Phosphonic acid, monoethyl ester, sodium salt (1:1)

Toxicity to reproduction

Administrative data

Endpoint:

three-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Sep 1977 - 25 May 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

Reproductive toxicity of the test substance was determined in a three-generation study. Parental generations were exposed orally to the test substance for at least 90 days prior to the first mating and were kept on the respective diets continuously throughout the study. Each parental generation was mated 2 times. The first litter was reared to 21 days post partum prior to sacrifice and macroscopic examinations. Approx. 10 days after weaning of the first offspring, animals were mated again to produce the second offspring of the respective generation, from which the following parental animals were selected. In addition, 5 P and 10 F1 dams of the second reproduction cycle were sacrificed on Day 20 of gestation for teratological examinations. Moreover, 10 animals per sex from the third generation were subjected to histopathology 21 days post partum.

GLP compliance:

no

Remarks:

Study start before GLP principles were introduced.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium triethyl triphosphonate

EC Number:

254-320-2

EC Name:

Aluminium triethyl triphosphonate

Cas Number:

39148-24-8

Molecular formula:

C2H7O3P.1/3Al

IUPAC Name:

aluminum tris(ethyl phosphonate)

Test animals

Species:

rat

Strain:

other: CFY (specific pathogen free)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Anglia Laboratory Animals, Alconbury, Huntingdon, Cambridgeshire, England
- Age at study initiation: (P) no specific information given; (F1 and F2) at least 90 days
- Weight at study initiation: (P) Males: 60 - 70 g; Females: 60 - 70 g; (F1): 79 g, (F2): 68 g
- Fasting period before study: no
- Housing:
pre-mating: 5 animals of the same sex per cage in suspended galvanised metal cages (Bowman R) equipped with solid sides and back and wire mesh front,
mating period: 1 male and 1 (P and F1 pairings) or 2 (F2 pairing) females in plastic breeding cages,
post-mating: males returned to the original cages and females stayed in the breeding cages until weaning of the first litter or final termination
- Diet: Spratt´s Laboratory Diet No. 2 - low fat, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 4
- Humidity (%): 50 ± 5
- Air changes (per hr): 12 - 14
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 20 Sep 1977 To: 25 May 1979

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): each week
- Mixing appropriate amounts with (Type of food): standard diet

Details on mating procedure:

- M/F ratio per cage:
P and F1 generation: 1/1,
F2 generation: 1/2
- Length of cohabitation: 20 - 21 days
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy
- Any other details on mating:
Animals from the P generation (F0) were mated two times. The first litter (F1A pups) was reared to Day 21 post partum prior to sacrifice for further post mortem examinations. Approx. 10 days after weaning of F1A pups, the F0 animals were re-mated for a period of 20 days employing different mating pairings to produce the second offspring (F1B).

F1 A pups examined and discarded at day 21 post partum, 5 litters of the second F0 mating (F1 B pups killed at day 20 of pregnancy for teratology examination. The remaining F0 dams were allowed to rear their offfspring until Day 21 post partum. At this time point, 25 males and females were selected from each group for the F1B generation. The remaining pups (F1B) and parent animals (F0) were sacrificed and included in macroscopically examinations.

F1B animals were reared on the respective diet until reaching the age of at least 90 days and were then mated for a period of 20 - 21 days. The resulting F2A generation was reared until Day 21 post partum followed by sacrifice and necropsy. Approx. 10 days after the weaning of F2A pups, the F1B animals were re-mated for a period of 20 - 21 days employing different mating pairings. 10 F1B litters (F2B pups) were sacrificed on Day 20 of gestation. 15 animals/sex (F2B pups) were reared on the respective diet for at least 91 days prior to scheduled sacrifice for necropsy. 12 males and 24 females (were reared on their diet for 91 days prior tothe matings. The remaining F2B pups and and F1B dams were killed after rearing and examined.
Mating of F2B animals occurred with 1 male cohoused with 2 females, employing different partners at the second mating. All pups of the F3A generation were examined and discarded 21 days post partum. 10 F3B animals per sex were subjected to organ weight determination 21 days post partum. Histopathology analysis were restricted to the control and high-dose groups except urinary bladder analysis, which was examined for all dose groups. Remaining F3B pups and F2B parental animals were examined and discarded 21 days post partum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dietary concentrations at weeks 1, 13, 26, 39, 52, 65 and 78 were tested to ensure homogeneity, stability and accuracy of diet concentrations via gas-liquid chromatography. Homogeneity data indicated a satisfactory mix of the test compound with the diet. Further, stability data showed that no significant decomposition occurred over a time period of 7 days. Moreover, with the exception of week 1 (6000 ppm: -21.8% from nominal concentration) and week 26 (6000 ppm: -17.3% and 12000 ppm: - 19.3%) diets, the actual concentrations were within 15% deviations of the nominal concentrations.

