Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.

REACH

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

EC number: 444-960-2 | CAS number: 39148-16-8

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Three-generation reproduction study, rat:

NOAEL (systemic toxicity, P0)≥ 24000 ppm (equivalent to 1958 mg/kg bw/day (lowest mean)).

NOAEL (systemic toxicity, P1 and P2): 6000 ppm (equivalent to 586 mg/kg bw/day (lowest mean)).

LOAEL (systemic toxicity, P1 and P2): 12000 ppm (equivalent to 1186 mg/kg bw/day (lowest mean)).

NOAEL (fertility, P0): 12000 ppm (equivalent to 954 mg/kg bw/day (lowest mean)).

LOAEL (fertility, P0): 24000 ppm (equivalent to 1958 mg/kg bw/day (lowest mean)).

NOAEL (fertility, P1 and P2): 12000 ppm (equivalent to 1186 mg/kg bw/day (lowest mean)).

LOAEL (fertility, P1 and P2): 24000 ppm (equivalent to 2999 mg/kg bw/day (lowest mean)).

NOAEL (fetal effects, F1): 12000 ppm (equivalent to 954 mg/kg bw/day (lowest mean)).

LOAEL (fetal effects, F1): 24000 ppm (equivalent to 1958 mg/kg bw/day (lowest mean)).

LOAEL (fetal effects, F2 and F3): 6000 ppm (equivalent to 586 mg/kg bw/day (lowest mean)).

(Read-across to Fosetyl-Al, CAS 39148-24-8)

Link to relevant study records

Reference 1

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
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
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
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
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
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
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
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.
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: 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.
: 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
: 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
: 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
: 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
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: 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.
: 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
: 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
: 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
: 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
: 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
: 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
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
Behaviour (functional findings):: not examined
Developmental immunotoxicity:: not examined
: 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.
: 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.
: Key result
Critical effects observed:: no
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.
Behaviour (functional findings):: not examined
Developmental immunotoxicity:: not examined
: 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.
: 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
: 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
: 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
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.

Reference 2

Endpoint:: three-generation reproductive toxicity
Type of information:: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:: key study
Justification for type of information:: refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:: read-across source
: 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: Rat, RA source CAS 39148-24-8 (M-203019-01-1)
Remarks:: 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
: Key result
Dose descriptor:: NOAEL
Remarks:: fertility
Effect level:: 12 000 ppm
Based on:: test mat.
Sex:: female
Basis for effect level:: other: no adverse effect observed at this dose level
Remarks on result:: other: Rat, RA source CAS 39148-24-8 (M-203019-01-1)
Remarks:: 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
: 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 corpura lutea)
Remarks on result:: other: Rat, RA source CAS 39148-24-8 (M-203019-01-1)
: 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
: 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: Rat, RA source CAS 39148-24-8 (M-203019-01-1)
Remarks:: 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
: 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: Rat, RA source CAS 39148-24-8 (M-203019-01-1)
Remarks:: 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
: 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 effects observed at this dose level
Remarks on result:: other: Rat, RA source CAS 39148-24-8 (M-203019-01-1)
Remarks:: 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
: Key result
Dose descriptor:: LOAEL
Remarks:: fertility
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 corpura lutea)
Remarks on result:: other: Rat, RA source CAS 39148-24-8 (M-203019-01-1)
Remarks:: 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
: 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
: 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
: 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 observed at this dose level
Remarks on result:: other: Rat, RA source CAS 39148-24-8 (M-203019-01-1)
Remarks:: 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.
: 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
Remarks on result:: other: It should be noted that a lower number of corpora lutea was evident at the same dose. Rat, RA source CAS 39148-24-8 (M-203019-01-1)
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.
: Key result
Critical effects observed:: no
: 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. Rat, RA source CAS 39148-24-8 (M-203019-01-1)
Remarks:: equivalent to 586 mg/kg bw/day (lowest mean), 586 mg/kg bw/day in F1B males 649 mg/kg bw/day in F1B females and 629 mg/kg bw/day in F2B males 721 mg/kg bw/day in F2B females.
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 586 mg/kg bw/day (nominal)
System:: hepatobiliary
Organ:: liver
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: not specified
: 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
: 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
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.

