ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Aug 1997- 27 April 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

Current version adopted in 2001

Deviations:

yes

Remarks:

Thyroid weights were not measured for F0 and F1 parental animals. Enumeration of cauda epididymal sperm reserve was not done.

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

Guideline in place during study conduct: adopted in 1983

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.35 (Two-Generation Reproduction Toxicity Test)

Version / remarks:

Adopted in 1987

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

Adopted in 1996 and 1997

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: "Reproductive Study," Japanese Ministry of Agriculture, Forestry and Fisheries, 59 NohSan No. 4200

Version / remarks:

Adopted in 1985

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

443-870-0

EC Name:

-

Cas Number:

163520-33-0

Molecular formula:

C18H17NO3

IUPAC Name:

ethyl 5,5-diphenyl-4,5-dihydro-1,2-oxazole-3-carboxylate

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, USA
- Age at receipt (P): 33 days
- Age at start of dosing (F1): 3 weeks
- Body weight at study initiation: (P) males: 161 - 230 g, females: 126 - 180 g; (F1, prior to mating) males: 392 - 607 g, females: 206 - 342 g
- Housing: individually in wire-mesh cages suspended above cage board until selection or pairing; after successful mating males stayed in wire mesh cages until scheduled necropsy, females were transferred to plastic maternity cages with nesting material (Bed-O'Cobs; The Andersons, Industrial Products Division, Maumee, USA) and stayed there until weaning on lactation Day 21. Females returned to wire mesh cages until scheduled necropsy and weaned F1 pups were housed together until PND 28 when pups were individually housed as described above.
- Diet: grounded Certified Rodent LabDiet® 5002 (PMI Nutrition International, Inc.), ad libitum
- Water: tap water in drinking water quality (reverse osmosis-treated on-site), ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 Aug 1997 To: 27 Apr 1998

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: grounded diet

Remarks:

Certified Rodent LabDiet® 5002

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with: standard diet
- Storage temperature of food: room temperature, in the dark

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy
- Further matings after one unsuccessful attempt: no
- After successful mating each pregnant female was caged: individually in plastic maternity cages with nesting material

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first day of dosing, diet formulations were prepared for each dose group. Six samples were collected for each group, including the control group, from the top, middle and bottom (homogeneity sample) or middle (concentration and stability samples) of the preparations. Two sets of each were designated for homogeneity, concentration and stability samples and stored deep frozen. A formulation the portion allocated for stability sampling was stored under laboratory animal room conditions and sampled after 0, 1, 2, 4, 8 and 15 days. A 100 g sample from the middle of each formulation, including the control, was collected weekly throughout the study and stored frozen. Samples of diet from each dosage level, including controls, were analysed for test article concentration at approximately 4-week intervals for the first 3 months of the study and then at 3-month intervals thereafter. The test substance was extracted from the diet by shaking with acetonitrile for determination by high performance liquid chromatography using UV detection. The mean test substance content for the test diets sampled at the time of preparation and analysed were within the range 87.3 to 101.1% of nominal (acceptable range +10% to -15% of nominal). Homogeneity was shown to be satisfactory at all levels (i.e. mean values obtained for top, middle and bottom samples were within the range 90 - 110% of nominal and these mean % values differed by <10%). Stability was shown to be satisfactory over the time of use of the diet, i.e. % nominal values declined by <10% over 15 days storage at room temperature. Data from the analyses therefore confirmed that the diet preparations used for administration to the animals were stable for 15 days at room temperature, were homogeneous and contained the amounts of test substance specified in the protocol.

Duration of treatment / exposure:

(P) Males: 10 weeks before mating and 9 weeks during mating and thereafter until necropsy (19 weeks in total)
(P) Females: 10 weeks before mating, up to 2 weeks during mating, 3 weeks during gestation, 3 weeks during lactation till weaning of their F1 offspring on post-natal day (PND) 21 and further 2 weeks until necropsy (approx. 20 weeks in total)
(F1) Males: 18 - 19 weeks after weaning, during growth into adulthood, mating and production of F2 generation, until weaning on PND 21 of the F2 generation.
(F1) Females: 18 - 19 weeks after weaning, during growth into adulthood, mating and production of F2 generation, until weaning on PND 21 of the F2 generation.

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

- F1 parental animals not mated until 10 weeks after being selected from the F1 litters.
- Selection of parents from F1 generation when pups were 28 days of age.
- Age at mating of the mated animals in the study: 17 weeks (F0), 14 - 15 weeks (F1)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 ppm

Remarks:

actual test substance intake:
P0 generation: males: 1.2 mg/kg bw/day, females: 1.8 mg/kg bw/day
F1 generation: males: 1.5 mg/kg bw/day, females: 2.0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

200 ppm

Remarks:

actual test substance intake:
P0 generation: males: 12.6 mg/kg bw/day, females: 18.3 mg/kg bw/day
F1 generation: males: 14.5 mg/kg bw/day, females: 20.0 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

4 000 ppm

Remarks:

actual test substance intake:
P0 generation: males: 249.8 mg/kg bw/day, females: 363.2 mg/kg bw/day
F1 generation: males: 300.6 mg/kg bw/day, females: 406.0 mg/kg bw/day

No. of animals per sex per dose:

30

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Results of a previous subchronic toxicity study showed that 4000 ppm caused a 10 - 11% reduction of body weight gain in both sexes, therefore it was expected to be a maximum tolerated dose. The 200 ppm dose level was expected to be a NOEL. The lowest dose level of 20 ppm was expected to be a certain NOEL.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily for appearance, behaviour, pharmacotoxic signs, moribundity and mortality. Females that delivered were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labour, delayed labour) or other difficulties.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were recorded weekly for all parental animals throughout the study period.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual F0 and F1, male body weights were recorded weekly throughout the study and prior to the scheduled necropsy. Individual F0 and F1, female body weights were recorded weekly until evidence of mating was observed. Once evidence of mating was observed, female body weights were recorded on gestation Days 0, 4, 7, 10, 14 and 20 and on lactation Days 1, 4, 7, 14 and 21. After weaning (lactation Day 21), weekly body weights were recorded for these females until the scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE :
Individual F0 and F1, male and female food consumption was measured weekly until pairing. Food intake was not recorded during the mating period. Male food consumption was measured after mating on a weekly basis until the scheduled necropsy. Female food consumption was recorded on gestation Days 0, 4, 7, 10, 14 and 20 and lactation Days 1, 4, 7, 14 and 21.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OTHER: food efficiency

Oestrous cyclicity (parental animals):

Vaginal smears were prepared daily to determine the stage of estrus for each female, beginning 21 days prior to pairing and continuing until evidence of mating was observed. For females with no evidence of mating, smearing was continued until termination of the mating period. Vaginal smears were also performed on the day of necropsy.