Duration of treatment / exposure:

(P/F0) Males: 90 days before mating, continuously until weaning of F1B offspring.
(P/F0) Females: 90 days before mating, continuously until weaning of F1B offspring
(F1) Males: 21 days at weaning, during growth into adulthood (at least 90 days), mating and production of the F2A and F2B generation
(F1) Females: 21 days at weaning, during growth into adulthood (at least 90 days), mating and production of the F2A and F2B generation, until weaning of the F2B generation.
(F2) Males: 21 days at weaning, during growth into adulthood (at least 90 days), mating and production of the F3A and F3B generation
(F2) Females: 21 days at weaning, during growth into adulthood (at least 90 days), mating and production of the F3A and F3B generation, until weaning of the F3B generation.
(F3): until sacrifice on PND 21

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

- F1 parental animals not mated until at least 90 days after selected from the F1B litters.
- Selection of parents from F1B generation when pups were 21 days of age.
- Selection of parents from F2B generation when pups were 21 days of age.
- Age at mating of the mated animals in the study:
P: not further specified
F1B: first mating: at least 111 days, second mating: approx. 141 days
F2B: first mating: at least 112 days, second mating: approx. 143 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

6 000 ppm (nominal)

Remarks:

P: 479 mg/kg bw/day; F1B: 546 mg/kg bw/day; F2B: 608 mg/kg bw/day
mean dose value as calculated from the reported body weight and food intake values determined during the first pre-mating period

Doses / concentrations 2

Dose / conc.:

12 000 ppm (nominal)

Remarks:

P: 933 mg/kg bw/day; F1B: 1134 mg/kg bw/day; F2B: 1212 mg/kg bw/day
mean dose value as calculated from the reported body weight and food intake values determined during the first pre-mating period

Doses / concentrations 3

Dose / conc.:

24 000 ppm (nominal)

Remarks:

P: 1890 mg/kg bw/day; F1B: 2813 mg/kg bw/day; F2B: 2721 mg/kg bw/day
mean dose value as calculated from the reported body weight and food intake values determined during the first pre-mating period

No. of animals per sex per dose:

25 P males and females
25 F1B males and females
27 F2B males and approx. 39 F2 females (including 15 non-mated animals/sex sacrificed at week 15 for macroscopic examination; 12 males and 24 females selected for mating)
10 F3 males and females

Control animals:

yes, plain diet

Positive control:

not included

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: regularly (not further described)
- Cage side observations included examination for signs of reaction to treatment.

BODY WEIGHT: Yes
- Time schedule for examinations:
P: weekly;
F1 - F3: weighing at birth and thereafter on Days 4, 8, 12 and 20;
P, F1 and F2 females: on alternate days during the mating period until pregnancy, thereafter on Days 7, 14, 17 and 20 of gestation and on Days 0, 7, 14 and 21 post partum

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule: weekly during the first pre-mating phase of each generation
- 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: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: during the first and penultimate week of the first pre-mating phase

OTHER:
- URINALYSIS: YES
Due to the observed kidney damage of 5 high-dose males which died prior to sacrifice, individual urine samples were collected overnight from 10 F1B control and high-dose pups during week 7.
Urinalysis included examination of: urinary volume, urinary pH, specific gravity, quantitative analysis of protein, reducing substances, glucose, ketones, bile pigments, urobilinogen, haemoglobin, microscopic analysis of spun deposit (epithelial cells, polymorphonuclear leucocytes, mononuclear leucocytes, erythrocytes, organisms, casts and abnormal constituents).

PROOF OF PREGNANCY:
- Time schedule: daily


FURTHER DETERMINATIONS:
pregnancy rate and length of gestation period

Sperm parameters (parental animals):

Parameters examined in P, F1 and F2 male parental generations which failed to induce pregnancy:
testis weight and histological examination of testes

Litter observations:

STANDARDISATION OF LITTERS
- Performed on Day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1B - F3B offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain (individually on Days 0, 4, 8, 12 and 21 post partum)

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

OTHER:
Selection of parental F1 and F2 generations: pups selected for parental generations were chosen from as many litters as possible by selecting the pups closest to the median weight of the respective sex in each litter. Brother to sister matings were avoided. For the F1B generation, 2 extra animals per sex were chosen to enable replacement if necessary. Unused replacement animals were sacrificed prior to the first mating.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: all surviving animals after weaning of the second offspring
- Maternal animals: all surviving animals after weaning of the second offspring
Furthermore, in addition to mated animals, 15 unmated animals/sex of the F2B generation were sacrificed at week 15 for macroscopic examination.
- 5 litters (F0 dams /F1B pups) and 10 litters (F1B dams/F2B pups) were sacrificed on Day 20 of gestation for teratological examinations.

GROSS NECROPSY
- After weaning of the second litters, the parental animals were sacrificed and examined macroscopically. Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
- In general, microscopic examination was restricted to animals of the F3B generation. Testes of parental males which failed to induce pregnancy and organs of the urinary tract showing abnormalities in gross pathology were included in histological examinations.