Effect on fertility: via oral route

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 954 mg/kg bw/day
Study duration:: subchronic
Species:: rat
Quality of whole database:: The available information comprises an adequate and reliable study (Klimisch score 2) from an analogue substance. Read across is justified based on (bio)transformation to common compounds. The selected study is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:: no study available

Effect on fertility: via dermal route

Endpoint conclusion:: no study available

Additional information

Reproductive Toxicity

No reliable data on reproductive toxicity is available for Fosetyl-sodium . However, there is reliable data available for the analogue substance Fosetyl-aluminium (Fosetyl-Al) which is considered for the evaluation of reproductive toxicity induced by Fosetyl-sodium (for further details on the read-across approach please refer to chapter 13 in the technical dossier).

Reproductive toxicity of Fosetyl-Al was determined in a three-generation study in rats (M-203019 -01 -1). Parental generations (P – F2B) were exposed to 6000, 12000 and 24000 ppm test substance continuously throughout the study (corresponding to mean nominal doses of 479, 933 and 1890 mg/kg bw/day (P), 546, 1134 and 2813 mg/kg bw/day (F1B) and 608, 1212 and 2721 mg/kg bw/day (F2B). Homogeneity, stability and accuracy of dietary concentrations were assured by gas-liquid chromatography. Control animals were included in the study and received plain diet.

In detail, parental generations were included in 3 reproduction cycles. The first litter (F1A –-F3A) were reared until Day 21 post partum followed by sacrifice and macroscopic examination. Approx. 10 days after sacrifice of the first litter, parental animals were mated again to produce the second offspring (F1B – F3B), from which the following parental generations were selected on Day 21 post partum. The selected parental animals were reared on the respective diet until reaching the age of at least 90 days, before they were mated to produce the next generation. In addition, 5 P and 10 F1B dams (litters)/group were sacrificed for teratological examinations on Day 20 of gestation. The remaining P and F1B dams were allowed to rear the second generation (F2B) from which parental animals were selected to produce the 3^rdgeneration. In addition, 15 animals/sex were included unmated in macroscopic examination. From the second litter of the 3^rdgeneration, 10 animals/sex were subjected to organ weight determination and microscopically examination, which was restricted to the control and high-dose group except for the urinary bladder, which was examined for all dose groups. The remaining young were macroscopically examined and discarded at Day 21 post partum.

Mortality was observed in animals from any test group with most of deaths occurring in F1B males of the high-dose group and to a lesser extent in F2B high-dose males, where 7/25 or 3/17 males died, respectively, during week 3 – 14. In the other dose groups, mortality rate of males was comparable among the groups including control animals. Furthermore, unscheduled death of female animals occurred at low incidences with up to 2 animals per group in the low (P and F1B generation), mid- (F2B) and high-dose group (P and F1B) without showing any dose-relationship. No overt signs of clinical toxicity were observed in any dose group. Autopsy of deceased males revealed alterations in the urinary tract including haemorrhage of the bladder wall, increased renal pelvic dilatation, interstitial nephritis and papillary necrosis. Similar alterations were observed at terminal examination of surviving animals. In females, an association between urinary tract alterations and mortality was only determined for one P high-dose female.

Group mean body weights of adults from the P generation were comparable among the groups throughout the observation periods including the pre-mating, mating and gestation periods. In P females, a reduced body weight about 11% was only observed in the high-dose group during the last 2 weeks of lactation of the second mating cycle. Due to the rather isolated appearance, the effect on body weight and weight gain was considered incidental and not related to treatment in that generation. Moreover, no mid-lactation peak in body weight was determined for high-dose females in both reproduction cycles.