Sperm parameters (parental animals):

Parameters examined in P and F1 male parental generations:
testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, sperm motility, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 post partum: yes
- If yes, maximum of 8 pups/litter (4 sex/litter as nearly as possible); excess pups were weighed, killed and discarded on PND 4

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities
Detailed physical examination and weighing: PND 1, 4, 7, 15 and 21
Developmental landmarks: Balanopreputial separation from PND 40 to PND 53, vaginal patency from PND 30 to PND 53

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, after the last litter of each generation was weaned
- Maternal animals: All surviving animals, after the last litter of each generation was weaned

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations of all euthanised or spontaneously dying animals. Examined were the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities including viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were collected and placed in 10% neutral buffered formalin.
adrenals , aorta, bone with marrow (sternebrae), brain (forebrain, midbrain, hindbrain), cervix, coagulating gland, eyes with optic nerve , gastrointestinal tract, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, heart, kidneys, liver (sections of two lobes), lungs (including bronchi, fixed by inflation with, fixative), lymph node (mesenteric), ovaries and oviducts, pancreas, peripheral nerve (sciatic), pituitary, prostate, salivary gland (submaxillary ), seminal vesicles, skeletal muscle (vastus medialis), skin with mammary gland, spinal cord (cervical), spleen, testes with epididymides and vas deferens, thymus, thyroids (with parathyroids), trachea, urinary bladder, uterus with vagina, all gross lesions

The tissues/ organs indicated below were weighed.
adrenals, brain, epididymides (total and cauda), kidneys, liver, ovaries with oviducts, pituitary, prostate, seminal vesicles with coagulating, glands (with accessory fluids), spleen, testes, thymus, uterus with cervix

The tissues/ organs indicated below were examined microscopically in P and F1 parental animals (10/sex/group) from the control and high dose group and for all animals found dead.
adrenal gland, brain, epididymis (right): caput, corpus and cauda; cervix, coagulating gland, kidneys, liver, ovaries, oviducts, pituitary, prostate, seminal vesicles, spleen, testis (right), thymus, uterus, vagina, vas deferens, all gross (internal) lesions

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and for examination of developmental landmarks and all F2 offspring were sacrificed at postnatal Day 21 days of age.
- These animals were subjected to postmortem examinations with emphasis on developmental morphology. One F1 and F2 pup/sex/litter was prepared for complete necropsy on PND 21

GROSS NECROPSY
- Gross necropsy with emphasis on developmental landmarks.

HISTOPATHOLOGY / ORGAN WEIGTHS
Complete necropsy was performed for one F1 and F2 pup/sex/litter as described for parental animals.

Statistics:

All analyses were conducted using two-tailed tests (except as noted below) for a minimum significance level of 5% comparing each treated group to the control group. Each mean was presented with the standard deviation and the number of animals used to calculate the mean. Data obtained from non-gravid animals were excluded from statistical analyses following the mating period. Statistical analyses were not performed when weekly food or body weight data for one or more animals were not available because the animals remained in the lactation phase. The following statistical tests were performed by a Digital® MicroVAX® 3400 computer (with appropriate programming) in this laboratory and are referenced on the report tables:
- Chi-square test with Yates' correction factor for Parental Mating and Fertility Indices
- One-way ANOVA with Dunnett's test for parental weekly body weights and weight changes, gestation and lactation body weights and body weight changes, parental food consumption, pre-coital interval, mean gestation length, number of pups born, pup body weights, absolute and relative organ weights, live litter size, sperm production rate, sperm numbers, mean day of acquisition of balanopreputial separation or vaginal perforation, mean body weight at acquisition.
- Kruskal-Wallis test with Mann-Whitney U test for sperm motility, sperm morphology, proportional postnatal survival, pup viability indices, live birth index, percent male at birth.
- Kolmogorov-Smirnov test (one-tailed test) for histopathological findings.

Reproductive indices:

Mating Index (%) for males and females = (No. of rats copulated / No. of mated pairs) x 100
Fertility Index (%) for females = (No. of females pregnant / No. of females used for mating) x 100
Fertility Index (%) for males = (No. of males siring a litter / No. of males used for mating) x 100
Gestation Index (%) = (No. of dams with live newborns / No. of pregnant dams) x 100

Offspring viability indices:

Life birth Index (%) = (No. of live offspring at Day 0 / No. of live offspring at birth) x 100
Viability Index (%) = (No. of live offspring at Day 4 / No. of live offspring at birth) x 100
Lactation Index (%) = (No. of live offspring at Day 21 / No. of live offspring at Day 4 post-selection) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related signs of toxicity observed. Clinical findings in the treated groups, such as scabbing and hair loss on various body surfaces, red material around the eyes, swollen ears and soft stool, occurred similarly in the control group or infrequently.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