TERATOLOGY
On Day 20 of the second gestation, 5 litters (F0 dams /F1B pups) and 10 litters (F1B dams/F2B pups) were sacrificed for the examination of congenital abnormalities and macroscopic changes in maternal organs. Ovaries and uteri were examined to evaluate the number of corpora lutea, number and distribution of live young and number and distribution of embryonic/fetal deaths. For each litter, pre-implantation and post-implantation loss was determined.

Postmortem examinations (offspring):

SACRIFICE
- The F1 and F2 offspring not selected as parental animals and all F3 offspring animals were sacrificed at Day 21 of age.
- Offspring animals were subjected to postmortem examinations as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
In general, micorscopic examination was restricted to animals of the F3B generation. Routinely microscopically examined organs from control and high-dose F3B animals: brain, eye, heart, lung, liver, spleen, kidney, stomach, duodenum, ileum, caecum, salivary gland, pancreas, lymph nodes, thymus, thyroid, pituitary, adrenals, testes, seminal vesicle, ovaries, uterus and any other tissue which appeared macroscopically abnormal. Furthermore, the urinary bladder was examined of 10 animals/sex from all dose groups of the F3B generation. Furthermore, organs with abnormal appearance in gross pathology from P, F1B and F2B offspring were included in histology (both matings).

Statistics:

Mean values and standard errors were calculated from the examined parameters. For litter data, group mean values were generally calculated in two ways: mean A generally includes all animals showing evidence of pregnancy at Day 20 of gestation (interim sacrificed animal) or at birth; mean B includes only dams with viable youngs either at Day 20 of gestation or at day 21 post partum.

Litter weights, mean pup weights and incidences of abnormality are only calculated as Mean B values. For all values expressed as a ratio e.g. pre-and post-implantation losses, group mean values are derived as the mean of percentages within individual litters.

As litter values do not follow a "normal" distribution, intergroup differences are analysed by non-parametric statistical methods using the litter as the basic sample unit.

Organ weights of the F3B generation were analysed by analysis of variance adjusting for body weight at sacrifice as covariate, provided there was found to be a significant relationship (F-test; P<0.1). Treatment means were compared with control values by the method of L.S.D. i n conjunction with Williams ‘test.

Offspring viability indices:

Offspring viability was calculated at birth and cumulatively at postnatal days 4, 8, 12 and 21. Group mean values were calculated from individual litter percentage values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

6000 ppm: 2 females died
12000 ppm: 1 male died
24000 ppm: 2 females died

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

24000 ppm: In females, a reduced body weight about 11% was only observed during the last 2 weeks of lactation of the second mating cycle. In males, overall weight gains were lower than those of controls.
Due to the rather isolated appearance, the effect on body weight and weight gain was considered incidental and not related to treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption determined during the first pre-mating period (week 1 - 13) was comparable between test groups and corresponding controls.

Food efficiency:

no effects observed

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

not specified

Description (incidence and severity):

Water consumption of both sexes at 24000 ppm was consistently greater than that of controls during the first week. In the week immediately prior to mating (week 12) the above pattern of differences in water consumption was not quite so consistent.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

24000 ppm: One female showed marked bilateral chronic renal pyelonephritis. Small quantities of purulent debris in the lumen of some tubules and the renal pelvis of one kidney. Due to the occurnec in single animals, the effect is not considered as treatment-related.

In unscheduled deaths, one male showed extensive deposits of lymphosarcoma (mediastinum) and deposits of lymphosarcomainvading adjacent connective tissues (cervical lymph node)

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Test substance intake: decreased nominal test substance intake in all dose groups prior to the first mating

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):

For the most part mating performance, as assessed by the chronological sequence of mating and numbers of females becoming pregnant, was comparable for all groups at both matings.
The mean duration of gestation was comparable for all groups at each mating.

Details on results (P0)

MATERNAL DEVELOPMENTAL TOXICITY
- Dead fetuses:
24000 ppm: An average of nine live fetuses were generated by dams, which is statistically significant from the control value (14.8)

- Pre- and post- implantation loss:
There was a dose-dependent increase in post- implantation loss at 6000 ppm, 12000 ppm, and 24000 ppm (7.0%, 13.2%, and 31.4%, respectively)

- Total littler losses by resorption: no adverse effects observed

- Early and late resorptions: no adverse effects observed

- Changes in the number of pregnant animals: no adverse effects observed

- Changes in pregnancy duration: not exaimed

- Other:
- Corpora lutea: effects observed, treatment related
24000 ppm: Significantly reduced compared to control
12000 ppm: Reduced number, not statistically significant

- Number of implantations: effects observed, treatment related
24000 ppm: Frequently statistically significant lower value compared to control
12000 ppm: Lower number of implantations per dam, not statistically significant

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 24 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: equivalent to 1958 mg/kg bw/day (lowest mean),1958 mg/kg bw/day in P0 males and 2125 mg/kg bw/day in P0 females , respectively

2

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: equivalent to 954 mg/kg bw/day (lowest mean), 954 mg/kg bw/day in P0 males and 1056 mg/kg bw/day in P0 females , respectively

