hour, room climate 22 ± 3°C, relative humidity at 30-70%, artificial lighting 12 h light/12 h dark
Hygiene status: Specified pathogen free (SPF) housing with permanent health monitoring of the barrier by sentinel animals (bedding sentinels) in accordance with FELASA recommendations1.
Caging: Groups of up to three male animals in open macrolon cages type 2000P, TechniPlast (size slightly larger than GV-SOLAS Type IV). Females, paired animals and single dams with their litters in open macrolon cages type III.
Bedding: Lignocel hygienic animal bedding (J. Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg), heat-treated
Nesting material: Narrow shavings, debarked aspen wood, width 2,5 mm, NBF E-11 (ABEDD LAB & VET Service GmbH), heat-treated
Cage enrichment: Wooden gnawing blocks, size medium 10x2x2 cm, debarked aspen wood, NGM E-022 (ABEDD LAB & VET Service GmbH), heat-treated
Diet: Maintenance diet for rats and mice, No. 1324 TPF, ad lib. Dams: Breeding diet for rats and mice No. 1314 TPF (Altromin Spezialfutter GmbH & Co. KG, 32791 Lage), ad lib.
Water: Sterilised community tap water, ad lib.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test item was administered at three graduated doses in an application volume of 5 ml per kg body weight. The individual dose volumes were calculated from the latest actual animal body weight data. 24 animals (12 males / 12 females) received pure corn oil as vehicle control via the same route.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In the quantitative re-analysis, the recovered test item content from the test item/corn oil preparations (suspensions & dilutions) was compared to the test item amount admixed to the corn oil vehicle. To assure valid results within the set acceptance limits (see rationale below), at least three replicates of test item-suspensions were re-analyzed per dose group and targeted date via High Performance Liquid Chromatography-UV (HPLC-UV; see phase study plan). As the test item-amount assigned for the low dose-preparation is still soluble in corn oil, two replicates per re-analysis were considered sufficient at this point. The concentration of the test item in the respective suspensions/solutions was determined once within pre-mating phase, at the beginning of the gestation phase and at the end of gestation/ beginning of lactation phase.

A quantitative re-analysis was performed by PI1 to compare the measured test item content in the test item/corn oil preparations (suspension and dilutions) with the preset test item amount admixed to the corn oil vehicle.
The recovery rate is defined as:
recovery rate = measured test item content (mean)/admixed amount of test item [%]

Recovery rates between 80% and 120% were set as acceptance limits (see rationale below).

Rationale for chosen acceptance limits:
In the validation identical replicates of each test item concentration such as 200 g/L, 50 g/L and 3 g/L, respectively, were prepared in corn oil and subsequently analyzed via HPLC-UV methodology (ID-Nr. 16041102G926; see appendix).
It was noted that especially the 200 g/L (high conc.) and 50 g/L (medium conc.) concentrations form dense suspensions in corn oil. The dense suspensions revealed in the re-analysis maximum variances in the recovery of 31% (for high conc.) or 17% (for med conc.) between the measured individual replicates.
The dose levels of 200 mg/kg (high dose); 50 mg/kg and 15 mg/kg were nominated as test item doses in this study and were administered to animals in respective test item concentrations of 40 g/L, 10 g/L and 3 g/L.
With respect to evaluated variances measured for dense test item suspensions and in regard to the re-analysis of high dose suspensions in the course of the study in-life phase, the acceptance limits for a successful re-analysis were set at 100 ± 20%.

Details on mating procedure:

During acclimatisation, pre-exposure and pre-mating, male rats were caged in groups of three animals per cage; females were housed individually. Throughout the mating period, animals were kept in pairs of one female and one male rat. The female was placed with the same male until pregnancy occured or two weeks had elapsed. In case a male died during the mating period or successful mating was not be detectable after 14 days, re-mating of females with proven males (sires) of the same group was considered. After the mating period, male rats were housed in groups of three originally formed during pre-mating phase; female rats were housed individually.

Duration of treatment / exposure:

Males were dosed daily for 43 days, including the day before the scheduled termination of the in-life phase. This included two weeks of dosing prior to mating and continued throughout the mating period until approximately two weeks post-mating. Females were dosed two weeks prior to mating, covering at least two complete oestrous cycles, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled termination of the in-life phase. Therefore the duration of the study following acclimatisation and pre-dosing oestrus-cycle evaluation depended on the female performance (14 days pre-mating, up to 14 days until mating, an average of 22 days of gestation, and a minimum of 13 days of lactation) and was at least 63 days (thereof 51 dosing days at minimum).