At 4000 ppm, 2 parental (P0) males died during the study; one male died during Week 7 and another died during Week 18. The cause of death for both of these animals was determined to be urogenital tract inflammation.
At 200 ppm, 1 P0 male died during week 10; the cause of death could not be determined.
At 0 ppm, 1 control group female died during Week 4; the cause of death could not be determined.
All other parental (P0) animals survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 4000 ppm group, absolute body weights of P0 males were consistently slightly below (5%) control group values from Week 6 and cumulative body weight change was 7 - 9% lower from Week 0 - 1 throughout the study period (Table 5 under "Any other information on results incl. tables"). In the 4000 ppm group, P0 males, mean body weight gains during weeks 0 - 1 through 9 - 10 were slightly lower than the control group values. The differences during weeks 0 - 1, 3 - 4, 5 - 6 and 8 - 9 were statistically significant (p <0.05 or p <0.01). Throughout the remainder of the generation (Weeks 10 - 11 through 18 - 19), mean body weight gains in the 4000 ppm group males were similar to the control group values. Cumulative body weight gains from Week 0 were reduced by 7 - 9% throughout the generation. The majority of the differences was statistically significant (p <0.05 or p <0.01). Mean body weights in the 4000 ppm group P0 males were consistently slightly lower (5%) than the control group values from Weeks 6 through 19. The difference during Week 10 was statistically significant (p <0.05).
Mean weekly body weights and body weight gains in the 20 and 200 ppm group males and females and the 4000 ppm group females were unaffected by test substance administration. The only statistically significant differences in these groups from the control values were a reduced mean body weight in the 4000 ppm group females for Week 17 (p <0.05) and a reduced mean body weight gain in the 20 ppm group males during Week 3 - 4 (p <0.05). These transient changes were not attributed to test substance administration.
During gestation, mean body weights and body weight gains in the 20, 200 and 4000 ppm groups were similar to the control group values; differences were slight and were not statistically significant.
During lactation, there was no treatment-related effect on P0 female body weight or body weight change at any dose level. In the 4000 ppm group P0 females, an increased mean body weight gain during lactation Day 1 - 4 was followed by a reduced mean body weight gain during lactation Day 4 - 7. The difference from the control group value during lactation Day 4 - 7 was statistically significant (p <0.01). No trends were apparent, and these differences during the first 6 days of lactation were attributed to biological variation. Mean body weight gain in the 4000 ppm group females was comparable to the control group value during lactation Day 7 - 14. During lactation Day 14 - 21, a mean body weight loss in the 4000 ppm group was greater than the loss observed in the control group. The difference was not statistically significant. When the entire lactation period (lactation Day 1 - 21) was evaluated, a reduced mean body weight gain in the 4000 ppm group was statistically significant at p <0.05 when compared to the control group value. Mean lactation body weights in the 4000 ppm group were similar to the control group values on lactation Days 1, 4, 7 and 14. On lactation Day 21, mean body weight in this group was 5% lower than the control group value; the difference was not statistically significant. Because the P0 female mean body weights and body weight gains were similar to the control group values throughout the remainder of the treatment period (pre-mating, gestation and post-weaning), the reduced body weights and body weight gains during the latter portion of the lactation period were attributed to biological variation. Mean lactation body weights and body weight changes in the 20 and 200 ppm group P0 females were unaffected by test substance administration. The only statistically significant differences (p <0.05 or p <0.01) from the control group were reduced mean body weight gains in the 20 and 200 ppm groups during lactation Days 4 - 7. However, mean body weight gains in these groups were greater than the control group value during lactation Days 1 - 4, and no dose-response relationship was apparent. Therefore, the differences were attributed to biological variation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption, evaluated as g/animal/day and g/kg bw/day, was generally unaffected by test substance administration in all treatment groups (at 20, 200 and 4000 ppm) in both sexes.
A statistically significant (p <0.05) reduction in food consumption was observed in the 4000 ppm group males during the first week of administration (Week 0 - 1). However, because the difference was slight (4.2% when evaluated as g/animal/day), the reduction was considered not to be treatment-related. The only other statistically significant differences (p <0.05 or p <0.01) from the control group were increased g/animal/day and/or g/kg bw/day values in the 4000 ppm group females during Week 2 - 3, 7 - 8 and 17 - 18. No trends were apparent, and the differences were attributed to biological variation.
During gestation, food consumption, evaluated as g/animal/day and g/kg bw/day, was unaffected by test substance administration in the 20, 200 and 4000 ppm group P0 females. No statistically significant differences from the control group values were observed.
During lactation, food consumption was unaffected by treatment. Food consumption, assessed as g/animal/Day and g/kg bw/Day, in the 4000 ppm group P0 females was comparable to that in the control group during lactation Day 1 - 4, 4 - 7 and 7 - 14. During lactation Days 14 - 21, food consumption (g/animal/day and g/kg bw/day) in the 4000 ppm group was slightly lower than that in the control group. The differences were statistically significant (p <0.05). When the entire lactation period (lactation Day 1 - 21) was evaluated, food consumption in the 4000 ppm group females was slightly lower than that in the control group. The differences were not statistically significant. Because P0 food consumption values were similar to the control group values throughout the remainder of the treatment period (pre-mating, gestation and post-weaning), the reduction in food consumption in the latter part of the lactation period was attributed to biological variation. Food consumption in the 20 and 200 ppm group P0 females was unaffected by test substance administration throughout the lactation period. No statistically significant differences from the control group were observed.
For details on test substance intake see Table 1 - 3 under "Any other information on results incl. tables".

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food efficiency was reduced in the 4000 ppm group males, mainly due to the reduction in body weight gains. Food efficiency was reduced during Week 3 - 4 and between Week 5 - 6 and 9 - 10 and generally correlated to reduced body weight gain data during these weeks of treatment for the 4000 ppm group males. The reductions were statistically significant (p <0.05 or p <0.01) during Week 3 - 4, 5 - 6, 7 - 8, and 8 - 9. No treatment-related effects were observed in 20 and 200 ppm group males and females or the 4000 ppm group females. Occasionally statistically significant differences from the control group values were not observed in a dose-dependent manner.
During gestation, as for food consumption, food efficiency was unaffected by test substance administration in the 20, 200 and 4000 ppm group P0 females. No statistically significant differences from the control group values were observed.
During lactation, food efficiency was increased in the 20, 200 and 4000 ppm groups relative to the control group during lactation Day 1 - 4. During lactation Day 4 - 7, food efficiency was decreased (p <0.05) in these same groups relative to the control group. Because no trends were apparent, these differences were considered not to be test substance-related. Food efficiency in the 20 and 200 ppm groups were similar to the control groups values throughout the remainder of lactation (lactation Day 7 - 14 and 14 - 21) and when the entire lactation period (lactation Day 1 - 21) were evaluated. Food efficiency in the 4000 ppm group was similar to the control group value during lactation Day 7-14 and slightly reduced (p <0.05 or p <0.01) during lactation Day 14 - 21 and 1 - 21. Because food efficiency in the P0 females in this group was similar to the control group throughout the remainder of the treatment period (pre-mating, gestation and post-weaning), the reduction in food efficiency in the latter part of the lactation period was considered not to be test substance related.

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

Behaviour (functional findings):

not examined

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):

At terminal necropsy of P0 animals, the incidence and/or severity of several kidney findings in the 4000 ppm group were increased compared to the control group in female animals only (Table 4 under "Any other information on results incl. tables"). These findings included nephropathy, epithelial (urothelial) hyperplasia, medullary/papillary cysts, medullary fibrosis/degeneration, papillary tubular epithelial hyperplasia and inflammation in the medulla/papilla. The increases in nephropathy and medullary/papillary cysts were statistically significant (p <0.05). The nephropathy in the 4000 ppm group females was characterized by tubular basement membrane thickening, basophilic cortical tubular epithelium and/or interstitial infiltration of mononuclear cells. The medullary/papillary cysts in these animals were lined by cuboidal tubular epithelium, often contained cellular or amorphous debris (not casts) and were located primarily in the outer medullary stripe with some extension into the inner medulla and/or papilla. Fibrosis and degenerating tubules were often present adjacent to the cysts but were also observed within the medulla and/or papilla without cyst formation. Papillary tubular epithelial hyperplasia, characterized by increased numbers of basophilic epithelial cells along the tubular basement membrane, and minimal acute inflammation of the papilla, were observed only in the 4000 ppm group females. In the 4000 ppm group females, the medullary/papillary cysts, medullary degeneration/fibrosis and inflammation were generally present in the animals with more severe nephropathy, but were also present in some animals with minimal nephropathy. In the 4000 ppm group males and females, there was an increase in the incidence and severity of hyperplasia of the epithelium lining the renal pelvis (urothelial hyperplasia). In the 4000 ppm group females, the epithelium along the papilla was most often affected, and it was usually observed in animals without evidence of pyelitis, pelvic mineralization or calculi, suggesting a test article-related effect. For the males in this group, the urothelial epithelial hyperplasia was often secondary to inflammation (pyelitis or pyelonephritis) or pelvic mineralization, and correlated with calculi observed macroscopically. Furthermore, there was no dose-response relationship. For this reason, the hyperplasia was considered not to be a test substance-related effect in the males. The incidence and severity of the kidney lesions in the 20 and 200 ppm groups were similar to those in the control group and therefore, were considered not to be test substance-related. No test substance-related microscopic findings were observed in any other tissue examined in the 4000 ppm group animals.
No test substance-related microscopic lesions were observed in the P0 animals that died during the study.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The regularity and duration of estrous were not affected by treatment. Individual variation in the estrous cycle occurred sporadically in all study groups without toxicological relevance.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Administration of the test substance at concentrations of 20, 200 and 4000 ppm had no apparent effects on P0 spermatogenic endpoints (mean testicular and epididymal sperm numbers, sperm production rate and sperm motility and morphology). Differences between the control and treated groups were slight, without statistical significance and of none toxicological relevance.