3

Key result

Dose descriptor:

LOAEL

Remarks:

fertility

Effect level:

24 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Decreased corpora lutea (statistically significant) and reduced implantation sites (considered secondary based on the effect on corpora lutea)

Remarks on result:

other: equivalent to 1958 mg/kg bw/day (lowest mean),1958 mg/kg bw/day in P0 males and 2125 mg/kg bw/day in P0 females , respectively

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 958 mg/kg bw/day (nominal)

System:

female reproductive system

Organ:

ovary

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

P1B
6000 ppm: 1 female died (non treatment-related, non-adverse)
12000 ppm: 1 male died (non treatment-related, non-adverse)
24000 ppm: 7 males and 1 female died (treatment-related, adverse)

P2B:
12000 ppm: 1 male and 2 females died (non treatment-related, non-adverse)
24000 ppm: 3 males died (treatment-related, adverse)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

P1B
24000 ppm: Significantly reduced body weights in males during pre-mating (-39% week 1 and -12.7% week 13) and first and second mating. In females, a similar but weaker effect on reduced body weight was observed. In females, reduced body weights were observed at 8.8%, 8.2%, and 14.5% at the end of pre-mating and first and second mating, respectively. Statistically reduced body weight in females during pre-mating (32.5% week 1 and 10.5% week 13), gestation (7.4%, Day 0) and lactation (16.9%, Day 21).


P2B
6000 ppm: In females, during the last two weeks of the second mating phase (weeks 29 and 30) reduced body weights were observed at 2.5% and 5.6%, respectively.
12000 ppm: In males, reduced body weights about 8.4 ± 5.5%, 9.4 ± 0.45% and 10.2 ± 0.34% during the pre-mating phase (14.5% week 1) and the first and second mating period, respectively, were observed. In females, reduced body weights were observed at 3.9%, 3.7%, and 12.1% at the end of the pre-mating phase (12% week 1) and the first and second mating period, respectively.
24000 ppm: In males, reduced body weights were observed at 4.2%, 12.1%, and 12.5% at the end of pre-mating (23.6% week 1 and 8.8% week 13), first and second mating phase, respectively. In females, reduced body weights were observed at 3.2%, 7.3%, and 16.6% at the end of pre-mating (20% week 1), first and second mating phase, respectively.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption determined during the first pre-mating period (week 1 - 13 for P2B and week 1 - 10 for P1B generation) was comparable between test groups and corresponding controls except a lower food consumption of P1B males during the initial rearing phase and a lower food consumption of P1B and P2B females during the first weeks of rearing in the 24000 ppm group. As food consumption was not affected through all different periods of the study, the effect is rather considered non-treatment-related.

Food efficiency:

no effects observed

Description (incidence and severity):

No effects observed neither in P1B nor in P2B.

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

not specified

Description (incidence and severity):

For both P1B and P2B generations, water consumption of both sexes at 24000 ppm was consistently greater than that of controls during the first week. In the week immediately prior to mating (week 12, P2B generations; week 9, P1B generation) the above pattern of differences in water consumption was not quite so consistent.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis was performed on 10 control and 10 24000 ppm males of P1B
A possible indication of the onset of early urinary tract damage at 24000 ppm was provided by the significantly (P<0.01) lower specific gravity and the recording of epithelial or polymorphonuclear cells in the urine of 4/10 test group males compared with 0/10 for controls.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

P1B
6000 ppm: In two males, pitted kidneys observed. Two females showed (slightly) pitted kidneys. One female each showed alopecia on ventral surface and kidneys with slight indentations
12000 ppm: In five males each, observations were made in kidney, testis, seminal vesicles, upper incisors (broken). In females, four showed effects in the kidneys (pitted, cyst, dilation). Further four females each showed effects in upper incisor (broken), on face (scab), lungs (speckled), and gut (filled with blood).
24000 ppm: Three males showed effects in kidneys (left kidney with cortical pit, right kidney misshapen at pelvis, right kidney with irregular cortical surface, left kidney slightly pitted). Three males showed reduced testes. One male of these also showed anterior caudate liver lobe apparently absent. In females, ten animals showed effects in kidneys (pitted, dilation, enlarged, "patchy" appearance).

P2B
MATED SACRIFICED WEEKS 28-31
6000 ppm: Three males failed to mate, one male showed enlarged right testis, another male had broken upper incisors. In females, one animal had broken upper incisors and a sore right eye
12000 ppm: Two males failed to mate and one of them also had slightly reduced testes. In females, one animal showed slighlty enlarged cecum, one showed sore eye, and another showed lesion on left median liver lobe
24000 ppm: One male failed to mate and showed red-rimmed eyes. In females, one animal showed growth around right salivary gland, four animals showed effects in kidneys (dilation, grossly misshapen, enlarged, and pitted), one animal had a swollen ureter, and another female showed alopecia of right ventral surface. Of the females with effects in kidney, one animal also showed effects in ureter (swollen).


