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REACH

reaction products of 1,4-butanediol with 1,3-chloro-2,3-epoxypropane, esters with prop-2-enoic acid

EC number: 701-230-0 | CAS number: -

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

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Administrative data

Key value for chemical safety assessment

Toxic effect type:: dose-dependent

Effects on fertility

Description of key information

A Combined 28 -day repeated dose and reproduction / developmental screening (2013) is available on butane-1,4-diylbis(oxy-2-hydroxypropane-3,1-diyl) bisacrylate.

Based on this study, the no observed adverse effect level (NOAEL) for general systemic toxicity was 50 mg/kg bw/d for male animals and 150 mg/kg bw/d for female animals.

The NOAEL for reproductive performance, fertility and developmental toxicity was set to 300 mg/kg bw/d in male and female Wistar rats.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted March 1996

Deviations:

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, adopted July 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

Species:

rat

Strain:

Wistar

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, male and female animals were derived from different litters
- Age at study initiation: 10-11 wks;
- Weight at study initiation: Males: 321-352 g; Females: 184-214 g
- Fasting period before study: no
- Housing: individually in Makrolon type M III cages (floor area app. 800cm²)
- Diet (e.g. ad libitum): Kliba maintenance diet mouse-rat "GLP" ad lib.
- Water (e.g. ad libitum): ad lib.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light):12h/12h

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The suspension of Laromer 8765 in corn oil was prepared daily by weighing the appropriate amount of test substance and adding corn oil up to the desired volume, which was subsequently released with a magnetic stirrer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility of the test substance
- Amount of vehicle (if gavage): 4 ml/kg b.w.

Details on mating procedure:

- M/F ratio per cage: 1:1 (except for 3 males of the high dose group, which were mated at 1:2 ratios due to the premature deaths of 3 males in this group)
- Length of cohabitation: overnight, for a maximum of 2 weeks or until mating was detected
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after unsuccessful attempt: no
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The various analyses confirmed
- the stability of the test substance in corn oil at room temperature over a period of 7 days
- the homogeneous distribution of the test substance in corn oil
- the correctness of the prepared concentrations of the test-substance preparations in corn oil was determined at start, end and once throughout the study (see report 01Y0446/01X037)

Duration of treatment / exposure:

males: 35 days
females: 55 days

Frequency of treatment:

daily (no administration to animal being in labor)

Dose / conc.:

300 mg/kg bw/day

Remarks:

500mg/kg (up to day 5), 300mg/kg (from day 6 onward)

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on range finding study (10C0446/01S014): no pathological changes in 3 female Wistar rats treated with 500 mg/kg for three weeks, animals moribund after 1 week exposure to 1000mg/kg due to ulcerations in the fore and glandular stomach.

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yess
- Time schedule: before, within 2h and 2-5h after treatment, additional check in the afternoon on workdays
- Cage side observations: morbidity, pertinent behavioral changes, signs of overt toxicity, littering and lactation bevhavior

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
- Observations: abnormal behavior during “handling”, 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: males and females without evidence of mating: weekly; females with evidence of mating: weekly prior to mating, on GD0, 7, 14, 20, on parturition days 0 and 4

FOOD CONSUMPTION:
- Food consumption for each animal determined was determined weekly, except during mating

WATER CONSUMPTION:
- Time schedule for examinations: monitored daily by visual inspection

OTHER: FOB, urinalysis, hematology / clinical chemistry (for details see entry in the repeated dose section)

Sperm parameters (parental animals):

Parameters examined in male parental animals:
testis weight, epididymis weight, stages of spermatogenesis in the male gonads

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, clinical symptoms, weight gain

GROSS EXAMINATION OF DEAD PUPS:
yes, externally and organs were assessed macroscopically

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on day 36
- Maternal animals: All surviving animals on day 56
- 6 males and 3 females died prematurely and were necropsied as soon as possible after their death and assessed by gross pathology.

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
The following tissues were weighed
- in all animals: epididymides, testes
- in 5 males and females of each group: adrenal glands, brain, heart, kidneys, liver, spleen, thymus
The uteri of all cohabited female parental animals were examined for the presence and number of implantation sites. The uteri of apparently non-pregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations.
The following tissues were examined histotechnically in 5 animals per sex of the control and high dose group, unless noted otherwise, and in all animals that died prematurely. Reproductive organs were examined in all animals of the control and high dose group:
adrenal glands, gross lesions (all affected animals in all dosage groups), bone marrow (femur), brain, cecum, cervix, coagulation glands, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (auxillary and mesenteric), ovaries, oviducts, prostate gland, peyer's patches, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, stomach (all animals from all dose groups), testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina

Postmortem examinations (offspring):

NECROPSY
- All surviving pubs were subjected to postmortem examinations: external examination and macroscopic examination of organs. Animals with notabel findings or abnormalitites were evaluated on a case-by-case basis.

Reproductive indices:

For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:
Male mating index (%) = number of males with confirmed mating (vaginal sperm detected in females) / number of males placed with females x100
Male fertility index (%) = number of males proving their fertility (implants in utero) / number of males placed with females x100

For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:
Female mating index (%) = number of females mated (vaginal sperm detected) / number of females placed with males x100
Female fertility index (%) = number of females pregnant (implants in utero) / number of females mated (vaginal sperm or implants in utero) x100
Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant x100

Offspring viability indices:

Live birth index (%) = number of liveborn pups at birth / total number of pups born x100
Viability index (%) = number of live pups on day 4 after birth / number of live pups on the day of birth x100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Respiration sounds were observed in 3 males of the mid dose, in all males of the high dose group, and in 1 high dose female on study days 7 (only males) and 21. Piloerection was observed in each 2 high dose males and females and one mid dose male and female on different days throughout the study.
Immediately after dosing, animals from all treatment groups showed salivation due to bad taste or local irritation of the upper digestive tract. This finding was assessed as non-adverse.

Mortality:

mortality observed, treatment-related

Description (incidence):

6 males and 2 females were found dead on different days (days 4, 5, 6, 16, 34, 34 for the males, and days 51 and 53 for the females, 1 female was sacrificed in moribund condition on day 55.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For the surviving high dose and mid dose male animals mean body weight was significantly lower on post-mating day 7 (-8.6% and -4.3%, respectively). In addition, body weight change values of high dose male animals were significantly lower during the pre-mating days 0-7 (-85.3%) and 0-13 (-42.1%). These deviations to the control values were regarded to be treatment-related. No changes in body weight parameters were determined for female animals in all groups.
No test substance-related, adverse findings were noted with regard to food consumption during the entire study period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see the section 7.5.1 for details.

Food efficiency:

not specified

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

effects observed, non-treatment-related

Description (incidence and severity):

see the section 7.5.1 for details.

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see the section 7.5.1 for details.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

see the section 7.5.1 for details.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

see the section 7.5.1 for details.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

see the section 7.5.1 for details.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related erosions/ ulcers were observed in many treated animals often correlating to the macroscopic finding. In many animals, an inflammatory or hyperplastic reaction to an erosion or ulcus was observed, characterized by varying degrees of edema in the submucosa, inflammatory cell
infiltrates of varying composition (neutrophils, lymphocytes, plasma cells) and hyperplasia of the squamous epithelium of the forestomach forming both elongating rete ridges as well as papillary projections into the lumen of the stomach. The hyperplasia was accompanied by hyperkeratosis.

Foci on the pancreatic lymph node observed macroscopically in some male animals of mid dose group and one high dose male correlated histologically to cyst and/ or lympho- reticular hyperplasia.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

In the mating pairs that failed to produce offspring, no lesions in the reproductive tract were detected.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Female mating index as 90% for control and high dose females, and 100% for all other groups.The mean duration until sperm was detected varied between 2.1 and 3.6 days. One rat each of the low and mid dose group did not become pregnant, leading to a female fertility index of 90% in these groups and 100% in the control and high dose group. One female each in the control and low dose group and 2 females in the high dose group did not deliver pubs. Thus the gestation index were 88.9% (control), 77.8% (low dose), 100% (mid dose), and 77.8% (high dose).
Male mating index was 100% for all test group and 90% for the control group, male fertility index ranged between 90% (control, low and mid dose group) and 100% (high dose group), taking into account that only 7 high dose males could be used as mating partners due to permature deaths.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: lower terminal body weight and premature deaths, most likely due to the irritant properties of the test substance

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Premature deaths, most likely due to the irritant properties of the test substance.

Dose descriptor:

NOAEL

Remarks:

reproductive performance, fertility and developmental toxicity

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

6 males and one female pub in the mid dose and one male pub of the high dose group showed reduced nutritional condition. As no dose-response relationship occurred these findings were assessed as being incidental and not related to treatment.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 100% for control group and high dose animals, 94% for low dose animals, and 97.3% in the mid dose. Single stillborn pups were seen in low and mid dose females, one low dose female had only stillborn pups. As no dose-response relationship was observed the finding was assessed as being incidental and not related to treatment.
All liveborn pubs in all dose groups survived until PND 4 (scheduled sacrifice).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

5 male and 3 female runts were seen in the mid dose group, which is within the biological variation of this rat strain.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test substance-related effects were observed.