Frequency of treatment:

daily

Duration of test:

06.09.2016 - 28.12.2016

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

The following parameters were observed and documented during the in-life period:
1. Viability / mortality Twice daily
2. General clinical signs / behaviour Daily
3. Body weight At least once weekly (including once before beginning of application)
4. Group/Individual food consumption At least once weekly (including once before beginning of application)
5. Group/Individual water consumption At least once weekly (including once before beginning of application)
6. Individual collection of vaginal smears Daily (Beginning of pre-exposure phase until evidence of mating + on day of necropsy)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes [all per litter]

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Over the course of the study, animals of the test item-treated groups displayed occasional wiping of their mouth with the cage bedding and increased salivation shortly after test item administration. With the exception of four high dose group females, only males and sires showed this particular behavioral sign, predominately at the high dose. The role of the test item on these effects could not be fully excluded. These observed effects were considered not adverse as they were mild in severity and had a minor impact on the experimental animals during the course of the study.
One female (E390/0) showed an increased respiration immediately after test item administration and died shortly thereafter (see chapter 4.2.2).
Other findings in the female dose groups (bleeding nose) were regarded as individual observations within the first two dosing weeks (pre-mating phase) and were considered related to animal stress caused by the gavage procedure.
One female (E409/0) showed signs of severe discomfort post birth (brushy fur; cold, pale skin at snout and mouth) most likely due to complications at birth. The animal recovered fully within the following four days.
In summary, during the course of the study some minor behavioral signs of animal discomfort, predominately in males and females of the high dose group, were noted. In this regard a minor test item-related effect can be considered.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

On premating day 1, one female of the high dose group (E390/0) was found dead shortly after test item administration. Necropsy did not identify a plausible cause for the death. Based on the animal’s behavior immediately after the last dose administration [animal showed increased frequency of respiration], it is assumed that the gavage error was most likely the cause of death. While there were no necropsy findings to support this conclusion, this effect was not considered test item-related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Over the course of the study no statistical differences in the mean body weights of female test groups and the respective control group were detected. Correspondingly, the mean body weight gain was comparable between all female groups either treated with the test item or the vehicle control item. All values were within normal range for female rats of this strain and age. Occasional differences especially towards the end of the gestation phase were assumed to be within the physiological variation caused by the pregnancy status of the animals.
For male animals, no statistical differences in mean body weights were observed between the test groups and the respective control group over the course of the study. Correspondingly, the mean body weight gain was comparable between all male animal groups either treated with the test item or the vehicle control item. All values were within normal range for rats of this strain and age.
In summary, no significant differences regarding absolute body weight and body weight gain were detected between the test group (neither female nor male) and the respective vehicle control group, hence, no test item related-effect was observed.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In the monitored pre- and post-mating phases (until day 20), female test groups showed as a tendency a slightly lower food consumption (between -17,9% and -2,2%), when compared to the vehicle control group. A dose-related effect was not observed in this regard. From day d0 post partum until day of termination some variability (±16,2% at maximum) in food consumption was noted. After all, the food consumption of all experimental groups was within a normal range for animals of this sex and age during the course of the study.
Differences in monitored food consumption between in the life phases of late gestation (d20-0pp) and lactation (d0-d13 post partum) and in-life phases monitored before (premating, mating and early gestation) were most likely caused by the specific physiological conditions of the females during late gestation and subsequent lactation. Hence, a test item-related effect on food consumption could not be detected during the course of the study.
The food consumption of all male test groups and vehicle control groups was within normal range for male rats of this strain and age. Besides minor varying, no test item-related tendency could be observed in regard to the food consumption of the male test groups when compared to the respective control group.
In summary, the monitored time intervals did not reveal any changes of toxicological relevance in regard to food consumption between the animal test groups and their respective vehicle control groups throughout male and female in-life phases. Some variances were observed for the female animal groups, which could be associated with the specific animal condition during phases of late pregnancy and subsequent lactation.