UNMATED SACRIFICED NOMINAL WEEK 15
6000 ppm: Eight males showed effects on epididymis (2), lung (2), colon (1), and lymph nodes (3). In females, five animals showed effects on uterus (1), lung (2), lymph nodes (1), and ovaries (1)
12000 ppm: Ten males showed effects on lung (8), kidney (1), and lymph nodes (1). In females, three animals showed effects on lung (2) and lymph nodes (1).
24000 ppm: Eight males showed effects on urinary bladder (2), kidney (2), lung (2), and lymph nodes (2). Four animals showed effects on lung (1), liver (1), cecum (1), and incisors (1).
(Testis of unmated animals processed but not examined.)

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

P1B
6000 ppm: Of the males that failed to mate (3), no abnormalities of the reproductive tract were detected
12000 ppm: Of the male that failed to mate (1), no abnormalities of the reproductive tract were detected
24000 ppm: In one male, moderate dilatation of the renal pelvis associated with dilated medullary collecting tubules in both kidneys, small scattered mineral deposits in the medulla and areas of chronic interstitial nephritis in the cortex of both kidneys. Apparent truncation of the papilla in one kidney although this may possibly be due to the plane of section observed. In another male, minimal deposits of mineral in the lumina of medullary collecting ducts and beneath the transitional epithelium of the renal pelvis in one kidney. Focal wedge-shaped areas of chronic interstitial nephritis in the cortex of both kidneys. Occasional cortical tubules contain eosinophilic colloid and are lined by basophilic epithelium.

Histopathological examination of two males with reduced testes that failed to mate revealed no abnormalities for one male and bilateral testicular atrophy for the other. No all animals that failed to mate revealed pathological abnormalities in the reproductive tract, incl testes. Thus, no clear correlation between an adverse effect on mating success exists to abnormalities on reproductive tissues and organs exists. Further, pregnancy rates were comparable among all generations and matings and hence, the effects on the testes do not indicate adverse effects.
In females, histopathological examination revealed moderate numbers of dilated cortical tubules in both kidneys, apparent foreshortening of the papilla in one kidney in one female. In two females, small groups of basophilic cortical tubules associated with chronic inflammation of the interstitium of the kidney were found. In a third female, marked pyelonephritis with adhesion of renal capsule to the capsule of the liver was seen. In two further females, moderate dilatation of the renal pelvis with or without areas of interstitial nephritis; occasional mineral costs in medullary tubules of one kidney

P2B
MATED SACRIFICED WEEKS 28-31
6000 ppm: Two males that failed to mate were examined histopathologically and no abnormalities were detected
24000 ppm: Of the male that failed to mate, no abnormalities were detected of the reproductive tract. In females, the animal with the effect on salivary gland revealed marked sialo-adenitis and inflammation of adjacent adipose tissue. One female revealed large areas of interstitial nephritis in the cortex associated with a truncated pyramid indicating post papillary necrosis. One animal with swollen right ureter revealed moderate mixed inflammation of the wall associated granulation tissue in adjacent adipose tissue. The animal with effects in kidneys and ureter showed several moderate areas of interstitial nephritis in one kidney and small areas of sub-epithelial fibrosis beneath the transitional epithelium in the pelvis of both kidneys.

Summary of urinary tract changes:
Parental effects: Autopsy of P/F1B and P/F2B males (high dose) that died unscheduled showed changes in the urinary tract (haemorrhage, of the bladder wall, increased renal pelvic dilatation, interstitial nephritis and papillary necrosis). A similar clustered distribution of urinary tract changes was also seen for both sexes at terminal examination of surviving rats. The incidences of urinary tract lesions are statistically significantly elevated only in high -dose P/F1B animals of both sexes.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

P2B
6000 ppm: lower pregancy rate (leading to an increased median pre-coital time) for both matings.
This was considered coincidental in the absence of a similar reduction in pregnancy rate at higher concentrations.

The mean duration of gestation was comparable for all groups at each mating of P1 and P2 generation.

Details on results (P1)

P1B
MATERNAL DEVELOPMENTAL TOXICITY
- Dead fetuses: effects observed, non-treatment related
12000 ppm: One female was without viable young. Average of live young reduced, not statistically significant
24000 ppm: One dam generating four viable pups was excluded from calculation due to mistimed sacrifice and possible ill health. However, among the nine animals examined, average of live young was reduced, not statistically significant

- Pre- and post- implantation loss: effects observed, non-treatment related
24000 ppm: Reduced pre- implantation loss (3.7% 24000 pm and 5.5% control group), not statistically significant

- Total littler losses by resorption: no effects observed

- Early and late resorptions: no effects observed

- Changes in the number of pregnant animals: no effects observed

- Changes in pregnancy duration: not exaimed

- Other:
- Corpora lutea: effects observed, treatment related
12000 ppm: Reduced number, not statistically significant
24000 ppm: Significantly reduced compared to control

- Number of implantations: effects observed, treatment related
12000 ppm: Lower number of implantations per dam, not statistically significant
24000 ppm: Frequently statistically significant lower value compared to control

(This effect might be caused by the reduced number of corpora lutea. Thus, this effect is rather interpreted as secondary effect caused by a lower number of corpora lutea.)