Overall group mean body weight was reduced in high-dose F1B males during the pre-mating phase (-39% ) and at later time points (week 13: -12,7%)): overall mean group body weight was reduced about 25.3 ± 9.8% compared to controls during the pre-mating phase. During the first and second mating, overall group mean body weight was reduced in F1B males about 12.3 ± 1 and 10.9 ± 0.6% compared to controls, respectively. Body weight development of F1B males of the low- and mid-dose group was slightly increased compared to the control group. In the F2B generation, a similar trend in body weight was observed in high-dose males: during the pre-mating phase, an overall reduction in body weight about 13.9 ± 6.44% in comparison to controls was observed. This effect appeared even stronger during the first week of pre-mating, where a reduction in body weight about 23.6% was evident. During the 1st and 2nd mating, overall body weight was reduced about 13.0 ± 0.58 and 13.1 ± 0.74% in comparison to controls, respectively. A similar trend was observed in mid-dose F2B males, which revealed a reduced body weight around 8.4 ± 5.5, 9.4 ± 0.45 and 10.2 ± 0.34% compared to controls during the pre-mating phase and the first and second mating period, respectively. Body weight of F2B low-dose males was comparable to the control group.

Mean body weight of F1B females was time-dependently decreased leading to a reduction of the overall group mean body weight about 15.8 ± 10.51% compared to controls for the pre-mating phase, with a reduction in body weight about 32.5% during the first week of pre-mating. Through the 1st and 2nd mating periods, a reduction about 7.4 ± 1.42 and 9 ± 4.46% compared to controls was determined, respectively. During gestation and lactation periods, body weights were still reduced in the mid- and high-dose group with the strongest effects present post partum (-16.9% Lactation day 21). Moreover, due to the decreased body weight, no mid-lactation peak in body weights was observed in high-dose females. In F1B low-dose females, body weight was slightly increased or unremarkable compared to the controls.

In F2B high-dose females, decreases in body weight were obtained for the 1st and 2nd mating phases (-7.7 ± 3.63 and -10.8 ± 6.28% compared to controls, respectively. Furthermore, during the pre-mating phase, an overall decrease in body weight about 8.5 ± 6.1% compared to controls was observed. This effect appeared even stronger during the first week of pre-mating, where a reduction in body weight about -20% was evident. In addition, mid-dose F2B females showed a slightly decreased body weight during the pre-mating, first and second mating phase (-5.6 ± 3.39 (-12% during the first week of pre-mating), -2.2 ± 1.73 and -6.3 ± 2.87% compared to controls, respectively), whereas the body weight development of low-dose F2B females was comparable to controls during the pre-mating and mating phases. At the end of the 1st gestation period and enduring until the end of the observation period low-dose females revealed an increased body weight. In contrast, F2B high-dose females revealed reduced body weights during gestation and lactation periods. Similar to the previous generation, no mid-lactation peak in body weight was observed in high-dose females due to the decreased body weight. Overall, the effects on body weights in mid and high dose F1 and F2 parental animals are considered treatment-related.

A lower food consumption was determined for F1B males during the initial rearing phase. Moreover, a lower food consumption of F1B and F2B females was observed during the first weeks of rearing in the high-dose group. During the rearing period prior to the first mating (week 1 - 13 for P and F2B generations and week 1 - 10 for F1B generation), a continuous decrease in test substance intake was determined for each sex in any dose group about at least 50%.

Fertility in males remained unremarkable among all generations except low-dose males of the F1B and F2B generation, in which 4/25 and 3/12 males failed to mate compared to control incidences of 1/25 (F1B) and 1/12 (F2B). Due to 1) the low incidence of infertile males present in all test groups including controls and 2) the normal appearance of reproductive organs also in infertile males and 3) the missing dose-dependency, a treatment-related effect on male fertility is excluded.