Histopathological findings:

not examined

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

Effect level:

>= 300 mg/kg bw/day

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

Reproductive effects observed:

Conclusions:

The NOAEL for reproductive performance, fertility and developmental toxicity was set to 300 mg/kg bw/d in male and female Wistar rats.

Executive summary:

Test substance was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 50 mg/kg bw/d (test group 1), 150 mg/kg bw/d (test group 2) and 500 mg/kg bw/d (test group 3). Because of clinical findings and the premature death of 3 male animals in test group 3, the dose was reduced from 500 to 300 mg/kg bw/d from study day 6 onwards.

The objective of the study was to detect possible effects of the test substance on the integrity and performance of male and female reproductive systems including gonadal function, mating behavior, conception, gestation and parturition. Furthermore, it was intended to obtain information about the general toxicological profile including target organs and the no observed adverse effect level (NOAEL) after repeated oral administration. The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice.

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 was detected in the vaginal smear.

Note: As a result of the premature death in 3 male animals of test group 3 (500 and 300 mg/kg bw/d) 3 male animal were mated in a 1:2 ratio, i.e. male animal No. 31 was mated with female animal Nos. 131 and 132, male No. 34 with female Nos. 133 and 134 and male animal No. 39 with Nos. 139 and 140. As a result of the premature death of male animal No. 39, female animal No. 140 was mated with male animal No. 36 from study day 16 onwards.

A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4. Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating and mating. During gestation and lactation period, F0

females were weighed on gestation days (GD) 0, 7, 14 and 20, after the day of parturition (postnatal day [PND] 0) and on PND 4.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia with CO2 and examined

macroscopically for external and visceral findings.

Clinicochemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period. Towards the end of the administration period a functional observational battery was performed and motor activity was measured in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) 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.

study, labored respiration was observed in 2 male animals and 1 female animal on few study days and gasping respiration was observed in 1 male animal on day 6 of the postmating phase and in 1 female animal on day 20 of the gestation phase.

days 0-7 (-85.3%) and 0-13 (-42.1%). Increased absolute and relative neutrophil counts in males and dcreased relative lymphocyte counts in males were observed. At pathology : mean terminal body weight was lower in male animals, i.e. -8%. Erosion/ ulcer and/or related squamous hyperplasia or inflammation/edema in the forestomach were observed in 9 of 10 male and all female animals. Erosions or ulcers in the glandular stomach were observed in 3 of 10 males.

In the intermediate group, respiration sounds were observed in 3 male and 2 female animals on several days during the study, labored respiration was observed in 3 male animals on few study days during the mating phase. Piloerection were in 1 male animal on day 8 and 9 of the mating phase and in 1 female animal on day 26 of the gestation phase. Mean body weight was significantly lower in male animals on mating day 8 (-4.3%). No test substance-related adverse findings were observed in clinical pathology. Erosion/ ulcer and/or related squamous hyperplasia or inflammation/edema in the forestomach were observed in 9 of 10 male and in 7 of 10 female animals.

Erosions or ulcers in the glandular stomach were observed in 1 of 10 female animals.

In th low dose group, only erosion/ ulcer and/or related squamous hyperplasia or inflammation/edema in the forestomach were observed in 6 of 10 male and 1 of 10 female animals.

No test substance-related, adverse findings were observed on reproduction performance, on clinical Examinations/Gross Findings of pups at 50, 150 and 300/500 mg/kg/day.

stomach at a dose level of 50 mg/kg bw/d and above. This finding was related to the irritating potential of the test substance.

Signs of systemic toxicity were observed at a dose level of 150 mg/kg bw/d (impaired body weight data in male animals) and above (premature deaths in both sexes), which were assumed to be related to the test substance administration.

Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 50 mg/kg bw/d for male animals and 150 mg/kg bw/d for female animals. With regard to effects in the stomach the NOAEL for local effects could not be set.

The NOAEL for reproductive performance, fertility and developmental toxicity was set to 300 mg/kg bw/d in male and female Wistar rats.

Effect on fertility: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 300 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: The study is considered as reliable with a klimisch score of 1

Effect on fertility: via inhalation route

Endpoint conclusion:: no study available

Effect on fertility: via dermal route

Endpoint conclusion:: no study available

Additional information

Combined 28 -day repeated dose and reproduction / developmental screening (2013)

In the high dose group, six male animals and 2 female animals were found dead during the study; in addition, 1 female animal was sacrificed moribund on gestation day 27. Respiration sounds were observed in 5 male and 3 female on several days during the study, labored respiration was observed in 2 male animals and 1 female animal on few study days and gasping respiration was observed in 1 male animal on day 6 of the postmating phase and in 1 female animal on day 20 of the gestation phase.

Piloerection were observed in 2 male animals and 2 female animals on several days during the study. Encrusted left eye was observed in 1 male animal on post-mating day 6. Blood in bedding and inability to deliver was seen in 1 female animal during the gestation phase from day 25 to day 27, light-brown vaginal discharge was observed on gestation day 27. Mean body weight was lower in male animals on post-mating day 7 (-8.6%). Mean body weight change values were decreased in male animals during the premating days 0-7 (-85.3%) and 0-13 (-42.1%). Increased absolute and relative neutrophil counts in males and dcreased relative lymphocyte counts in males were observed. At pathology : mean terminal body weight was lower in male animals, i.e. -8%. Erosion/ ulcer and/or related squamous hyperplasia or inflammation/edema in the forestomach were observed in 9 of 10 male and all female animals. Erosions or ulcers in the glandular stomach were observed in 3 of 10 males.

Erosions or ulcers in the glandular stomach were observed in 1 of 10 female animals.

In th low dose group, only erosion/ ulcer and/or related squamous hyperplasia or inflammation/edema in the forestomach were observed in 6 of 10 male and 1 of 10 female animals.

No test substance-related, adverse findings were observed on reproduction performance, on clinical Examinations/Gross Findings of pups at 50, 150 and 300/500 mg/kg/day.

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration by gavage of test substance to male and female Wistar rats revealed local pathological findings in the stomach at a dose level of 50 mg/kg bw/d and above. This finding was related to the irritating potential of the test substance.

The NOAEL for reproductive performance, fertility and developmental toxicity was set to 300 mg/kg bw/d in male and female Wistar rats.

Effects on developmental toxicity

Description of key information

A developmental toxicity study is available in rats on 1,4-butanediylbis[oxy(2-hydroxy-3,1-propanediyl)] diacrylate.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Sep 2020 - 02 Nov 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

Species:

rat

Strain:

Sprague-Dawley

Remarks:

RjHan:SD (Rats CD®), Indemn of Organism Pathogen Specific Han (IOPS Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, le Genest-Saint-Isle, France
- Age at study initiation: 10-11 weeks old
- Weight at study initiation: Mean body weight of 296 g (range: 243 g to 349 g)
- Females: Sexually mature and primigravid
- Fasting period before study: No
- Housing: Individually in polycarbonate cages (Tecniplast 2154, 940 cm²) with stainless steel lids and containing autoclaved sawdust (Le comptoir des sciures, Meyzieu, France). Individual housing was chosen as group housing can adversely affect gestation. Each cage contained nylabone and cocoon for the environmental enrichment of the animals.
- Diet: SSNIFF rat/mouse pelleted maintenance diet, batch No. 57266070 (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Tap water (filtered with a 0.22 µm filter), ad libitum
- Contaminant analyses: The batches of diet and sawdust were analyzed by the Suppliers for composition and contaminant levels. Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants were present in the diet, drinking water or sawdust at levels which could be expected to interfere with, or prejudice, the outcome of the study.
- Acclimation period: 4 or 5 days before the beginning of the treatment period (arrival of the females on Days 1 or 2 p.c.).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C (set to maintain)
- Humidity (%): 50 ± 20% (set to maintain)
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 01 Oct 2020 - 02 Nov 2020

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

FORMULATION PROCEDURE
- Type of test item formulation (visual observation): Emulsion in the vehicle
- Preparation procedure: According to Study No. 48225 VAS (Magaud, 2020) (homogeneity testing) describing the preparation procedure for a range of concentrations covering the lowest and highest used in this study (i.e. the dose formulations prepared at 1 mg/mL and 100 mg/mL in corn oil were found to be homogenous).
- Frequency of preparation: Test item dose formulations: on the days of treatment. Control dose formulation: according to the vehicle.
- Storage and delivery conditions (control and test item dose formulations): At room temperature and protected from light. Test item dose formulations were used within 4 hours after the end of their preparation.

ADMINISTRATION
The dose formulations were administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, at approximately the same time. The quantity of the dose formulation administered to each animal was adjusted according to the most recently recorded body weight. A constant dosage volume of 5 mL/kg bw/day was used. The dose formulations were maintained under delivery conditions (at room temperature and protected from light) throughout the administration procedure. The control dose formulation was stirred just before administration and the test item dose formulations were vigorously stirred for at least 30 minutes before administration. The formulations were maintained under continuous magnetic stirring throughout the administration procedure (vigorously for test item dose formulations).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analytical technique: High Performance Liquid Chromatography with tandem Mass Spectrometry detection (HPLC/MS MS)
- Principle and validation of the method: Analytical method provided by the Sponsor and validated at Charles River Laboratories Evreux (Study No. 48225 VAS (Magaud 2020)) prior to dose formulation analysis. Checked parameters, acceptance criteria and obtained results are detailed in the validation report.
- Determination of test item concentrations in dose formulations: In the first and last weeks of treatment of the study (two controls). A sample was taken from control and test item dose formulations and analyzed using the validated method.
- Acceptance criterion: Measured concentration = nominal concentration ± 15%.