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

The female dose groups showed a moderate variability in all monitored post-mating intervals, when compared to the respective control group. However, with the exception of the time period d20-0pp (-9,2% to +19,6%) all other time intervals revealed
differences within the range of ±15%, and therefore within the limits of variation frequently observed for female rats of this strain and age. No dose-related tendency in water consumption was observed for any experimental dose group during the course of the study.
The differences in water consumption between monitored intervals of the late gestation (d20-0pp)/lactation period (d0-d13pp) and the previously monitored intervals (pre-mating, mating and early gestation) were most likely caused by the specific physiological conditions of the animals during late pregnancy and lactation. No test item-related effect was noted during the course of the study.
The water consumption of all male test groups and vehicle control groups was within normal range for male rats of this strain and age. Although slightly varying (±10%), no test item-related tendency could be observed in regard to observed water consumption of the male test groups when compared to the respective control group.
In summary, male and female dose groups did not reveal any test item-related differences in water consumption, when compared to the respective vehicle control group. Some variances were observed for the female animal groups, which could be associated with the specific animal condition during phases of late pregnancy and subsequent lactation.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The statistical analysis of animal T4-hormone data by “Dunnett’s post hoc t-test after Oneway ANOVA” revealed a significant difference in total T4-hormone content in blood plasma in the Sire medium dose group but not in the Sire high- and low dose groups, when compared to the respective vehicle control group. A further box plot analysis identified all measured individual medium dose T4-values as within the limits (50%-percentile to lower whisker) measured for the vehicle control group. Therefore the statistical results were considered to reflect an unequal distribution (variation) of individual values between medium dose group and vehicle control group. For this reason differences detected between the Sires medium dose group and vehicle control group were regarded as a statistical but not as a test item-related effect. Furthermore, in regard to effects caused by endocrine disruptor activity, there were no positive test item-related correlates identified - neither in macroscopic (gross necropsy) nor microscopic (histopathology) male organ analysis between the test groups and the respective vehicle control group. In summary, no test item-related changes in total T4-hormone content in blood plasma could be detected in the experimental animals.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Animal behavioral signs of wiping of nose and mouth through cage bedding or salivation after dose application were observed in the test item-treated male and female animal groups, but the findings were predominantly observed at the high dose.
Although the observed behavioral signs were probably related to the test item, they were considered to be of minor toxicological importance. No adverse clinical signs were observed during the course of the study.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The male dose groups revealed no statistical significant differences in organ weights between the test item groups and the vehicle control group.
For one female of the medium dose group the student’s t-test analysis indicated a difference in the relative weight of the uterus associated with the cervix. No significant differences were identified in the subsequent statistical analysis by “Dunnett’s
post hoc t-test after One-way ANOVA” and “Dunn’s post hoc test after Krusker-Wallis*”.
*: was performed in addition due to a significance detected in the Bartlet’s test indicating an inequality of residue distribution between the test groups.
The microscopic analysis of the uterus in association with the cervix revealed no test itemrelated findings.
Neither the male nor the female test groups did show any significant weight changes of the thyroid when compared to the vehicle control group after t-test analysis. Hence, a test itemrelated effect on the endocrine organ weight was not detected.
In summary, all macroscopic observations or measured organ weight differences evaluated in adult animals or in d13 pp litters were regarded as not related to the administration of the test item.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathology findings:
According to guideline OECD 421, tissue samples from a selective panel of reproductive organs and macroscopic lesions as well as vaginal smears prepared at necropsy from dams and sires of the experimental high dose group and respective vehicle control group were correlated and conclusively analyzed by the histopathologist.
Based on macroscopic and microscopic observations, it was concluded that the test item 4-Oxo-4-p-tolylbutyric acid, adduct with 4-ethylmorpholine (Halox 570) did not have an adverse effect on male or female reproduction, up to the highest tested dose level of 200 mg/kg.
From the combination of results, obtained from clinical biochemistry (T4-hormone analysis), from macroscopic and from microscopic analysis, it was concluded that no test item-related effect relevant for the animal reproduction could be detected. Therefore the additional analysis of blood plasma samples and histological reproductive organ samples from experimental dams and associated d4 pp pups were regarded as obsolete.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic observations
There were no test item-related microscopic observations in testis, epididymis and ovary.
A sperm granuloma was found in one animal treated with 200 mg/kg (animal no. E378/0; correlating to a macroscopic nodule at the epididymal caput). This is a common spontaneous finding that may develop as a consequence of congenital ductual aplasia (Creasy et al. 2012). Due to the low incidence, it was not considered to be related to the test item. A sperm granuloma was also found in a control animal (E389/2).
All examined ovaries showed numerous large eosinophilic corpora lutea, typical of post parturition. Most animals had undergone a post parturition estrus cycle and were in the diestrus phase. Both in the vehicle control and in the test item group, a few animals were in metestrus. There was general concordance between the estrus cycle determined on ovaries and the estrus cycle determined on vaginal smears.
Germ cells in the testis were evaluated by identifying tubules with respect to their stage in the spermatogenic cycle. Neither germ cell depletion nor spermatid retention were found. The population of Sertoli cells was normal and interstitial Leydig cells did neither show atrophy or hyperplasia. Epididymides did not show desquamated germ cells, cell debris or reduced number of sperm.
All other recorded microscopic observations in single animals from various groups represent common spontaneous findings in Wistar rats of this age.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Macroscopic observations
There were no macroscopic observations attributed to the test item.
A nodule at the caput of the epididymis was found in one animal treated with 200 mg/kg of the test item. This nodule correlated to sperm granuloma. Such granulomas are common spontaneous findings in male rats. It is considered to represent a spontaneous finding not related to the test item.
All recorded macroscopic observations in single animals from various groups represent common spontaneous findings in Wistar rats of this age. White nodules described on the kidneys from various male animals did not show a microscopical correlate and most likely represent portions of perirenal fat.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