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic (P1 and P2 generations)

Effect level:

6 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed at this dose level

Remarks on result:

other: equivalent to 586 mg/kg bw/day (lowest mean), 586 and 629 mg/kg bw/day in P1 and P2 males and 649 and 721 mg/kg bw/day in P1 and P2 females, respectively

2

Key result

Dose descriptor:

LOAEL

Remarks:

systemic (P1 and P2 generations)

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Remarks on result:

other: equivalent to 1186 mg/kg bw/day (lowest mean), 1203 and 1288 mg/kg bw/day in P1 and P2 males and 1297 and 1186 mg/kg bw/day in P1 and P2 females, respectively

3

Key result

Dose descriptor:

NOAEL

Remarks:

fertility (P1 and P2 generations)

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed at this dose level

Remarks on result:

other: equivalent to 1186 mg/kg bw/day (lowest mean), 1203 and 1288 mg/kg bw/day in P1 and P2 males and 1297 and 1186 mg/kg bw/day in P1 and P2 females, respectively

4

Key result

Dose descriptor:

LOAEL

Remarks:

fertility (P1 and P2 generations)

Effect level:

24 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Decreased corpora lutea (statistically significant), frequently statistically significant lower implantations compared to control (considered secondary based on the effect on corpora lutea)

Remarks on result:

other: equivalent to 2999 mg/kg bw/day (lowest mean), 3256 and 3066 mg/kg bw/day in P1 and P2 males and 2999 and 3030 mg/kg bw/day in P1 and P2 females, respectively

Target system / organ toxicity (P1)

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2 999 mg/kg bw/day (nominal)

System:

female reproductive system

Organ:

ovary

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2 999 mg/kg bw/day (nominal)

System:

urinary

Organ:

bladder

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

24000 ppm: An average of nine live fetuses were generated by dams, which is statistically significant from the control value (14.8). However, it should be noted that a lower number of corpora lutea was evident at the same dose.

24000 ppm: slightly increased cumulative loss of litters about 20% after the first mating at Day 4 post partum. No treatment related effect was seen on total litter loss (which was scattered throughout the various groups and
generations). (non treatment-related, non adverse)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

FIRST MATING
24000 ppm: decreased pup weight starting from Day 8 until day 21 post partum

SECOND MATING
24000 ppm: decreased pup weight (not significant) on day 12 post partum. Significant on day 21 post partum

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:

no effects observed

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:

effects observed, non-treatment-related

Description (incidence and severity):

F1A
6000 ppm: One pup was found dead with craniorachischisis. Another pup had a left eye missing.
12000 ppm: Five pups showed effects on eyes (hemorrhage spot, ocular opacities), one of them also showed right renal pelvic dialtion
24000 ppm: One pup showed anophtalmia and microphtalmia, another showed ocular opacity of the left eye, two pups showed black tail which broke off, and one pup had a missing right eye

F1B
12000 ppm: One pup showed pale area on margin of median liver lobe
24000 ppm: One pup had cysts on urinary bladder.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1A
6000 ppm: Of the pup with the left missing eye: Histological examination revealed no evidence of eye tissues in the left orbit at any of the six levels examined. Normal Harderian gland occupied the majority of the orbit . The righteye and associated orbital tissues were within normal limits.
12000 ppm: Two pups with effects on the eyes did not reveal abnormalities histopathologically
24000 ppm: No microscopic change detected in the pup with ocular opacity of the left eye. Of the pup with the right eye missing, histological examination could not detect an eye in the right orbit. An area of retinal dysplasia was seen in the left eye.

F1B
12000 ppm: Histological examination revealed a small group of degenerate hepatocytes beneath the hepatic capsule.
24000 ppm: Histological examination revealed moderate acute Inflammation of the bladder wall and adjacent prostate. Several erosions and areas of moderate hyperplasia in the urinary epithelium. Minimal oedema of the prostate.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

24000 ppm: a slight, but not statistically significant decrease in litter size after first mating

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

FETAL DEVELOPMENTAL TOXICITY
- Changes in sex ratio: no effects observed

- Changes in litter size and weights: effects observed, treatment-related
12000 ppm: Litter weight reduced, not statistically significant
24000 ppm: Litter weight statistically reduced
However, the reduced litter weight may be considered as result of the lower corpora lutea count.

- Changes in postnatal survival: no effects observed

- External malformations: effects observed, non-treatment related
Cleft palate, Small pup with moderate subcutaneous oedema, and diaphragmatic hernia observed in one fetus at 6000 ppm, 12000 ppm, and 24000 ppm, respectively. Due to the occurence in only one fetus at each concentration, the effect is not considered to be related to treatment.