Details on mating procedure:

The females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as Day 0 p.c.

Duration of treatment / exposure:

From Day 6 to Day 20 p.c., inclusive

Frequency of treatment:

Once daily

Duration of test:

Day from arrival of animals to the test facility (Day 1 or 2 p.c.) till scheduled euthanasia and hysterectomy (Day 21 p.c.)

Dose / conc.:

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

Remarks:

Low-dose group

Dose / conc.:

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

Remarks:

Mid-dose group

Dose / conc.:

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

Remarks:

High-dose group

No. of animals per sex per dose:

24 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

ALLOCATION TO GROUPS
During the acclimation period, the animals were allocated to the groups, according to a computerized stratification procedure, based on body weight recorded on Day 2 p.c., so that the average body weight of each group was similar.

ADMINISTRATION ROUTE RATIONALE
The oral route was selected since it is a route of administration which is recommended by the Regulatory Authorities for this type of study.

DOSE SELECTION RATIONALE
The dose levels were selected by the Sponsor based on the results of previous studies as described below.
In an OECD 422 study (BASF SE, project number 85R0446/01S019), the test item, 1,4 butanediylbis[oxy(2-hydroxy-3,1-propanediyl)] diacrylate, was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 50, 150 and 500 mg/kg bw/day. Because of clinical findings and the premature death of 3 males at 500 mg/kg bw/day, the dose was reduced at 300 mg/kg bw/day from Day 6 of the study. Females were treated for 2 weeks before mating, throughout mating and gestation, and until Day 13 post partum inclusive.
At 500/300 mg/kg bw/day, female parental toxicity consisted of premature deaths for 3 females (one female was found dead on Day 23 p.c., one female was prematurely euthanized for delivery difficulties on Day 27 p.c. and one female died on Day 12 post-partum during the lactation period) and clinical signs (i.e. transient respiration sounds, laboured respiration and/or gasping respiration in 5/10 females at the end of the gestation period along with hypersalivation and/or piloerection). There were no relevant changes in body weight or body weight gain when compared to controls. Erosion/ulcer and/or related squamous hyperplasia or inflammation/edema was observed in the forestomach in 10/10 females. At 150 and 50 mg/kg bw/day, there were also erosion/ulcer and/or related squamous hyperplasia or inflammation/edema in the forestomach of some animals due to the irritation potential of the test item.
In absence of severe effects on the mean body weight change and considering that respiratory difficulties and mortality occurred after approximately 5 weeks of treatment at 500/300 mg/kg bw/day, 500 mg/kg bw/day was selected as the high-dose level in the preliminary study for effects on embryo-fetal development.
In the preliminary study for effects on embryo-fetal development by the oral route in rats (Study No. 48227 RSR (Papineau, 2020)), three groups of eight pregnant female Sprague-Dawley rats received the test item, 1,4-butanediylbis[oxy(2-hydroxy-3,1-propanediyl)] diacrylate, daily by oral administration (gavage) throughout gestation (Day 6 to Day 20 p.c.) at dose levels of 100, 300 or 500 mg/kg bw/day. An additional group of eight pregnant females received the vehicle control, corn oil, under the same experimental conditions. The dosing volume was 5 mL/kg bw/day.
At 500 mg/kg bw/day, there were:
• ptyalism in all females and transient loud breathing in 1/8 females,
• high net body weight change,
• slight to marked adverse erosion/ulcer, slight to marked inflammation/edema and/or moderate to marked squamous cell hyperplasia in the forestomach that correlated with white discoloration in 7/8 animals. Non-adverse erosion (grade 1) was also noted in the stomach of one animal,
• no effects on pre- and post-implantation losses, number of viable fetuses, fetal body weight and pre-natal development.
At 300 mg/kg bw/day, there were:
• ptyalism in all females,
• slight to moderate adverse erosion/ulcer in 2/8 animals in the forestomach,
• minimal to moderate inflammation/edema and minimal to moderate squamous cell hyperplasia in the forestomach that correlated with white discoloration in 3/8 animals.
At 100 mg/kg bw/day, ptyalism was noted in 1/8 females.
Therefore, in the absence of clinical signs and bodyweight change, 500 mg/kg bw/day was selected as the high-dose level even if ulcers in the forestomach, considered as adverse, were observed. The low dose and mid dose were selected using a ratio representing approximately a 2- or 3 fold interval (i.e. 100 and 300 mg/kg bw/day). Mortality is expected at doses higher than 500 mg/kg bw/day due to the severity grade of the ulcer (i.e. grade 4, corresponding to marked ulcer) observed at this dose level in 3/8 females.

Maternal examinations:

MORBIDITY AND MORTALITY: Yes
- Time schedule: Each animal, once a day before and after the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

CLINICAL SIGNS: Yes
- Time schedule: From arrival, each animal was observed once a day as part of routine examinations.
From the start of the treatment period, each animal was observed at least twice a day
- Observations made: clinical signs (including evidence of abortion)

BODY WEIGHT: Yes
- Time schedule: each animal, on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c.

FOOD CONSUMPTION: Yes
- Time intervals: Days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: By inhalation of carbon dioxide gas followed by cervical dislocation.
- Macroscopic post-mortem examinations: principal thoracic and abdominal organs (prematurely and scheduled euthanized females).
- Preservation of tissues: Macroscopic lesions observed in females were sampled and kept preserved in 10% buffered formalin. Since remarkable macroscopic lesions were observed in test item treated group animals, the corresponding tissues of five control animals were sampled and preserved. For all study animals, thyroids with parathyroids and stomach with forestomach were preserved in 10% buffered formalin.

MICROSCOPIC EXAMINATIONS AND ORGAN WEIGHTS: Yes
- Dose groups examined: Control and low-, mid- and high-dose, all animals
- Preservation of tissue and microscopic examination: Macroscopic lesions, stomach with fore stomach, thyroids with parathyroids
- Preservation: 10% buffered formalin
- Organ weights (after fixation): Thyroids with parathyroids
- Preparation of histological slides: All tissues required for microscopic examination were trimmed based on the RITA guidelines, when applicable (Ruehl-Fehlert et. al., 2003; Kittel et. al., 2004; Morawietz et. al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately 4 microns and stained with hematoxylin eosin.

Ovaries and uterine content:

EXAMINATION OF OVARIES AND UTERINE CONTENT: Yes, after termination (21 p.c.)
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number and distribution of dead and live fetuses: Yes
- Number and distribution of early and late resorptions: Yes
- Number and distribution of uterine scars: Yes
- Number and distribution of implantation sites: Yes
A gross evaluation of placentas was also undertaken. The placenta weight was recorded for each fetus and the fetal weight/placental weight ratio was calculated.

CLASSIFICATION USED TO RECORD
- Uterine scar: uterine implantation without implant
- Early resorption: evidence of implant without recognizable embryo
- Late resorption: dead embryo or fetus with degenerative changes
- Dead fetus: dead fetus with no degenerative changes

Blood sampling:

BLOOD SAMPLING
- Time schedule: At termination on Day 21 p.c., between 7.30 and 9.30 am, from all females
- Anaesthesia: Yes, isoflurane
- Blood sampling: From the orbital sinus
- Processing: Blood was centrifuged within 2 hours after sampling. The plasma was transferred into 2 separate tubes and frozen at -80°C (no pooling of blood/plasma).

THYROID HORMONES ANALYSIS
- Hormone levels determined: T3, T4 and TSH
- Method: LC MS/MS method and those of Thyroid Stimulating Hormone (TSH) by Luminex MAP® technology.

Fetal examinations:

POST-MORTEM EXAMINATIONS
- Sacrifice: With sodium pentobarbital administered into the buccal cavity.
- Body weight: Yes, of each fetus.
- Sex: Yes, of each fetus. Sex was determined at the time of hysterectomy by measurement of the anogenital distance (AGD) and was confirmed by internal examination of sexual organs at detailed dissection of the soft tissues or at evisceration. The individual AGDs was normalized to the cube root of the body weight of each fetus.
- External examinations: Yes, each fetus, which included the observation of all visible structures, surfaces and orifices.
- Soft tissue examinations: Yes. Approximately half of the fetuses in each litter were subjected to a detailed dissection of the soft tissues, which included the observation of all the organs and structures of the neck, thorax and abdomen and indication of incomplete testicular descent/cryptorchidism in males fetuses. The fetuses were then eviscerated and were fixed in Harrison’s fluid for examination of the structures of the head.
- Skeletal examinations: Yes. The remaining fetuses per litter were eviscerated and then fixed with ethyl alcohol.
A detailed examination of the skeleton (bones + cartilages) was performed after staining with alizarin red S and alcian blue. This examination included the observation of all the bones and cartilage structures of the skull, spine, rib cage, pelvis and limbs.

