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

Toxicity to reproduction

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

supporting 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

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, adopted July 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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
- 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 oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The suspension of the tesst substance 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.

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

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 (actual dose received)

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:

10

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 500mg/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

Observations and examinations performed and frequency:

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (16-20h)
- How many animals: 5 parental animals per sex and group
- The following parameters were examined: Leukocyte count, Erythrocyte count, hemoglobin, hemtocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, differential blood count, reticulocytes, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Animals fasted: Yes (16-20h)
- How many animals: 5 parental animals per sex and group
- The following parameters were examined: ALT, AST, ALP, GGT, sodium, potassium, chlorid, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: day31 (males), day 50 (females)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes during collection
- How many animals: 5 parental animals per sex and group
- The following parameters were examined: pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment, color, turbity, volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: day 29 (males), day 53 (females)
- Dose groups that were examined: 5 animals per sex and group (only females with litter)
- Battery of functions tested: home cage and open field observation, motor activity, sensory motor test / reflexes (including: Reaction to an object being moved towards the face, touch sensitivity, vision (Visual placing response), pupillary reflex, pinna reflex, auditory startle response, righting response, behavior during handling, vocalization, pain perception (Tail pinch), grip strength of forelimbs and hindlimbs, landing foot-splay test)

Sacrifice and pathology:

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 following tissues were examined histotechnically in at least 5 animals per sex of the control and high dose group, unless noted otherwise, and in all animals that died prematurely:
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

Other examinations:

Reproductive performance: see entry in chapter 7.8.1

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.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test substance-related adverse findings were noted with regard to food consumption during the entire study period.

Food efficiency:

not specified

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

no effects observed

Description (incidence and severity):

No test substance-related adverse findings were noted.

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In mid and high dose males, absolute and relative neutrophil counts were increased whereas relative lymphocyte counts were decreased. Total white blood cell counts were not significantly altered in the mentioned individuals. However, in the mid dose males absolute and relative neutrophil accounts as well as relative lymphocyte counts were within historical control ranges. Therefore, in the mentioned alterations in this test group of the differential blood cell counts were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among urinalysis parameters were observed.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No test substance related effects were observed.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance related effects were observed.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Most of the animals having died spontaneously showed erosions/ ulcers on the forestomach, in one animal the macroscopic finding was not confirmed histologically. 7 males and all females of the high dose group showed erosions / ulcers in the forestomach, in one animal of each sex this finding was observed in the glandular stomach. One high dose female had a mass in the forestomach, that correlated to severe inflammation/ edema, as confirmed during histopathologic examination. In the mid dose group, 9 males and 6 females had erosions / ulcers in the forestomach, and again this finding was observed in one animal of each sex in the glandular stomach. In the low dose, 5 males and 2 females were affected.

Neuropathological findings:

not examined

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.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

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

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 effects

Effect level:

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

Sex:

female

Basis for effect level:

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

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

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

Sex:

male/female

Basis for effect level:

other: erosions / ulcers in the stomach

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

Conclusions:

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.
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.

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.

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.

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.

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.

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

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:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, adopted July 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of butane-1,4-diol and 1-chloro-2,3-epoxypropane, esters with acrylic acid

EC Number:

701-230-0

Cas Number:

52408-42-1

Molecular formula:

C16H26O8

IUPAC Name:

Reaction products of butane-1,4-diol and 1-chloro-2,3-epoxypropane, esters with acrylic acid

Test material form:

liquid

Test animals

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

Administration / exposure

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)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 mg/kg bw/day

Remarks:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

1

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

2

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.

3

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

Target system / organ toxicity (P0)

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

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 300 mg/kg bw/day

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

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.

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.

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.

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.

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.