- Skeletal malformations: effects observed, non-treatment related
One, two, three, and none fetuses showed minor skeletal anomalies at control, 6000 ppm, 12000 ppm, and 24000 ppm, respectively.

- Visceral malformations: effects observed, non-treatment related
Two, three, three, and one fetuses showed minor anomalies at control, 6000 ppm, 12000 ppm, and 24000 ppm, respectively

Effect levels (F1)

1

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

24 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

other: litter weight and number of live offspring

Remarks on result:

other: It should be noted that a lower number of corpora lutea was evident at the same dose.

Remarks:

equivalent to 1958 mg/kg bw/day (lowest mean), 1958 mg/kg bw/day in F0 males 2125 mg/kg bw/day in F0 females.

2

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect at this dose level

Remarks on result:

other: equivalent to 954 mg/kg bw/day (lowest mean), 954 mg/kg bw/day in F0 males 1056 mg/kg bw/day in F0 females.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No mortality was observed in F2 and F3 offspring

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F2
FIRST MATING
24000 ppm: decreased pup weight on day 21 post partum

SECOND MATING
12000 ppm: decreased pup weight on day 21 post partum
24000 ppm: decreased pup weight on day 12 and 21 post partum

F3
FIRST MATING
24000 ppm: decreased pup weight starting from Day 12 until day 21 post partum

SECOND MATING
12000 ppm: decreased pup weight starting from Day 12 until day 21 post partum
24000 ppm: decreased pup weight starting from Day 12 until day 21 post partum

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:

no effects observed

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

F3B GENERATION (Third pup generation)
6000 ppm: Statistically significant decrease in relative liver, spleen, and thymus weight in males. In females, statistically significant decrease in relative thymus weight.
12000 ppm: Statistically significant decrease in relative liver, spleen, and thymus weight in males. In females, statistically significant decrease in relative thymus weight.
24000 ppm: Statistically significant increase in relative lung weight and statistically significant decrease in relative liver, spleen, and thymus weight in males. In females, statistically significant decrease in relative thymus weight.
All effects excluding the spleen observations are considered non-treatment-related, due to missing dose-relationship.
The reduced spleen weight was dose-dependent and therfore considered treatment-related. However, in the absence of correlated histopathological findings, this should not be considered as a specific effect of developmental toxicity .

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F2A
6000 ppm: 12 pups had enlarged hearts, one pup of which had pale lungs and liver, one pup had forepaw bent backwards and with curved back
12000 ppm: 23 pups showed effects in kidney (pale, pelvic dilation), 25 pups showed effects in liver (pale, lesion on right lobe), 21 pups had enlarged heart, and ten pups showed yellow intestine.
24000 ppm: Nine pups trembling and with no locomotor activity. Five pups showed renal pelvic dilation, three pups showed effects on eyes (dry, hemorrhage)

F2B
6000 ppm: One pup with scab on tip of tail, one pup with enlarged stomach, two pups with pelvic dilation, and one pup with bilateral opacity
12000 ppm: Five pups showed effects on eyes (dull, encrusted), six pups showed effects on kidneys (pale, cysts), five pups showed pale liver, 24 pups had enlarged hearts, one pup had swollen and fluid-filled intestine, and another pup had forepaw (went backwards)
24000 ppm: One pup with enlarged and pale kidney, one pup with dry and crusty left eye, one pup unable to bend forelimb

F3A
6000 ppm: Four pups with effects on kidneys (dilation, cysts), one pup with daphragmatic surface in liver, one pup with bent tail
12000 ppm: Five pups with effects on kidneys (dilation, cysts), one pup showed effects on liver (necrosis, irregular shape), two pups had hard lumps on bladder, one pup had microphtalmia, one had a bruised head and face at birth
24000 ppm: 20 pups showed effects on kidneys (dilation, enlarged/swollen, absent), two pups showed lumps in urinary bladder

F3B
6000 ppm: Three pups with effects on kidneys (dilation), eight pups with effects on eyes (dry, cloudy, crusted), one pup had injured hind leg
12000 ppm: Four pups showed renal pelvic dilation, one pup had cloudy eye
24000 ppm: 26 pups showed efects on kidneys (dilation, cysts, absent), three pups showed dry and/or cloudy eyes

In general, occasional macroscopic changes were observed and the incidence of affected animals was low over the three generations and showed no consistent relationship to dietary concentration. Further enlarged hearts are considered rather to be an artifact than a specific treatment-related developmental effect.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

F2A
12000 ppm: Of the ten pups that showed yellow intestine, pale liver and kidneys, and enlarged heart; Histological examination revealed 2 with several cyitic tubules associated with a tear in one kidney; no other abnormalities detected in tissues examined from 3 pups. Of the pup with the lesion on the liver; histological examination revealed a focal subcapsular area of flbrosis, mineralisation andmultlnucleate foreign body giant cell infiltration.
24000 ppm: Of the pups with no locomotor activity; no abnormalities detected at histological examination of brains of two pups.