CLASSIFICATION USED TO RECORD
- Malformation: A permanent structural change that is likely to adversely affect the survival or health.
- Variation: A change that occurs within the normal population under investigation and is unlikely to adversely affect the survival or health (this might include a delay in growth or morphogenesis that otherwise followed a normal pattern of development).

Statistics:

Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).
The sequences followed for performing the statistical analysis of organ weight data and hormones, fetal Body weight/placental weight ratio and anogenital distance are pictured in section 6.11.2 and 6.11.3 of the original study report, respectively.

Indices:

Data are recorded and calculated using computerized validated software. Data of non-pregnant females are not included in group mean calculations (body weight, body weight change, food consumption and litter data).

The following parameters were calculated:
For each pregnant female:
• body weight change for different intervals,
• net body weight (presented as carcass weight): Body weight on Day 21 p.c. - gravid uterine weight
• net body weight change: Body weight on Day 21 p.c. - body weight on Day 6 p.c. - gravid uterine weight
For each litter:
• total number of resorptions: Sum of uterine scars + early resorptions + late resorptions
• total number of dead fetuses: Sum of dead fetuses
• % of dead fetuses per litter: (Total number of dead fetuses / Number of implantation sites) x 100
• total number of live fetuses: Sum live male + live female fetuses
• % of live fetuses per litter: (Total number of live fetuses / Number of implantation sites) x 100
• % of pre-implantation loss: ((Number of corpora lutea - Number of implantation sites) / Number of corpora lutea) x 100
• % of post-implantation loss: ((Number of implantation sites - Number of live fetuses) / Number of implantation sites) x100
• average fetal body weight: Sum of individual fetal weights / Number of live fetuses
• AGD/cube root of fetal body weight (ratio calculated with Excel): AGD / ³vBody weight

Parameters calculated for each group are given in section "Any other information on materials and methods incl. tables".

Historical control data:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item-related clinical signs were noted with a dose-related incidence and consisted of ptyalism observed in females from 100 mg/kg bw/day, piloerection, loud breathing and/or abdominal breathing observed in females from 300 mg/kg bw/day. Ptyalism observed for 1 to 14 days was considered to be non-adverse as it is commonly observed when oily dose formulations are administered by gavage. Piloerection observed for 1 to 3 days was considered to be non-adverse as it was observed only episodically and was no longer observed after 1 or 3 days. Loud and/or abdominal breathing considered to be adverse effects of the test item. The duration of the effects increased with the dose level. The other clinical signs (i.e. chromorhinorrhea and/or scab) were considered to be unrelated to the test item, as they were not dose-related and they were transient and/or reported in isolated animals.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

At 500 mg/kg bw/day, on Day 10 p.c., one pregnant female was found dead. Prior to death, ptyalism was observed from Day 8 p.c. and no relevant changes were observed in the body weight gain and food consumption. The cause of death of this female was not evident. There were no macroscopic findings at necropsy. Microscopic findings (minimal erosion/ulcer, squamous cell hyperplasia and inflammation/edema in the forestomach) in this animal were considered to be test item-related but did not explain the death given their low magnitude.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effects were noted on mean body weights or mean body weight changes at any dose level.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no effects on mean food consumption at any dose level.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

The mean thyroid hormone levels (T3, T4 and TSH) were considered to be unaffected by the test item as statistically significant changes are not dose-related and of low magnitude.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no relevant changes in the weights of thyroid glands.
There were no effects on mean gravid uterus weight, carcass weight or mean net body weight change at any dose level. At 500 mg/kg bw/day and when compared with controls, there was a slight decrease in mean net body weight change (-14%), mainly due to the contribution of 2 females. As this finding was not statistically significant, was not associated with a lower mean body weight gain and was of slight magnitude, a test item treatment relationship was considered to be unlikely.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item related macroscopic observations were observed in the forestomach and stomach. In the forestomach, many white masses, correlating microscopically with squamous cell hyperplasia and inflammation/edema, were observed in all females treated from 300 mg/kg bw/day. In the stomach, brown discoloration was noted in 3/23 surviving females at 500 mg/kg bw/day. This change correlated (at microscopic examination) with erosion in two of these females.
The other macroscopic findings in treated females (enlarged placenta in one female at 100 mg/kg bw/day and white discoloration in the adrenals in one female at 300 mg/kg bw/day) had no histologic correlates and were considered to be incidental.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No test item-related microscopic changes were observed in the thyroid glands. In the stomach, minimal mucosal hypertrophy (pyloric region) was present with dose-related incidence from 300 mg/kg bw/day and was associated at 500 mg/kg bw/day with minimal (five females) or slight (one female) erosion.
Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the rat.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

There were no abortions reported.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no test item-related effects on pre- and post-implantation loss.

Total litter losses by resorption:

not specified

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

At 100 mg/kg bw/day and when compared with controls, there was a slight increase in the mean number of early resorptions (2.5-fold with p<0.01 for number of early resorptions). Although these differences were statistically significant, they were not considered to be test item related as they occurred at the low dose level only.

Dead fetuses:

no effects observed

Description (incidence and severity):

There was no test item-related effect on the number or percentage of dead fetuses.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

At hysterectomy on Day 21 p.c., 24/24, 23/24, 24/24 and 23/24 females were pregnant with live fetuses in the groups treated at 0, 100, 300 and 500 mg/kg bw/day, respectively.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

clinical signs

histopathology: neoplastic

histopathology: non-neoplastic

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no effects on mean fetal body weight, placenta weight or fetal body weight/placental weight ratio at any dose level.

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Mean percentage of male fetuses (%) were 49.5, 47.3, 50.3, and 45.2 for the control, low-, mid-, and high-dose group, respectively. There were no effects on sex ratio at any dose level.

Changes in litter size and weights:

not examined

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

There were no effects on anogenital distance at any dose level.

Changes in postnatal survival:

not examined

External malformations:

effects observed, treatment-related

Description (incidence and severity):

VARIATIONS
There were no test item-related increases in the incidence of external variations. At 300 mg/kg bw/day, one litter had a fetus with paw hyperflexion and at 100 mg/kg bw/day, one litter had a fetus with curled tail. As these findings were isolated, not associated with additional findings at external examination and not observed at higher doses, they were not considered to be test item related.

MALFORMATIONS
At 500 mg/kg bw/day, one litter had three fetuses with cleft palate that correlated with absent palate (at visceral or skeletal observation). These malformations were observed with higher fetal and litter incidences than those noted in controls and Historical Control Data.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

CARTILAGE
The presence of cartilages in the above-mentioned structures is interpreted with the following incidences of variations.

VARIATIONS
The distribution of significant differences in fetal and litter incidences of skeletal variations is presented in Table 14 of section "Any other information on results incl. tables".
When compared with controls, there was a tendency towards increased incidences of litters and fetuses with incomplete ossification of the centrum of thoracic vertebra(e) from 300 mg/kg bw/day, reaching statistical significance at 500 mg/kg bw/day (mean fetal incidence of 7%: p<0.05). However the incidences were below in the historical control mean (14.7%).
At 500 mg/kg bw/day and when compared with controls, there were higher fetal and/or litter incidences of fetuses with incomplete ossification of frontal, nasal and parietal bones, incomplete ossification of the centrum of cervical vertebra(e), incomplete ossification of 1st to 4th sternebra(e), incomplete ossification of 2nd to 5th metacarpal bone and presence of 27 pre sacral vertebra(e).
The corresponding incidences of presence of cartilages for cervical and thoracic vertebra, sternebra and metacarpal confirmed the presence of the partially or non ossified structure. The above differences of incomplete ossification were noted at 300 and/or 500 mg/kg bw/day, they were considered to be test item-related although some of them were within the range of the Historical Control Data but non-adverse as cartilages were present.
Other skeletal variations [i.e. dumbbell ossification of the centrum of thoracic vertebra(e), incomplete ossification of arch or unossification of the centrum of caudal vertebra(e), unossification of 5th sternebra, ossification point on 14th thoracic vertebra(e) and/or short supernumerary 14th rib] were isolated, not dose-related and/or were observed with a similar or minimally different incidence in control and/or test item treated groups. Therefore, they were considered to be unrelated to the test item treatment. The relevance of the findings is presented in the section “Details on embryotoxic / teratogenic effects”.
When compared with controls and/or the HCD, the global litter and fetal incidences of skeletal variations were below in all test item-treated groups.