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproductive performance of P0 animals was unaffected by test substance administration at dose levels of 20, 200 and 4000 ppm (Table 6 under "Any other information on results incl. tables"). Fertility indices were 79.3%, 86.7%, 86.7% and 86.2% for the P0 males and 79.3%, 86.7%, 86.7% and 86.7% for the P0 females in the control, 20, 200 and 4000 ppm groups, respectively. Mating indices were 100%, 93.3%, 100% and 100% for both the P0 males and females in the same respective dose groups. Males, which did not sire a litter numbered 6, 4, 4 and 4 in the control, 20, 200 and 4000 ppm groups, respectively. In the same respective dose groups, 6, 2, 4 and 4 females had evidence of mating but did not deliver; all of these females were non-gravid. 2 and 4 females in the control and 20 ppm groups, respectively, had no evidence of mating; with the exception of 2 females in the 20 ppm group, all of these females were gravid. The mean numbers of days between pairing and coitus in the treated groups were comparable to the control group values.
The mean lengths of gestation were comparable between the P0 treated groups and the control group. No differences were statistically significant. The mean gestation lengths in the 20, 200 and 4000 ppm groups were 22.0, 22.0 and 21.9 days, respectively, compared to mean gestation lengths of 21.9 days in the concurrent control group and the reproductive historical control data of the laboratory. No signs of dystocia were noted.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: actual test substance intake (P0 generation): males: 12.6 mg/kg bw/day, females: 18.3 mg/kg bw/day, combined sexes: 15.5 mg/kg bw/day

2

Key result

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food efficiency

histopathology: non-neoplastic

Remarks on result:

other: actual test substance intake (P0 generation): males: 249.8 mg/kg bw/day, females: 363.2 mg/kg bw/day, combined sexes: 306.5 mg/kg bw/day

3

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: actual test substance intake (P0 generation): males: 249.8 mg/kg bw/day, females: 363.2 mg/kg bw/day, combined sexes: 306.5 mg/kg bw/day

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

4 000 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs of toxicity were noted at any concentration for F1 parental animals. Clinical findings noted in the treated groups, such as hair loss and scabbing on various body surfaces and swollen ears, were noted infrequently and/or at similar frequencies in the control group.

Mortality:

no mortality observed

Description (incidence):

All F1 parental animals survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 4000 ppm group, mean body weights of F1 parental animals were statistically significantly lower (10 - 11%) than the control group values at the beginning of the pre-mating phase (Table 5 under "Any other information on results incl. tables"). In males, although the difference from the control group values gradually reduced to about 5% in Week 37, values were often statistically significantly different. Cumulative body weight gain in the males was reduced throughout the generation. Mean body weight of female animals was only reduced initially.
In detail, mean body weight gains of F1 males at 4000 ppm were lower than the control group values during Week 18 - 19 and 19 - 20 ; the differences were statistically significant (p <0.01). Throughout the remainder of the generation (Week 20 - 21 through 36 - 37), mean body weight gains in the 4000 ppm group F1 males were generally slightly lower than or comparable to the control group values. The majority of the differences were not statistically significant. Cumulative body weight gain from Week 18 onwards was reduced throughout the generation. Many of the differences were statistically significant (p <0.05 or p <0.01). Mean body weights in the 4000 ppm group males were lower than the control group values, and often statistically significant (p <0.05 or p <0.01), from Week 18 through Week 37. Initially, the values were about 11% below the control group values but the difference gradually reduced to about 5% by Week 37. In the 4000 ppm group F1 females, the mean body weight for Week 18 was reduced by about 10%, and statistically significant (p <0.05), when compared to the control group value. This decrease was attributed to the significantly reduced PND 21 mean body weight in these females (p <0.01). Mean body weight gains in the 4000 ppm group females were comparable to the control group values throughout the pre-breeding period, and mean body weights for these females were within 5% of the control group value by Week 20 and were within 3% of the control group value just prior to breeding (Week 28). Mean weekly body weights and body weight gains in the 20 and 200 ppm group F1 males and females were unaffected by test article administration. Several statistically significant differences from the control group values were observed. However, the changes were transient and/or did occur without a dose-response relationship. Therefore, the changes were attributed to biological variation.
During gestation, mean body weights and body weight gains were unaffected by test substance administration in the F1 parental females in the 20, 200 and 4000 ppm groups. The only statistically significant differences from the control group included an increased mean body weight gain in the 20 ppm group during Gestation Day (GD) 14 - 20 (and consequently during GD 0 - 20), a reduced mean body weight in the 4000 ppm group on GD 14 and an increased mean body weight in the 20 ppm group on GD 20 (p <0.05). However, the differences were slight, transient and/or did not occur in a dose-dependent manner. Therefore, they were attributed to biological variation.
During lactation, mean body weights and body weight changes were unaffected by test substance administration at all dose levels. The only statistically significant differences from the control group values were reduced mean body weights in the 4000 ppm group on lactation Days 4 and 7 (p <0.05). The differences were slight (5 - 6%) and were attributed to biological variation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 4000 ppm, food consumption, evaluated as g/animal/day, of F1 parental males was reduced (11%) during Week 18 - 19 through Week 21 - 22 when compared to the control group values. The differences were statistically significant (p <0.05 or p <0.01). Throughout the remainder of the generation (Week 22 - 23 through Week 36 - 37), g/animal/day food consumption values in the 4000 ppm group males was comparable to that in the control group. On a g/kg bw/day basis, food consumption values in the 4000 ppm group F1 males were often increased and statistically significant (p <0.05 or p <0.01). These increases were attributed to the differences in body weights observed between the control and 4000 ppm group males. Food consumption, evaluated as g/animal/day and g/kg bw/day, was unaffected by test substance administration in the 20 and 200 ppm group males and females and the 4000 ppm group females. Values of treated animals (g/kg bw/day) were occasionally increased and statistically significant (p <0.05 or p <0.01) when compared to the control group values. However, the corresponding g/animal/day values were comparable to the control group values, and the increases were attributed to differences in body weights between the control and treated groups.
During gestation, food consumption, evaluated as g/animal/day and g/kg bw/day, was unaffected by test substance administration at all dose levels throughout the gestation period. Statistically significant differences from the control group were increased g/animal/day values in the 20 ppm group during GD 0 - 4 and GD 14 - 20 (p <0.05). Similar increases were not observed at the higher concentrations of 200 and 4000 ppm, and no dose-response relationship was observed. Therefore, this finding was attributed to biological variation.
During lactation, food consumption (g/animal/day and g/kg bw/day) in the F1 females was unaffected by treatment at all dose levels. No statistically significant differences from the control group were observed.
For details on test substance intake see Table 1 - 3 under "Any other information on results incl. tables".