not examined

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical biochemistry

clinical signs

dead fetuses

effects on pregnancy duration

food consumption and compound intake

gross pathology

histopathology: neoplastic

maternal abnormalities

mortality

necropsy findings

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

water consumption and compound intake

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

not examined

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

changes in litter size and weights

external malformations

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

A daily oral administration of the test item 4-Oxo-4-p-tolylbutyric acid, adduct with 4-ethylmorpholine (Halox 570) to male and female Wistar rats at dose levels of 200 mg, 50 mg and 15 mg/kg body weight over a time period of 43 days for males and 52-58 days for females resulted in some minor animal behavioral changes observed in male and female animals predominately of the high dose groups. The findings were neither considered adverse nor toxic. As the findings could not be associated with a substantial impact on the animal’s health, the NOAEL regarding the subchronic toxicity was set to 200 mg/kg body weight. Furthermore, no pathological evidence for toxic effects on the reproduction performance of female and male rats was found. Regarding the overall time period of gestation/lactation and after birth until end of in-life phase no evidence for a toxic effect on the pup development could be detected. The NOAEL regarding reproduction and development of 4-Oxo-4-p-tolylbutyric acid, adduct with 4-ethylmorpholine (Halox 570) under these study conditions was estimated to be 200 mg/kg body weight for the female and male animals.

Executive summary:

In the present study toxic effects of the test item 4-Oxo-4-p-tolylbutyric acid, adduct with 4- ethylmorpholine (Halox 570) at a maximum dose of 200 mg/kg body weight on the development and reproduction of Wistar rats after oral administration were under examination.

General clinical and behavioral signs:

Animal behavioral signs of wiping of nose and mouth through cage bedding or salivation after dose application were observed in the test item-treated male and female animal groups, but the findings were predominantly observed at the high dose.

Although the observed behavioral signs were probably related to the test item, they were considered to be of minor toxicological importance. No adverse clinical signs were observed during the course of the study.

Body weight, food and water consumption:

Regarding the body weight and the body weight gain, no significant differences were observed between all test item-treated animal groups (male and female) and their respective vehicle control groups. Occasional differences observed for the female animals at specific time intervals of the in life phase could be correlated with the animal’s pregnancy and lactation status and were therefore strongly assumed to be of natural origin.

The monitoring of food and water consumption revealed no test item-related difference between all female and male experimental dose groups and the respective vehicle control groups. The observed variations showed no dose-dependency and were within limits frequently observed for animals of this sex and age and could therefore not be associated with a test item-related adverse effect.

The differences generally observed in female food and water consumption between late pregnancy/ lactation phases and the earlier in life-phases were related to the specific physiological condition of the animals and therefore considered not adverse or toxicological relevant.

T4-hormone analysis from blood plasma:

The clinical biochemistry of blood plasma from experimental animal groups (males and d13 pp pups) did not reveal a test item-related significant difference in absolute T4-hormone content between test groups and respective control groups.

Necropsy:

The necropsy of sires, dams and older pups (day 15-17pp) did not reveal any findings, which could be regarded as adverse or toxicologic relevant for animal reproduction or development. None of the findings could be associated with the administration of the test item.

Histology:

The histopathological analysis (on macroscopic and microscopic level) revealed no evidence for a test item-related effect on the reproductive organs from experimental adult animals.

Reproduction and Development:

Regarding reproduction and development (achievement and duration of pregnancy, offspring losses, survival rate of pups and development of pups) no difference was observed for all test item-treated animals, when compared to the vehicle control animals. There was no evidence for any test item-related effect.