MALFORMATIONS
At 500 mg/kg bw/day, five malformed fetuses were seen in three litters for which a test item relationship could not be ruled out as litter and/or fetal incidences were above those recorded in controls and Historical Control Data:
• One litter had a multi-malformed fetus with split frontal bone, fused cervical vertebra(e) arch(es) and fused rib(s) along with a low fetal weight (3.93 g),
• One litter had a three multi-malformed fetus:
o One fetus with split frontal bone, supernumerary lumbar vertebra(e) and fused rib(s) along with a low fetal weight (4.24 g),
o One fetus with split frontal bone and fused rib(s) along with a low fetal weight (4.32 g),
o One fetus with absent palate that correlated with external malformation (cleft palate) (5.11 g).
• one litter had a malformed fetus with supernumerary lumbar vertebra(e) with a low fetal weight (4.78 g),
At 100 mg/kg bw/day, one litter had a multi-malformed fetus with cervical vertebra(e) rib, fused cervical vertebra(e) arch(es), split frontal bone, fused centrum of thoracic vertebra(e), fused sternebra(e), fused ribs and low fetal weight (3.13 g). As these skeletal findings were recorded with lower fetus and litter incidences, and/or with no dose-relationship and/or as they were of isolated occurrence, a test item-related effect was considered to be excluded. These malformations were observed with higher fetal and litter incidences than those noted in controls and Historical Control Data. The relevance of the findings is presented in the section “Details on embryotoxic / teratogenic effects”.
No skeletal malformations were observed at 0 or 300 mg/kg bw/day.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

VARIATIONS
There were no test item-related increases in the incidence of visceral variations. At 500 mg/kg bw/day and when compared with controls, there were high fetal incidence of fetuses with dilated renal pelvis and/or dilated ureter. As these differences were not statistically significant and/or were within the range of the Historical Control Data, these findings were considered to be randomly distributed within the groups without any biological significance. Other soft tissue variations (i.e. enlarged spleen, absent innominate artery, and/or fluid-filled abdomen) were isolated, not dose-related and/or were observed with a similar or minimally different incidence in control and/or test item treated groups. Therefore, they were considered to be unrelated to the test item treatment.

MALFORMATIONS
At 500 mg/kg bw/day, one litter had two fetuses with cleft palate that correlated with cleft palate noted at external observation. These malformations were observed with higher fetal and litter incidences than those noted in controls and Historical Control Data.
No soft tissue malformations were observed at 0, 100 or 300 mg/kg bw/day.

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

At fetal examination and when compared with controls and/or Historical Control Data, a tendency towards increased incidences of skeletal variations indicative of delayed ossification was observed from 300 mg/kg bw/day [i.e. incomplete ossification of the centrum of thoracic vertebra(e)] and at 500 mg/kg bw/day [i.e. incomplete ossification of frontal, nasal and parietal bones, incomplete ossification of the centrum of cervical vertebra(e), incomplete ossification of 1st to 4th sternebra(e), incomplete ossification of 2nd to 5th metacarpal bone and presence of 27 pre sacral vertebra(e)].
Increases in the incidences of malformations at 500 mg/kg bw/day involved mainly the head bones and the axial skeleton and were observed in seven fetuses from three different litters:
• cleft palate (external and/or visceral) in three fetuses from one litter corresponding to absent palate at skeletal examination;
• split frontal bone and fused ribs in three fetuses from two litters associated with low fetus weights (< 4.32 g);
• supernumerary lumbar vertebra(e) in two fetuses from two litters associated with low fetus weights (<4.78 g);
• fused cervical vertebra(e) arch(es) in one fetus associated with a low fetus weight (3.93g).
Overall, at 500 mg/kg bw/day, the incidences of malformations observed at external, visceral and skeletal examination were above the highest range of the controls and Historical Control Data. Therefore, a test item-relationship could not be ruled out.
These malformations were observed in presence of marked irritation of the forestomach in the females at this dose level (moderate to marked hyperplasia and/or inflammation/edema and/or slight to marked erosion/ulcer). Indeed at this dose level, 14/23 and 6/23 females showed a moderate or marked erosion/ulcer of forestomach respectively.

Key result

Dose descriptor:

NOAEL

Remarks:

Embryo-fetal toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

skeletal: rib

skeletal: vertebra

other: cleft palate

Description (incidence and severity):

At 500 mg/kg bw/day, one litter had three fetuses with cleft palate that correlated with absent palate (at visceral or skeletal observation). At 500 mg/kg bw/day, five malformed fetuses were seen in three litters for which a test item relationship could not be ruled out as litter and/or fetal incidences were above those recorded in controls and Historical Control Data: One litter had a multi-malformed fetus with split frontal bone, fused cervical vertebra(e) arch(es) and fused rib(s) along with a low fetal weight (3.93 g); One litter had a three multi-malformed fetus: One fetus with split frontal bone, supernumerary lumbar vertebra(e) and fused rib(s) along with a low fetal weight (4.24 g), One fetus with split frontal bone and fused rib(s) along with a low fetal weight (4.32 g), One fetus with absent palate that correlated with external malformation (cleft palate) (5.11 g); One litter had a malformed fetus with supernumerary lumbar vertebra(e) with a low fetal weight (4.78 g).

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

CHEMICAL ANALYSIS OF THE DOSE FORMULATION

The test item concentrations in the administered dose formulations analyzed in the first and last weeks of the treatment period remained within an acceptable range of variations (+3.2% to +10.1%) when compared with the nominal values (± 15% of the nominal concentrations). No test item was detected in the control dose formulation.

CLINICAL OBSERVATIONS

Test item-related clinical signs (surviving females) are summarized in Table 1.

Table 1: Clinical Observations

Dose level (mg/kg bw/day)	0	100	300	500
Piloerection	0	0	1	2
Loud breathing	0	0	4	8
Abdominal breathing	0	0	2	4
Ptyalism	0	1	23	23
Number of affected animals	0/24	1/24	23/24	23/23

BODY WEIGHTS AND BODY WEIGHT GAINS

Mean body weights and mean body weight changes recorded in control and test item-treated animals are summarized in Table 2.

Table 2: Mean Body Weights/Mean Body Weight Changes (g)

Dose level (mg/kg bw/day)	0	100	300	500
Body weight (g)
Day 6 p.c.	298	296	295	294
	-	(-1)	(-1)	(-1)
Day 9 p.c.	314	313	309	308
	-	(0)	(-2)	(-2)
Day 12 p.c.	338	338	335	330
	-	(0)	(-1)	(-2)
Day 15 p.c.	361	362	357	355
	-	(0)	(-1)	(-2)
Day 18 p.c.	411	409	402	399
	-	(0)	(-2)	(-3)
Day 21 p.c.	464	460	460	451
	-	(-1)	(-1)	(-3)
Days 6 - 9 p.c.	+17	+17	+13	+14
Days 9 - 12 p.c.	+24	+25	+26	+22
Days 12 - 15 p.c.	+23	+24	+22	+24
Days 15 - 18 p.c.	+49	+47	+45	+45
Days 18 - 21 p.c.	+54	+51	+58	+52
Days 6 - 21 p.c.	+167	+164	+165	+157
	-	(-2)	(-1)	(-6)

p.c.: post-coitum; -: not applicable; ( ): in brackets, percentage difference vs. controls; No statistical significance.

FOOD CONSUMPTION

Mean food consumption values recorded during the study are summarized in Table 3.

Table 3: Mean Food Consumption (g/animal/day)

Dose level (mg/kg bw/day)	0	100	300	500
. Days 6 - 9 p.c.	25	24	23	24
. Days 9 - 12 p.c.	26	27	26	25
. Days 12 - 15 p.c.	28	28	27	26
. Days 15 - 18 p.c.	30	30	30	30
. Days 18 - 21 p.c.	32	32	33	31

p.c.: post-coitum; No statistical significance.

NECROPSY FINDINGS

Test item-related macroscopic observations are presented in Table 4.

Table 4: Group Incidences of Test Item-Related Macroscopic Observations at Main Necropsy

	Female Dose level (mg/kg bw/day)
	0	100	300	500
No. animals	24	24	24	23
Forestomach
White mass	-	-	24	23
Brown discoloration	-	-	-	3

-: not observed.

MICROSCOPIC EXAMINATION

Test item-related microscopic observations are presented in Table 5.

Table 5: Group Incidences (with Severities) of Test Item-Related Microscopic Observations at Main Necropsy

	Female Dose level (mg/kg bw/day)
	0	100	300	500
No. animals	24	24	24	23
Forestomach
Hyperplasia; squamous cell
Minimal (grade 1)	-	3	-	-
Slight (grade 2)	-	-	3	-
Moderate (grade 3)	-	-	16	6
Marked (grade 4)	-	-	5	17
Erosion/Ulcer
Slight (grade 2)	-	-	8	3
Moderate (grade 3)	-	-	12	14
Marked (grade 4)	-	-	-	6
Inflammation/Edema
Minimal (grade 1)	-	-	2	-
Slight (grade 2)	-	-	4	-
Moderate (grade 3)	-	-	17	15
Marked (grade 4)	-	-	-	8
Stomach
Hypertrophy; mucosa; pylorus
Minimal (grade 1)	-	-	6	19
Erosion
Minimal (grade 1)	-	-	-	5
Slight (grade 2)	-	-	-	1

-: not observed.

THYROID HORMONES

Mean thyroid hormone levels (T3, T4 and TSH) in females are presented in Table 6.