Food efficiency:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on food efficiency at all dose levels in both sexes, when comapred to control.
During gestation, as for food consumption, food efficiency was unaffected by test substance administration at all dose levels throughout the gestation period.
During lactation, in the F1 females food efficiency was unaffected by treatment at all dose levels. No statistically significant differences from the control group were observed.

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

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):

No test substance-related effects were observed on organ weight data (absolute or relative to final body weight) for the F1 males and females at any concentration.
Mean absolute kidney weight in the 4000 ppm group males was significantly (p <0.01) lower than the control group value. However, the corresponding relative weight was similar to the control group value, and the difference was attributed to differences in final body weights between the two groups. Mean absolute and/or relative thymus gland weights in the 200 and 4000 ppm group females were higher than the control group values; the differences were statistically significant (p <0.05). However, no dose-response relationship was apparent, and similar increases were not observed in the 200 and 4000 ppm group males. Therefore, the changes were attributed to biological variation. The mean absolute right epididymis weight in the 20 ppm group males was significantly (p <0.05) higher than the control group value. However, similar increases were not observed at the higher concentrations of 200 and 4000 ppm, and no dose-response relationship in correlation to the test substance-treatment was apparent. Mean relative testes, epididymides and spleen weights were significantly increased (p <0.05 or p <0.01) in the 4000 ppm group males and/or females when compared to the control group values. However, similar increases were not observed in the corresponding absolute weights, no test substance-related microscopic findings were observed in these tissues, and the changes were attributed to differences in final body weights between the control and 4000 ppm groups. Mean relative liver weights were significantly increased (p <0.01) in the 4000 ppm group males and females when compared to the control group values. However, the corresponding absolute liver weights in the 4000 ppm group were only slightly increased compared to the control group values and there were no microscopic findings correlated with the weight increases. Therefore, the increased liver weights were considered not to be an adverse effect of test substance administration.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At scheduled necropsy of F1 parental animals, no treatment-related internal findings were observed at any concentration. Findings in the treated groups, such as dilated renal pelves, dark red areas on the lungs, cystic oviducts, small testes, dark red contents of the ileum and reddened/enlarged lymph nodes were noted infrequently (generally in isolated animals) and/or at similar frequencies in the control group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the F1 parental generation, treatment-related changes in the kidney were similar to those observed in the P0 parental generation, but also occurred in the males (Table 4 under "Any other information on results incl. tables"). Both males and females in the 4000 ppm group showed increases in the incidences of medullary cysts and medullary degeneration/fibrosis. The increases were statistically significant (p <0.05). The severity of nephropathy was very slightly increased in these males and females. Additionally, the incidence of urothelial hyperplasia that was not secondary to pelvic mineralization or pyelitis/pyelonephritis was slightly increased in the 4000 ppm group males. The cysts, degeneration/fibrosis and urothelial hyperplasia were often observed in animals with moderate to severe nephropathy in both the males and females in this group (this trend was also observed for the 4000 ppm group P0 females). In females, inflammation of the medulla/papilla was observed only in the 4000 ppm group but in males it was observed in the 20 and 4000 ppm groups in a manner that was not dose-related. Papillary tubular hyperplasia was not observed in the F1 generation animals.
Generally, test substance-related lesions were consistently observed in the kidneys in the 4000 ppm group P0 females and F1 males and females. The most consistent change was the occurrence of medullary/papillary cysts and degeneration/fibrosis in the medulla. Other changes observed (urothelial hyperplasia, papillary tubular hyperplasia, inflammation of the papilla/medulla) did not occur consistently in a dose-related manner, or were not consistent within each sex and between generations. However, these changes occurred more frequently in kidneys that contained cysts or degeneration/fibrosis, and were considered to be probable effects of the test substance. Many of the changes in the kidney were suggestive of age-related nephropathy, which is a spontaneous lesion in rats of this age and strain. The increase in the incidence and/or severity of these findings in the 4000 ppm group suggests that the test substance exacerbated the spontaneous lesions. Although cysts are often observed as part of advanced nephropathy, the increase in medullary/papillary cysts in this study is noteworthy because of their number, size, location and somewhat unusual morphologic appearance. The incidence and/or severity of these kidney lesions in the 20 and 200 ppm groups were similar to those in the control group or were not dose-responsive and were not considered to be test substance-related. No test substance-related microscopic findings were observed in any other tissue examined in the 4000 ppm group animals.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The regularity and duration of estrous were not affected by treatment. Individual variation in the estrous cycle occurred sporadically in all study groups without toxicological relevance.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Administration of the test substance at concentrations of 20, 200 and 4000 ppm had no apparent effects on F1 spermatogenic endpoints (mean testicular and epididymal sperm numbers, sperm production rate and sperm motility and morphology). Differences between the control and treated groups were slight, without statistical significance and of none toxicological relevance.

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproductive performance of F1 parental animals was unaffected by test substance administration at dose levels of 20, 200 and 4000 ppm (Table 6 under "Any other information on results incl. tables"). Fertility indices were 90.0%, 93.3%, 96.7% and 86.7% for both the F1 males and females in the control, 20, 200 and 4000 ppm groups, respectively. Mating indices were 100%, 96.7%, 96.7% and 96.7% for both males and females in the same respective dose groups. Males, which did not sire a litter numbered 3, 2, 1 and 4 in the control, 20, 200 and 4000 ppm groups, respectively. Females, which had evidence of mating but did not deliver numbered 4, 1, 0 and 3 in the same respective dose groups; with the exception of one control group female, all of these animals were non-gravid. 1, 1 and 3 females in the 20, 200 and 4000 ppm groups, respectively, had no evidence of mating; 2 of the 4000 ppm group females delivered litters, and the remaining 3 females were non-gravid. The mean numbers of days between pairing and coitus in the treated groups were similar to the control group values; differences were slight and were not statistically significant.
The mean lengths of gestation were comparable between the F1 treated groups and the control group. No differences were statistically significant. The mean gestation lengths in the 20, 200 and 4000 ppm groups were 21.9, 21.9 and 21.8 days, respectively, compared to mean gestation lengths of 21.9 days in the concurrent control group and the reproductive historical control data of the laboratory. No signs of dystocia were noted.