F2B
6000 ppm: Of the pup with ocular opacities, no abnormalities detected at histological examination
12000 ppm: Of the four pups with right dull eye; no abnormalities detected at histological examination. Of the pups with enlarged hearts, pale liver and kidneys; no abnormalities detected at histological examination. Of the pup with the encrusted eye; histological examination revealed moderate right keratitis.

F3A
12000 ppm: Of the two pups with hard lumps in bladder; histological examination revealed one pup with minimal generalised hyperplasia of the transitional epithelium with minimal mixed inflammation and oedema of the submucosa; and the second pup with moderate generalised hyperplasia of the transitional epithelium with associated minimal mixed inflammation and oedema in the submucosa; mineral deposits were noted in the lumen. Of the pup with median live rlobe of irregular shape; histological examination revealed several small areas of past necrosis and early scarring in the parenchyma associated with adhesions in the hepatic capsule
24000 ppm: Of the two with lumps in urinary bladder; histological examination of one of these revealed moderate generalised hyperplasia of the transitional epithelium associated with minimal mixed inflammation and oedema in the submucosa; minimal deposits were noted in the lumen.

F3B
6000 ppm: Of the one pup with renal pelvic dilation; histological examination revealed free blood in the pelvic lumen

F3
12000 ppm: In 1/10 males there was minimal epithelial hyperplasia and/or hypertrophy of the transitional epithelium, sometimes associated with small papillary projections and/or desquamation of epithelial cells in the lumen
24000 ppm: Test substance related microscopic changes in the urinary tract were seen in the F3B pups (minimal epithelial hyperplasia and/or hypertrophy of the transitional epithelium,sometimes associated with small papillary projectionsand/or desquamation of the epithelial cells; presence of crystalline or calcareous deposits): these changes were seen in 8/10 males and 8/10 females from the 24 000 ppm group and only in males (1/10) from the 12 000 ppm group.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

- Sex ratio (m/f) was statistically altered only at 12000 ppm in the F2 generation and only in second mating (7.5% males/4.7% females)
- No intergroup differences in litter size were observed neither in F2 nor in F3 generation.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

F2
FETAL DEVELOPMENTAL TOXICITY
- Reduction in number of live offspring: effects observed, non-treatment related
12000 ppm: One female was without viable young. Average of live young reduced, not statistically significant
24000 ppm: One dam generating four viable pups was excluded from calculation due to mistimed sacrifice and possible ill health. However, among the nine animals examined, average of live young was reduced, not statistically significant

- Changes in sex ratio: no effects observed

- Changes in litter size and weights: effects observed, treatment-related
12000 ppm: Litter weight reduced, not statistically significant
24000 ppm: Litter weight statistically reduced
However, based on the calculation of litter weight (the number of pups in one litter was not considered) , the reduced litter weight is considered as result of the lower corpora lutea count)

- Changes in postnatal survival: no effects observed

- External malformations: effects observed, non-treatment related
Inter-ventricular septal defects and craniorochyschisis was observed in one fetus at 12000 ppm, and 24000 ppm, respectively.

- Skeletal malformations: effects observed, non-treatment related
Two, four, four, and two fetuses showed minor skeletal anomalies at control, 6000 ppm, 12000 ppm, and 24000 ppm, respectively.

- Visceral malformations: effects observed, non-treatment related
Two, none, two, and one fetuses showed minor anomalies at control, 6000 ppm, 12000 ppm, and 24000 ppm, respectively

Effect levels (F2)

Key result

Dose descriptor:

LOAEL

Generation:

other: F2 and F3 generation

Effect level:

6 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: In regard to reduced litter weight, it should be noted that a lower corpora lutea count was observed at the same dose.

Remarks:

equivalent to 586 mg/kg bw/day (lowest mean), 586 mg/kg bw/day in F1B males and 649 mg/kg bw/day in F1B females, 629 mg/kg bw/day in F2B males and 721 mg/kg bw/day in F2B females.

Target system / organ toxicity (F2)

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

586 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2 999 mg/kg bw/day (nominal)

System:

urinary

Organ:

bladder

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 958 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

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

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Overall, the test substance induced systemic toxicity at the mid- and high-dose level in parental animals of the second and third generation (starting at approx. 586 and 1186 mg/kg bw/day in P1 and P2 generations, respectively) shown by reduced body weight and urinary tract changes (especially in high-dose males). Reproductive toxicity (fertility), indicated by reduced corpora lutea count, was observed at the highest dose level in the first and second parental generation, occurring at extreme high doses of approx. 1958 and 2999 mg/kg bw/day in P0 and P1 females, respectively). Consequent to the lower corpora lutea count the numbers of implantations per dam, live young and litter weight values were lower than those of controls at day 20 of pregnancy. As the effects on female fertility occurred at dose levels which also caused systemic toxicity, the effect on female fertility is considered as a secondary non-specific consequence of other toxic effects.
Applying the read-across approach, the target substance Fosetyl-sodium is not expected to differ in its reproductive toxicity potential.