Table 6: Mean Thyroid Hormone Levels (T3, T4 and TSH) in Females

	Thyroid hormones
Dose level (mg/kg bw/day)	0	100	300	500
T3 (ng/mL)	0.31	0.35	0.36	0.31
SD	0.130	0.070	0.079	0.148
	-	(+13)	(+16)	(0)
T4 (ng/mL)	13.65	16.55	16.26*	15.13
SD	3.033	6.940	3.501	4.021
	-	(+21)	(+19)	(+11)
TSH (pg/mL)	849	876	1158*	913
SD	357.8	366.9	530.2	454.2
	-	(+3)	(+36)	(+8)

-: not applicable; ( ): in brackets, percentage difference vs. controls; Statistically significant: *: p<0.05.

GRAVID UTERUS WEIGHT AND NET BODY WEIGHT CHANGE

Mean carcass weight, net weight change and gravid uterus weight are summarized in Table 7.

Table 7: Mean Carcass Weight, Net Body Weight Change and Gravid Uterus Weight (g) (weights are rounded values)

Dose level (mg/kg bw/day)	0	100	300	500
Gravid uterus weight	108 -	102 (-6)	107 (-1)	106 (-2)
Carcass weight	357 -	358 (0)	353 (-1)	345 (-3)
Net body weight change from Day 6 p.c.	+59	+62	+58	+51

p.c.: post-coitum; -: not applicable; ( ): in brackets, percentage difference vs. controls;^(a): weights are rounded values.

HYSTERECTOMY DATA

Hysterectomy data are presented in Table 8.

Table 8: Hysterectomy Data

Dose level (mg/kg bw/day)	0	100	300	500
Number of pregnant females at hysterectomy	24	23	24	23
Number of females with live fetuses at termination	24	23	24	23
Number of females with total resorption	0	0	0	0
Mean number of corpora lutea	15.4	15.7	15.7	15.1
Mean number of implantation sites	13.9	14.1	14.1	13.7
Mean pre-implantation loss (%)	8.6	9.9	9.3	9.4
Number of females with pre-implantation loss	13	14	13	15
Mean number of live fetuses	13.1	12.6*	13.1	13.1
Dead fetuses (%)	0.0	0.0	0.0	0.0
Mean number of implantation scars	0.0	0.0	0.0	0.0
Mean number of early resorptions	0.6	1.5**	0.8	0.6
Mean number of late resorptions	0.2	0.0	0.2	0.0
Mean number of resorptions + scars	0.8	1.6*	1.0	0.6
Number of females with resorptions	12	12	11	10
Mean post-implantation loss (%)	5.9	12.1	7.0	5.1
Number of females with post-implantation loss	12	13	11	10

Statistically significant: *: p<0.05 (for number of fetuses or number of resorptions+scars) and **: p<0.01 (for number of early resorptions).

FETAL EXTERNAL VARIATIONS

The distribution of the fetal and litter incidences of external variations is presented in Table 9.

Table 9: Mean litter (L) and Fetal (F) Incidences of External Variations (%)

Dose level (mg/kg bw/day)	0	100	300	500	HCD
Dams with live fetuses, n	24	23	24	23	235
Live fetuses, n	315	289	315	301	2896
Paw and digit
. paw hyperflexion, L(F)	0 (0)	0 (0)	4.2 (0.3)	0 (0)	0 (0)^(a)
Tail
. curled tail, L(F)	0 (0)	4.3 (0.3)	0 (0)	0 (0)	0 (0)^(a)
Litters affected, n (%)^(c)	0 (0)	1 (4.3)	1 (4.2)	0 (0)	6 (2.6) ^(b)
Fetuses affected, n (%)^(c)	0 (0)	1 (0.3)	1 (0.3)	0 (0)	7 (0.2) ^(b)

n: number; HCD: Historical Control Data (control data collected from ten studies covering 2018 and 2019); ^(a): upper litter (fetal) incidence per study; ^(b): mean number (percentage) of litters or fetuses affected; ^(c): all variations combined; No statistical significance.

FETAL EXTERNAL MALFORMATIONS

The distribution of the fetal and litter incidences of external malformations are presented in Table 10.

Table 10: Mean litter (L) and Fetal (F) Incidences of Main Soft Tissue Malformations (%)

Dose level (mg/kg bw/day)	0	100	300	500	HCD
Dams with live fetuses, n	24	23	24	23	235
Live fetuses, n	315	289	315	301	2896
Mouth, jaw, palate
. cleft palate, L(F)	0 (0)	0 (0)	0 (0)	4.3 (1.0)	2.6 (0.2) ^(a)
Litters affected, n (%)^(c)	0 (0)	0 (0)	0 (0)	1 (4.3)	8 (3.4) ^(b)
Fetuses affected, n (%)^(c)	0 (0)	0 (0)	0 (0)	3 (1.0)	10 (0.3) ^(b)
Affected Fetuses/Litter %	0.0	0.0	0.0	1.1	Not applicable

FETAL SOFT TISSUE VARIATION

The distribution of the fetal and litter incidences of soft tissue variations is presented in Table 11.

Table 11: Mean Litter (L) and Fetal (F) Incidences of Soft Tissue Variations (%)

Dose level (mg/kg bw/day)	0	100	300	500	HCD
Dams with live fetuses, n	24	23	24	23	235
Live fetuses, n	153	141	153	143	1385
Spleen
. enlarged, L(F)	0 (0)	4.3 (1.4)	0 (0)	0 (0)	0 (0)^(a)
Kidneys
. dilated renal pelvis, L(F)	20.8 (3.9)	21.7 (3.5)	16.7 (3.9)	21.7 (7.0)	30.8 (9.1) ^(a)
Ureters
. dilated ureter, L(F)	45.8(12.4)	52.2(14.2)	50.0(13.7)	47.8(18.2)	48.7(15.1)^(a)
Vessels
. absent innominate artery, L(F)	12.5 (2.6)	13.0 (2.1)	33.3 (6.5)	26.1 (4.2)	20.8 (3.6) ^(a)
General
. fluid-filled abdomen, L(F)	0 (0)	0 (0)	0 (0)	4.3 (0.7)	2.6 (0.4) ^(a)
Litters affected, n (%)^(c)	12 (50.0)	14 (60.9)	17 (70.8)	17 (73.9)	70 (29.8) ^(b)
Fetuses affected, n (%)^(c)	24 (15.7)	24 (17.0)	30 (19.6)	34 (23.8)	132 (9.5) ^(b)

n: number; HCD: Historical Control Data (control data collected from ten studies covering 2018 and 2019);

^(a): upper litter (fetal) incidence per study; ^(b): mean number (percentage) of litters or fetuses affected;

^(c): all variations combined; No statistical significance.

FETAL SOFT TISSUE MALFORMATION

The distribution of the fetal and litter incidences of soft tissue malformations is presented in Table 12.

Table 12: Mean Litter (L) and Fetal (F) Incidences of Soft Tissue Malformations (%)

Dose level (mg/kg bw/day)	0	100	300	500	HCD
Dams with live fetuses, n	24	23	24	23	235
Live fetuses, n	153	141	153	143	1385
Mouth, jaw, palate
. cleft palate, L(F)	0 (0)	0 (0)	0 (0)	4.3 (1.4)	0 (0) ^(a)
Litters affected, n (%)^(c)	0 (0)	0 (0)	0 (0)	1 (4.3)	0 (0) ^(b)
Fetuses affected, n (%)^(c)	0 (0)	0 (0)	0 (0)	2 (1.4)	0 (0) ^(b)
Affected Fetuses/Litter %	0.0	0.0	0.0	1.4	Not applicable

FETAL SKELETAL CARTILAGES

The distribution of significant differences in fetal and litter incidences of cartilage findings is presented in Table 13.

Table 13: Mean Litter (L) and Fetal (F) Incidences of Skeletal Cartilages

Dose level (mg/kg bw/day)	0	100	300	500	HCD
Dams with live fetuses, n	24	23	24	23	235
Live fetuses, n	162	148	162	158	1511
Cartilage of cervical vertebra(e)
present, L(F)	8.3 (1.2)	4.3 (0.7)	4.2 (0.6)	17.4 (2.5)	12.5 (3.4) ^(a)
Cartilage of thoracic vertebra(e)
present, L(F)	8.3 (1.9)	21.7 (3.4)	20.8 (4.3)	26.1 (7.0*)	66.7 (19.3) ^(a)
Cartilage of sternebra . present, L(F)	4.2 (0.6)	0 (0)	0 (0)	21.7 (3.8)	20.0 (4.3) ^(a)
Cartilage of metacarpal bone . present, L(F)	4.2 (0.6)	4.3 (0.7)	4.2 (0.6)	13.0 (1.9)	15.0 (2.6) ^(a)
Litters affected, n (%)^(c)	21 (87.5)	16 (69.6)	13 (54.2)	16 (69.6)	128 (54.5) ^(b)
Fetuses affected, n (%)^(c)	73 (45.1)	43 (29.1)	21 (13.0)	32 (20.3)	584 (38.6) ^(b)

n: number; *: p<0.05 for number of fetus; HCD: Historical Control Data (control data collected from ten studies covering 2018 and 2019); ^(a): upper litter (fetal) incidence per study; ^(b): mean number (percentage) of litters or fetuses affected; ^(c): all variations combined.

FETAL SKELETAL VARIATIONS

The distribution of significant differences in fetal and litter incidences of skeletal variations is presented in Table 14.