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: actual test substance intake (F1 generation): males: 14.5 mg/kg bw/day, females: 20.0 mg/kg bw/day, combined sexes: 17.3 mg/kg bw/day

2

Key result

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

Remarks on result:

other: actual test substance intake (F1 generation): males: 300.6 mg/kg bw/day, females: 406.0 mg/kg bw/day, combined sexes: 353.3 mg/kg bw/day

3

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: actual test substance intake (F1 generation): males: 300.6 mg/kg bw/day, females: 406.0 mg/kg bw/day, combined sexes: 353.3 mg/kg bw/day

Target system / organ toxicity (P1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

4 000 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Pups, which were found dead or euthanized in extremis numbered 10, 7, 27 and 26 in the control, 20, 200 and 4000 ppm groups, respectively. In the same respective dose groups, 4, 3, 11 and 4 pups were missing and presumed to have been cannibalized. Although the numbers of pups that were found dead, euthanized in extremis and/or missing in the 200 and 4000 ppm groups were higher than in the control group, no dose-response relationship was observed. The increases were due to 1 female in each group with total litter loss (4000 ppm group) or loss of nearly the total litter (15 of 19 pups in the 200 ppm group). Therefore, no relationship to test substance administration was apparent. The general physical condition of the F1 pups during lactation was similar in all groups, including the control group.

Mortality / viability:

no mortality observed

Description (incidence and severity):

The mean live litter size, number of pups born, and percentage of males per litter at birth were unaffected by parental exposure to the test substance at all dose levels tested (Table 6 under "Any other information on results incl. tables"). No statistically significant differences from the control group values were observed. Pup survival was not affected by parental exposure in the 20, 200 and 4000 ppm groups from birth to PND 0, PND 0 - 1, PND 1 - 4, PND 4 - 7, PND 7 - 14, PND 14 - 21, birth to PND 4, and PND 4 - 21. No statistically significant differences from the control group values were observed. Pup viability in the 200 and 4000 ppm groups was slightly reduced from birth to PND 0 when compared to the control group. This was mainly attributed to one female in each group, which lost almost the whole litter or the whole litter, respectively. However, viability from birth to PND 4 pre-selection in the 4000 ppm dose group was greater than that in the 200 ppm dose group (92.3% compared to 87.9%, respectively). Since there was no evidence of a dose-response relationship or statistical significance, this decrease was considered incidental and not to be a test substance-related effect.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 4000 ppm dose group, F1 pup body weight was significantly lower (11%) than the control group value on PND 21 although there was no treatment-related effect at birth (Table 6 under "Any other information on results incl. tables"). In detail, mean F1 pup body weights in the 4000 ppm group were comparable to the control group values on PND 1, 4 (before and after selection), 7 and 14. On PND 21, mean pup body weight in this group was 11% lower than the control group value. The difference was statistically significant at p <0.01. Mean F1 pup body weights were unaffected by parental treatment at concentrations of 20 and 200 ppm. No statistically significant differences from the control group were observed.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

In the 4000 ppm group, there was a slight delay in the achievement of balanopreputial separation, which was due to a significant reduction in body weight/body weight gain in these prepubertal male offspring (Table 6 under "Any other information on results incl. tables"). However, this is a well-established correlation demonstrating a secondary sign of systemic toxicity, not indicating inherent reproductive toxicity potential of the test substance.
In detail, a delay in the occurrence of balanopreputial separation was evident in the 4000 ppm group. The mean days of balanopreputial separation observed for males in the control, 20, 200 and 4000 ppm groups were PND 43.3, 44.1, 44.3 and 46.2, respectively. The difference between the control and 4000 ppm group values was statistically significant (p <0.01). The mean body weights on the day of acquisition of balanopreputial separation in the 20, 200 and 4000 ppm groups were similar to the control group value. However, weekly body weights in the 4000 ppm group during the period of achievement of balanopreputial separation (Week 19 - 21) were reduced by 9 - 11% and the reduction reached statistical significance (p <0.05 or p <0.01) compared to the control group value.
In the 4000 ppm group, there was a slight delay in the achievement of vaginal patency due to a significant reduction (10 - 11%) in body weight of these peripubertal F1 female offspring (Table 6 under "Any other information on results incl. tables"). However, this is a well-established correlation demonstrating a secondary sign of systemic toxicity, not indicating inherent reproductive toxicity potential of the test substance.
In more detail, a delay in the occurrence of vaginal patency was observed in the 4000 ppm group. The mean days of vaginal patency noted for females in the control, 20, 200 and 4000 ppm groups were 33.2, 33.2, 33.7 and 34.8, respectively. The difference between the control and 4000 ppm group values was statistically significant (p <0.01). The mean body weights on the day of acquisition of vaginal patency in the 20, 200 and 4000 ppm groups were similar to the control group value. However, weekly body weights in the 4000 ppm group during the period of achievement of vaginal patency (PND 21 - Week 19) were reduced by 7 - 11% compared to the control group value. The Week 18 value was statistically significantly different (p <0.05).
The day of acquisition of both vaginal patency and balanopreputial separation has been shown to be delayed by reduced body weight. Because both of these parameters were delayed in the 4000 ppm dose group only, the delay was attributed to the clearly reduced post-weaning body weights rather than being an endocrine-mediated event.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Organ weights (absolute and relative to final body weight) in the selected F1 weanlings were unaffected by treatment at all concentrations. Mean absolute brain weights in the 4000 ppm group males and females were lower than the control group values; the difference in the females was statistically significant (p <0.01). However, mean relative brain weights in the 4000 ppm group males and females were higher than the control group values; the differences were statistically significant (p <0.05 or p <0.01). Therefore, the differences in absolute weights were attributed to differences in body weights between the control and 4000 ppm groups. The mean absolute spleen weight in the 4000 ppm group females was reduced, and statistically significant (p <0.05), when compared to the control group value. However, mean relative spleen weight in these females was similar to the control group value, and the difference was attributed to differences in body weights between the groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No internal findings that could be attributed to parental treatment with the test substance were noted at the necropsies of pups that were found dead or were euthanized in extremis. Malformations were noted in 1, 1 and 2 pups in the control, 200 and 4000 ppm groups, respectively, and included the following. One control group pup had an enlarged right kidney and agenesis of the left kidney and ureter. In the 200 ppm group, one pup had anophthalmia and exencephaly without open eyelids. At a concentration of 4000 ppm, one pup had vertebral agenesis with a filamentous tail, and another had microphthalmia. Aside from the presence or absence of milk in the stomach, the only other internal findings in pups that died or were euthanized in extremis consisted of a submeningeal hemorrhage for 1 of the control group pups and hemorrhagic bladders for 3 pups in the 200 ppm group.
At the necropsy of surplus F1 pups on PND 21, no treatment-related internal findings were noted at any concentration. Dilated renal pelves were noted for 6, 5, 2 and 4 pups in the control, 20, 200 and 4000 ppm groups, respectively. Of these, 2 pups in the 20 ppm group and 1 pup in the 200 ppm group also had distended ureters and 1 pup in the 4000 ppm group also had a cystic kidney. 1 pup in each of the 20 and 4000 ppm groups had small testes. Malformations were noted for 1 pup in each of the control and 20 ppm groups; 1 control group pup was hydrocephalic (the anomaly consisted of increased cavitation of both lateral ventricles), and 1 pup in the 20 ppm group had agenesis of the left testis and epididymis.
At the necropsy of selected F1 weanlings on PND 21, no treatment-related internal findings were observed at any concentration. 2, 2 and 1 pups in the control, 20 and 200 ppm groups, respectively, had dilated renal pelves and/or distended ureters. One pup in the 20 ppm group had a subcutaneous hemorrhage in the scapular region. No other remarkable internal findings were noted in the selected F1 weanlings.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Generation:

F1

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: delayed sexual maturation due to reduced body weight (systemic toxicity) at 4000 ppm

Remarks on result:

other: actual test substance intake of F1 generation: males: 14.5 mg/kg bw/day, females: 20.0 mg/kg bw/day, combined sexes: 17.3 mg/kg bw/day

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related findings. Pups which were found dead numbered 7, 3, 9 and 17 in the control, 20, 200 and 4000 ppm groups, respectively. In the same respective dose groups, 3, 1, 6 and 1 pups were missing and presumed to have been cannibalized. The general physical condition of the F2 pups during lactation was similar in all groups, including the control group.

Mortality / viability:

no mortality observed

Description (incidence and severity):

The mean live litter size, number of pups born, and percentage of males per litter at birth were unaffected by the F1 parental exposure in the 20, 200 and 4000 ppm groups (Table 6 under "Any other information on results incl. tables"). The only statistically significant differences from the control group were an increased mean live litter size and an increased number of pups born in the 20 ppm group (p <0.01). However, similar increases were not observed at the higher dose levels of 200 and 4000 ppm, and no dose-response relationship to the test substance was apparent. Pup survival was not affected by parental exposure in the 20, 200 and 4000 ppm groups from birth to PND 0, PND 0 - 1, PND 1 - 4, PND 4 - 7, PND 7 - 14, PND 14 - 21, birth to PND 4, and PND 4 - 21. No statistically significant differences from the control group values were observed.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean F2 pup body weights were unaffected by parental treatment at concentrations of 20, 200 and 4000 ppm (Table 6 under "Any other information on results incl. tables"). No statistically significant differences from the control group were observed.

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:

no effects observed

Description (incidence and severity):

No adverse effects on mean organ weights (absolute and relative to final body weights) were observed in the F2 pups at any concentration. No statistically significant differences from the control group were observed.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In addition to the absence of milk in the stomach, remarkable necropsy findings for pups found dead during the postnatal period included the following. One pup in the 4000 ppm group had a malformation (microphthalmia). In the same group, one pup of another litter had a major blood vessel variation (a retroesophageal right subclavian artery). At the necropsy of surplus F2 pups on PND 21, no treatment-related internal findings were noted at any concentration. Dilated renal pelves were noted in 3, 8, 5 and 1 pups in the control, 20, 200 and 4000 ppm groups, respectively. One of the 20 ppm group pups also had a distended ureter. Malformations were noted for two pups in one litter in the 200 ppm group. Both of these pups were hydrocephalic (the anomaly consisted of increased cavitation of both lateral ventricles and the third ventricle). At the necropsy of the selected F2 weanlings on PND 21, no treatment-related internal findings were noted. Dilated renal pelves were noted in 1 pup in each of the 20 and 200 ppm groups. No other internal findings were observed.

Histopathological findings:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Generation:

F2

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: highest dose tested; actual test substance intake: P0 generation: males: 249.8 mg/kg bw/day, females: 363.2 mg/kg bw/day; F1 generation: males: 300.6 mg/kg bw/day, females: 406.0 mg/kg bw/day

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

4 000 ppm

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Any other information on results incl. tables

Table 1: Mean test substance intake in the male parental animals

Males	Compound intake [mg/kg bw/day]
dietary level [ppm]	Prior to breeding		After breeding		Overall mean
	P0	F1	P0	F1	P0	F1
20	1.4	1.7	0.9	1.0	1.2	1.5
200	14.4	17.4	9.5	9.8	12.6	14.5
4000	285.3	359.0	190.6	203.4	249.8	300.6

 

Table 2: Mean test substance intake in the female parental animals

Females	Compound intake [mg/kg bw/day]
dietary level [ppm]	Prior to breeding		Gestation		Lactation*		Overall mean
	P0	F1	P0	F1	P0	F1	P0	F1
20	1.7	1.9	1.4	1.4	3.1	3.2	1.8	2.0
200	16.7	19.7	13.4	14.3	31.5	31.2	18.3	20.0
4000	346.5	405.2	266.8	274.2	577.4	634.6	363.2	406.0

  * Values for the entire lactation period were elevated presumably due to the offspring diet consumption during the postnatal period.

Table 3: Combined test substance intake

	Compound intake [mg/kg bw/day]
Dietary level [ppm]	P0		F1		Combined generations
	♂	♀	♂	♀	♂	♀
20	1.2	1.8	1.5	2.0	1.35	1.9
200	12.6	18.3	14.5	20.0	13.55	19.15
4000	249.8	363.2	300.6	406.0	275.2	384.6