Table 14: Mean Litter (L) and Fetal (F) Incidences (%) of Skeletal Variations

Dose level (mg/kg bw/day)	0	100	300	500	HCD
Dams with live fetuses, n	24	23	24	23	235
Live fetuses, n	162	148	162	158	1511
Head skull
. frontal: incomplete ossification, L(F)	0 (0)	4.3 (0.7)	4.2 (0.6)	13.0 (1.9) 3 L / 3F	5.0 (0.9) ^(a)
. nasal incomplete ossification, L(F)	0 (0)	4.3 (0.7)	0 (0)	8.7 (1.3) 2L / 2F	0 (0)^(a)
. parietal: incomplete ossification, L(F)	0 (0)	4.3 (0.7)	0 (0)	8.7 (1.3) 2L / 2F	10.5 (1.6)^(a)
Cervical vertebrae . incomplete ossification of centrum, L(F)	8.3 (1.2)	0 (0)	4.2 (0.6)	17.4 (2.5) 4L / 4F	45.0 (14.5) ^(a)
Thoracic vertebra(e) . dumbbell ossification of centrum, L(F)	0 (0)	8.7 (1.4)	4.2 (0.6)	8.7 (1.3) 2L / 2F	5.0 (0.7) ^(a)
. incomplete ossification of centrum, L(F)	12.5 (1.9)	13.0 (2.0)	20.8 (4.9)	26.1 (7.0*) 6L / 11F	50.0 (14.7) ^(a)
Caudal vertebra(e)
. incomplete ossification of arch, L(F)	0 (0)	0 (0)	0 (0)	4.3 (0.6) 1L / 1F	15.0 (2.2) ^(a)
. unossified centrum, L(F)	0 (0)	0 (0)	0 (0)	4.3 (0.6)	5.6 (0.9) ^(a)
Sternebra . incomplete ossification of 1^st to 4^th sternebra, L(F)	0 (0)	0 (0)	0 (0)	8.7 (1.9) 2L/3F	15.0 (3.4) ^(a)
. unossified 5^th sternebra, L(F)	0 (0)	0 (0)	0 (0)	4.3 (0.6) 1L/1F	5.0 (0.7) ^(a)
Rib . . ossification point on 14^th thoracic vertebra, L(F)	4.2 (1.2)	17.4 (4.1)	4.2 (1.9)	13.0 (2.5) 3L/4F	21.1 (6.0) ^(a)
. short supernumerary 14^th rib(s), L(F)	0 (0)	0 (0)	0 (0)	4.3 (0.6) 1L/1F	0 (0)^(a)
Metacarpal Bone . incomplete ossification of 2^nd to 5^th metacarpal, L(F)	4.2 (0.6)	4.3 (0.7)	4.2 (0.6)	13.0 (1.9) 3L/3F	10.0 (1.7) ^(a)
Spine . presence of 27 pre-sacral vertebrae, L(F)	0 (0)	0 (0)	0 (0)	8.7 (1.3) 2L/2F	0 (0)^(a)
Litters affected, n (%)^(c)	23 (95.8)	18 (78.3)	15 (62.5)	16 (69.6)	194 (82.6) ^(b)
Fetuses affected, n (%)^(c)	77 (47.5)	49 (33.1)	29 (17.9)	32 (20.3)	742 (49.1) ^(b)

FETAL SKELETAL MALFORMATIONS

The distribution of the fetal and litter incidences of skeletal malformations is presented in Table 15.

Table 15: Mean Litter (L) and Fetal (F) Incidences of Skeletal Malformations

Dose level (mg/kg bw/day)	0	100	300	500	HCD
Dams with live fetuses, n	24	23	24	23	235
Live fetuses, n	162	148	162	158	1511
Head skull
. frontal: split, L(F)	0 (0)	4.3 (0.7)	0 (0)	8.7 (1.9)	0 (0) ^(a)
Head others
. absent palate, L(F)	0 (0)	0 (0)	0 (0)	4.3 (0.6)	0 (0) ^(a)
Cervical Vertebra(e)
. cervical rib, L(F)	0 (0)	4.3 (0.7)	0 (0)	0 (0)	0 (0) ^(a)
. fused arch(es), L(F)	0 (0)	4.3 (0.7)	0 (0)	4.3 (0.6)	10.3 (1.6)^(a)
Thoracic Vertebra(e)
. fused centrum, L(F)	0 (0)	4.3 (0.7)	0 (0)	0 (0)	0 (0) ^(a)
Lumbar Vertebra(e) . supernumerary, L(F)	0 (0)	0 (0)	0 (0)	8.7 (1.3)	0 (0) ^(a)
Sternebra . fused, L(F)	0 (0)	4.3 (0.7)	0 (0)	0 (0)	5.3 (0.9)^(a)
Rib . fused, L(F)	0 (0)	4.3 (0.7)	0 (0)	8.7 (1.9)	5.6 (0.9) ^(a)
Litters affected, n (%)^(c)	0 (0)	1 (4.3)	0 (0)	3 (13.0)	13 (5.5)^(b)
Fetuses affected, n (%)^(c)	0 (0)	1 (0.7)	0 (0)	5* (3.2)	15 (1.0) ^(b)
Affected Fetuses/Litter %	0.0	0.5	0.0	3.4	Not applicable

n: number; *: p<0.05; HCD: Historical Control Data (control data collected from ten studies covering 2018 and 2019); ^(a): upper litter (fetal) incidence per study; ^(b): mean number (percentage) of litters or fetuses affected; ^(c): all malformations combined.

DISTRIBUTION OF FETAL MALFORMATIONS

The overall litter and fetal incidences of the malformations recorded at external, soft tissue and skeletal examinationsare presented in Table 16.

Table 16: Distribution of Fetal Malformations

Dose level (mg/kg bw/day)	0	100	300	500	HCD
External
Litters affected, n (%)	0 (0)	0 (0)	0 (0)	1 (4.3)	8 (3.4) ^(a)
Fetuses affected, n (%)	0 (0)	0 (0)	0 (0)	3 (1.0)	10 (0.3) ^(a)
Soft tissue
Litters affected, n (%)	0 (0)	0 (0)	0 (0)	1 (4.3)	7 (3.0) ^(a)
Fetuses affected, n (%)	0 (0)	0 (0)	0 (0)	2 (1.4)	10 (0.7) ^(a)
Skeletal
Litters affected, n (%)	0 (0)	1 (4.3)	0 (0)	3 (13.0)	13 (5.5) ^(a)
Fetuses affected, n (%)	0 (0)	1 (0.7)	0 (0)	5* (3.2)	15 (1.0) ^(a)
Total
Litters affected/evaluated (%)	0/24 (0)	1/23 (4.4)	0/24 (0)	3/24 (12.5)	/
Fetuses affected/evaluated (%)	0/315 (0)	1/289 (0.3)	0/315 (0)	7/301 (2.3)	/

HCD: Historical Control Data (control data collected from ten studies covering 2018 and 2019); ^(a): mean incidences; /: not reported in HCD; Statistically significant: *: p<0.05 for the number of fetuses affected.

SUMMARY TABLE

Table 17: Summary table of relevant endpoints

Dose level (mg/kg bw/day)	0	15	50	130
Pregnant/total dams	24/24	23/24	24/24	23/24
Mean number of early resorptions	0.6	1.5**	0.8	0.6
Mean number of late resorptions	0.2	0.0	0.2	0.0
Dead fetuses (%)	0.0	0.0	0.0	0.0
Mean pre-implantation loss (%)	8.6	9.9	9.3	9.4
Mean post-implantation loss (%)	5.9	12.1	7.0	5.1
Body weight on day 21 p.c. (g) In brackets, percentage difference (%) vs. controls	464	460 (-1)	460 (-1)	451 (-3)
Body weight gain day 6-21 (g) In brackets, percentage difference (%) vs. controls	+167	+164 (-1)	+165 (-1)	+157 (-3)
Gravid uterine weight (g) In brackets, percentage difference (%) vs. controls	108	102 (-6)	107 (-1)	106 (-2)
Mean number of live fetuses per animal	13.1	12.6*	13.1	13.1
Mean fetal body weight (g) In brackets, percentage difference (%) vs. controls	5.97 -	5.85 (-2)	5.94 (-1)	5.91(-1)
Mean placental weight (g) In brackets, percentage difference (%) vs. controls	0.69 -	0.69 (0)	0.67 (-3)	0.68(-1)
Variations, fetus affected, n (%) - External - Soft tissue - Skeletal	0 (0.0) 24 (15.7) 77 (47.5)	1 (0.3) 24 (17.0) 49 (33.1)	1 (0.3) 30 (19.6) 29 (17.9)	0 (0.0) 34 (23.8) 32 (20.3)
Malformations, fetus affected, n (%) - External - Soft tissue - Skeletal	0 (0.0) 0 (0.0) 0 (0.0)	0 (0.0) 0 (0.0) 1 (0.7)	0 (0.0) 0 (0.0) 0 (0.0)	3 (1.0) 2 (1.4) 5* (3.2)
Cartilages, fetus affected, n (%)	73 (45.1)	43 (29.1)	21 (13.0)	32 (20.3)

Bold characters: Statistically significant difference vs. controls: *: p<0.05, **: p<0.01

Conclusions:

MATERNAL
The No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 100 mg/kg bw/day based on adverse findings recorded from 300 mg/kg bw/day (i.e. loud and/or abdominal breathing, minimal to marked erosion/ulcer and inflammation/edema associated with squamous cell hyperplasia in the forestomach of the dams),

EMBRYO-FETAL DEVELOPMENT
The No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 300 mg/kg bw/day based on fetal malformations at 500 mg/kg bw/day in a context of moderate maternal toxicity.

Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item, 1,4-butanediylbis[oxy(2-hydroxy-3,1-propanediyl)] diacrylate, on the pregnant female and on embryonic and fetal development, following daily oral administration (gavage) to pregnant female rats from implantation until the day before scheduled hysterectomy [Day 6 to Day 20 post-coitum (p.c.) inclusive]. This GLP study was carried out according to OECD test guideline No. 414 (25 June 2018).

Three groups of 24 time-mated female Sprague-Dawley rats received the test item, 1,4-butanediylbis[oxy(2-hydroxy-3,1-propanediyl)] diacrylate, by the oral route (gavage), at a dose level of 100, 300 or 500 mg/kg bw/day, once daily from Day 6 to Day 20 p.c. inclusive. Another group of 24 time-mated female rats received the vehicle only, corn oil, under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg bw/day was used. The test item concentrations in the dose formulations were determined on two occasions using a validated LC/MS-MS analytical method. The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. At termination, thyroid hormones (TSH, T3 and T4) were determined for all females.

On Day 21 p.c., the females were euthanized and a macroscopic post-mortem examination was performed. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, uterine scars, early and late resorptions, and live and dead fetuses were recorded. Placentas were observed and weighed. The fetuses were weighed, examined for external abnormalities and sexed by internal examination of the gonads. The anogenital distance was measured. The fetuses were subjected to detailed external, soft tissue and skeletal examinations (including cartilage). Macroscopic lesions, thyroids with parathyroids and samples of stomach with forestomach were collected, preserved in 10% buffered formalin and microscopically examined. Thyroids with parathyroids were weighed.

The concentrations of the test item in the dose formulations, determined in the first and last weeks of the treatment period, were +3.2% to +10.1% of the nominal concentrations and remained within an acceptable range of variations (± 15% of the nominal concentrations). No test item was detected in control dose formulations.

There was one death at 500 mg/kg bw/day on Day 10 p.c, for which the relation with test item was not clear. On Day 21 p.c., all females were pregnant with live fetuses, with the exception of one female at 100 mg/kg bw/day for which a test item relationship was excluded. Test item-related clinical signs consisted of ptyalism from 100 mg/kg bw/day (one single dam), and piloerection and loud and/or abdominal breathing from 300 mg/kg bw/day. Loud and abdominal breathing were considered to be adverse clinical signs. There were no effects on mean body weights, mean body weight changes or mean food consumption at any dose level. There were no effects on mean gravid uterus weight, carcass weight or mean net body weight change at any dose level. Thyroid hormone analysis did not reveal any disturbances at any dose level. No organ weight or macroscopic/microscopic changes were observed in the thyroid glands at any dose level. At 100 mg/kg bw/day, minimal squamous cell hyperplasia was observed in the forestomach in a few females only. No histopathological changes were observed in the stomach at this lowest dose level. From 300 mg/kg bw/day, adverse, minimal to marked erosion/ulcer and/or inflammation/edema, associated with squamous cell hyperplasia and correlating with white masses at necropsy, were present in the forestomach of all females, and non-adverse minimal mucosal hypertrophy (pyloric region) was noted in the stomach along with minimal or slight erosion in a small number of animals. There were no test item-related effects on hysterectomy data at any dose level.

No effects on the fetal body weight, placental weight, fetal body weight/placental weight ratio, sex ratio or anogenital distance were noted at any dose level. There were no test item-related increases in the incidence of external or visceral variations. At 500 mg/kg bw/day and when compared with controls and/or Historical Control Data, there were increases in the fetal and litter incidences of external malformations (i.e. cleft palate in three fetuses from the same litter), increases in the fetal and litter incidences of visceral malformations (i.e. cleft palate in two fetuses from the same litter corresponding to external malformations), increases in the fetal and litter incidences of skeletal variations [i.e.incomplete ossification of frontal, nasal and/or parietal bones, incomplete ossification of the centrum of cervical and/or thoracic vertebra(e) , incomplete ossification of 1st to 4th sternebra(e), incomplete ossification of 2nd to 5th metacarpal bone, presence of 27 pre-sacral vertebra(e)] and skeletal malformations mainly involving the head bones or the axial skeleton [i.e. split frontal bone, absent palate, fused arch(es) of cervical vertebra(e), supernumerary lumbar vertebra(e) and/or fused rib(s)] that affected five fetuses from three litters. At 300 mg/kg bw/day, and when compared with controls, there were increases in the fetal and litter incidences of skeletal variations [i.e. incomplete ossification of the centrum of thoracic vertebra(e)].

On the basis of the results obtained in this study:

the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 100 mg/kg bw/day based on adverse findings recorded from 300 mg/kg bw/day (e. loud and/or abdominal breathing, minimal to marked erosion/ulcer and inflammation/edema associated with squamous cell hyperplasia in the forestomach of the dams),

the No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 300 mg/kg bw/day based on fetal malformations at 500 mg/kg bw/day in a context of moderate maternal toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 300 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: The key study is considered to be reliable (klimisch score of 1) for the endpoint considered.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:: no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:: no study available

Additional information

Main developmental toxicity study in rat (CRL 2021)

On the basis of the results obtained in this study:

the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 100 mg/kg bw/day based on adverse findings recorded from 300 mg/kg bw/day (e. loud and/or abdominal breathing, minimal to marked erosion/ulcer and inflammation/edema associated with squamous cell hyperplasia in the forestomach of the dams),

the No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 300 mg/kg bw/day based on fetal malformations at 500 mg/kg bw/day in a context of moderate maternal toxicity.

Dose-range finding study in rat (CRL 2020)

A preliminary study was performed to evaluate the potential toxic effects of 1,4-butanediylbis[oxy(2-hydroxy-3,1-propanediyl)] diacrylate on the pregnant female and on embryonic and fetal development, following daily oral administration (gavage) to pregnant female rats from implantation to the day before scheduled hysterectomy [Day 6 to Day 20 post-coitum (p.c.)inclusive], in order to select dose levels for a further main study.

Three groups of eight time-mated female Sprague-Dawley rats received 1,4-butanediylbis[oxy(2-hydroxy-3,1-propanediyl)] diacrylate at 100, 300 or 500 mg/kg bw/day by the oral route (gavage), once daily from Days 6 to 20 p.c. Another group of eight mated females received the vehicle alone (corn oil) under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg bw/day was used. All animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 21 p.c., the females were euthanized and a macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed. Hysterectomy was also performed and the numbers of corpora lutea, implantations, uterine scars, early/late resorptions and live/dead fetuses were recorded. The fetuses were sexed, weighed, examined for oral cavity and external abnormalities and then discarded. Microscopic examination was performed on necropsy findings observed in the stomach of the dams.

On Day 21 p.c., all females were pregnant except for two females (one in each 300 and at 500 mg/kg bw/day groups). There were no unscheduled deaths. Ptyalism was transiently observed in 1/8 females at 100 mg/kg bw/day and in all females at 300 and 500 mg/kg bw/day. Loud breathing was transiently observed in 1/8 females at 500 mg/kg bw/day. There were no relevant effects on body weight, body weight change or food consumption. There were no adverse effects on gravid uterus, carcass weights or net body weight change. Adverse erosion/ulcer, inflammation/edema and/or squamous cell hyperplasia was observed in the forestomach at = 300 mg/kg bw/day and correlated with white discoloration in 3/8 animals at 300 mg/kg bw/day and in 7/8 animals at 500 mg/kg bw/day. Non-adverse erosion was also noted in the stomach of one animal at 500 mg/kg bw/day. No macroscopic lesions were observed at 100 mg/kg bw/day. There were no effects on hysterectomy parameters. There was a slight non-adverse decrease infetal body weight at 300 and 500 mg/kg bw/day (5.81 and 5.80 vs. 6.13 in controls, respectively). Non test item related lower percentages of male fetuses were noted at 300 and 500 mg/kg bw/day (36.2% and 26.0% vs. 49.2% in controls, respectively). There were no external fetal variations and no test-item related external fetal malformations.

In conclusion, although adverse histopathological findings related to the irritation potential of the test item were observed in the forestomach of females at 300 mg/kg bw/day and above, 500 mg/kg bw/day could be considered as a suitable high dose level for the main embryo-fetal developmental study with
1,4-butanediylbis[oxy(2-hydroxy-3,1-propanediyl)] diacrylate in the absence of excessive maternal toxicity.

Justification for classification or non-classification

Based on the available data, the test substance does not require classification for reproduction toxicity according to Regulation EC°1272/2008.

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