Table 4: Histopathological findings in the kidney of the parental animals

Parameter

Genera­tion

Controls

20 ppm

200 ppm

4000 ppm

Dose-response

♂

♀

♂

♀

♂

♀

♂

♀

♂

♀

Histopathology Kidneys

Incidence

Nephropathy

P0

27/30

9/29

26/30

12/30

27/29

10/30

22/28

20/30*

-

-

F1

24/30

17/30

26/30

16/30

28/30

19/30

28/30

21/30

-

-

Mineralization, tubular

P0

2/30

16/29

0/30

12/30

2/29

17/30

2/28

21/30

-

-

F1

5/30

15/30

1/30

18/30

1/30

20/30

2/30

15/30

-

-

Lymphoid infiltrate, interstitial

P0

1/30

4/29

2/30

4/30

0/29

5/30

1/28

2/30

-

-

F1

2/30

0/30

1/30

5/30

1/30

3/30

0/30

1/30

-

-

Mineralisation, papillary tubular

P0

3/30

2/29

1/30

6/30

3/29

5/30

3/28

2/30

-

-

F1

1/30

0/30

0/30

2/30

0/30

1/30

1/30

4/30

-

-

Mineralisation, pelvic

P0

2/30

2/29

1/30

0/30

0/29

0/30

1/28

1/30

-

-

F1

2/30

1/30

1/30

2/30

1/30

0/30

5/30

8/30

-

-

Hyperplasia, urothelial epithelial

P0

1/30

0/29

4/30

2/30

3/29

2/30

4/28

9/30

-

-

F1

4/30

3/30

3/30

0/30

1/30

7/30

13/30

7/30

-

-

Cyst medullary/papillary

P0

3/30

0/29

3/30

0/30

4/29

0/30

1/28

11/30*

-

-

F1

1/30

0/30

3/30

1/30

3/30

5/30

21/30*

14/30*

-

-

Inflammation medulla/papilla

P0

0/30

0/29

0/30

0/30

0/29

0/30

2/28

5/30

-

-

F1

0/30

0/30

1/30

0/30

0/30

0/30

1/30

2/30

-

-

Hyperplasia, epithelial, papillary tubular

P0

0/30

0/29

0/30

0/30

2/29

0/30

0/28

5/30

-

-

F1

n.d.

0/30

n.d.

0/30

n.d.

1/30

n.d.

0/30

-

-

Degeneration/Fibrosis, medullary

P0

3/30

4/29

4/30

1/30

4/29

0/30

5/28

12/30

-

-

F1

0/30

0/30

4/30

2/30

2/30

4/30

22/30*

15/30*

-

-

 * significantly different from control (p<0.05, Kolmogrov-Smirnov test)

** significantly different from control (p<0.01, Kolmogrov-Smirnov test)

 

Table 5: Body weight development in parental animals

Parameter			Genera­tion		Controls				20 ppm				200 ppm				4000 ppm		Dose-response
					♂		♀		♂		♀		♂		♀		♂	♀	♂	♀
Body weight gains		[g]
	Week 0-1		P0	64		27		66		24		66		26		59**		26	-	-
	Week 18-19		F1	58		39		54		41		55		37		52**		39	-	-
	Week 0-5		P0	224		97		222		90		228		93		209*		95	-	-
	Week 18-23		F1	282		135		280		137		271		128		260**		137	-	-
	Week 0-10		P0	322		134		316		125		325		129		292**		131	-	-
	Week 18-28		F1	414		186		413		189		402		182		392		185	-	-
	Week 0-19		P0	398		164		386		156		409		159		368*		158	-	-
	Week 18-37		F1	517		232		524		232		509		225		495		228	-	-

 * significantly different from control (p <0.05, Dunnett’s test)

** significantly different from control (p <0.01, Dunnett’s test)

 

Table 6: Reproductive performance

Parameter		Genera-tion	Controls		20 ppm	200 ppm	4000 ppm	Dose response
Mating index	%	P0	100.0		93.3	100.0	100.0	-
		F1	100.0		96.7	96.7	96.7	-
Fertility index	%	P0	79.3		86.7	86.7	86.7	-
		F1	86.7		93.3	96.7	86.7	-
Gestation length	Mean [days]	P0	21.9		22.0	22.0	21.9	-
		F1	21.9		21.9	21.9	21.8	-
Gestation index	%	P0	100.0		96.2	96.2	96.2	-
		F1	100.0		100.0	100.0	96.2	-
Live birth index	%	F1	98.8		99.2	95.1	94.3	-
		F2	99.4		100.0	98.4	94.2	-
Litter size	Mean	F1	13.5	12.3		14.2	13.6	-
		F2	12.5	14.4**		12.9	12.4	-
Pup weight PND1	Mean [g]	F1	6.7	6.8		6.4	6.6	-
		F2	6.8	6.7		6.9	6.7	-
Pup weight PND21	Mean [g]	F1	49.7	49.8		48.7	44.1**	-
		F2	47.1	47.2		47.8	45.6	-
Sex ratio	% Males	F1	55.0	48.6		53.0	53.1	-
		F2	48.7	49.7		51.2	51.1	-
Viability index	%	F1	97.0	96.5		87.9	92.3	-
		F2	97.6	99.3		96.1	93.8	-
Lactation index	%	F1	97.8	94.2		99.5	100.0	-
		F2	99.0	99.6		99.1	99.5	-
Sexual maturation	Males [PND]	F1	43.3	44.1		44.3	46.2**	-
	Females [PND]	F1	33.2	33.2		33.7	34.8**	-
Sperm characterization	% with normal appearance	P0	99.0	99.2		99.4	99.2	-
		F1	98.9	98.8		98.8	99.2	-
	% sperm motility	P0	83.0	81.9		85.0	81.8	-
		F1	80.2	79.6		77.8	80.0	-
	Sperm production rate [106/g/day]	P0	18.3	18.6		17.3	18.1	-
		F1	14.5	14.3		13.5	13.9	-

 ** significantly different from control (p <0.01, Dunnett’s test)

Applicant's summary and conclusion

Conclusions:

Dietary, sub-chronic administration of the test substance at a dose level of 4000 ppm (highest dose tested) to rats for 2 successive generations induced systemic toxicity (as evidenced by reductions in body weight gain and/or body weight and food intake/efficiency and an increase in microscopic kidney lesions) in the P0 and F1 generation parental males and females. Neonatal toxicity was observed as reduced F1 pup body weights in the 4000 ppm group. Delays in the achievement of vaginal patency and balanopreputial separation were observed in the F1 animals in the 4000 ppm group as secondary signs of systemic toxicity due to reduced body weight. The study provided no evidence of a reproductive toxicity potential of the test substance since the observed effects were in fact secondary to systemic toxicity.
The No Observed Adverse Effect Level (NOAEL) for both reproductive and systemic toxicity was 200 ppm, equivalent to a mean actual test substance intake of 12.6, 18.3 and 15.5 mg/kg bw/day in P0 males, females and combined sexes, respectively, and to 14.5, 20.0 and 17.3 mg/kg bw/day in F1 males, females and combined sexes, respectively. The Lowest Observed Adverse Effect Level (LOAEL) for both reproductive and systemic toxicity was 4000 ppm, equivalent to a mean actual test substance intake of about 306.5 mg/kg bw/day in P0 males and females, and to 353.3 mg/kg bw/day in F1 males and females combined. In the F2 generation, where no effects were evident within the observation period, the NOAEL for systemic and reproductive toxicity corresponded to the highest dose tested, i.e. 4000 ppm.