Moreover, mating performance correlated to the median pre-coital time was comparable among the groups except the low-dose F2B group, which revealed a prolonged pre-coital time around 3 – 4 fold compared to controls during the first and second mating. Furthermore, the number of pregnancies and the percentage of females delivering viable offspring were comparable among the groups except the low-dose group which revealed decreased pregnancy rates of 67, 54 or 58% for the F1B (second mating) and F2B generation (first and second mating), respectively. As most of the non-delivering low-dose females did not show any signs of mating including signs of sperm in the vaginal smear or vaginal plug the prolonged pre-coital time and decreased pregnancy rate might be correlated to the slightly increased number of infertile F1B and F2B males in the low-dose group. However, as the mid- and high-dose groups revealed pre-coital time intervals and pregnancy rates similar to the concurrent control groups, the biological significance of the effects in the low-dose group remain questionable. Thus, lower pregnancy rates, endured pre-coital time and slightly increased incidences of infertile males seen only in the low-dose group are therefore interpreted as coincidental especially as the reproductive organs of infertile low-dose males were unremarkable. At microscopic examination, abnormalities of the testes in F1B and F2B males included necrotic tissue around the epididymis, reduced seminal vesicles or reduced testes which were not associated to infertility. A correlation between infertility and microscopical findings was determined in only 1 male, which showed bilateral testicular atrophy. Due to the low incidence of the abnormalities, a correlation between pathological findings and exposure to the test substance may be excluded.

On Day 20 of gestation, 5 P and 10 F1 dams/litters were sacrificed. The number of corpora lutea was slightly reduced in mid- and high-dose dams, but significance was only reached at the high-dose for both generations.Consequent to the lower corpora lutea count, the numbers of implantations per dam (not statistically significant), litter size andlitter weight were lower than those of controls at day 20 of gestation. At 24 000 ppm but not at 12 000ppm differences from control values frequently attained statistical significance.However, only the P0 generation showed an association between the reduced number of corpora lutea with a decreased number of live young (statistically significant), increased post-implantation loss and increased embryonic deaths. The litter weight was significantly reduced in F0, F1B and F2B generations of the high dose group. The amount of pre-implantation loss, sex ratio and mean foetal weight at birth (but not on postnatal day 21) were comparable among the groups in both generations.

In gross pathology, occasional gross abnormalities were detected over all generations without dose-dependency in the lung, liver, spleen, lymph nodes and testes. Urinary tract changes including signs of haemorrhage in the bladder and renal pelvic dilatation, interstitial nephritis and papillary necrosis were evident in surviving and deceased animals starting occasionally at 12000 ppm. The incidence of urinary tract lesions was enhanced in high dose animals of the F1B and F2B generations, which reached statistical significance in F1B males (incidence = 9/25) and females (incidence = 10/25). In some animals, urinary tract changes were correlated with mineral deposits in the urinary tract.

Urinalysis performed on 10 control and 10 F1B high-dose males revealed a slight but statistically significant decrease in specific gravity. Furthermore, a small number of epithelial cells, erythrocytes and polymorphonuclear leucocytes was detected in urine samples of high-dose animals. Otherwise, no abnormalities were observed in the examined parameters.

The litter weight was significantly reduced in F1 and F2 generations. In the offspring, the following effects were observed:

In the F1 generation, a reduced number of live offspring was observed in the highest dose group. In detail, an average of 9 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.

Total litter loss, and sex ratio of the offspring were unremarkable in all 3 generations and both matings.

Moreover, body weight of the offspring (F1, F2 and F3) was comparable for all groups and generations until Day 4 post partum. Afterwards, during mid- and late-lactation, body weight gain was reduced in high-dose offspring, which might be related to the reduced body weight gain in maternal animals during lactation. A similar but less marked effect on body weight gain was evident in the mid-dose group. Body weight gain of the low-dose group did not show a consistent effect.

Macroscopic examination was performed with the offspring of the P (F1A and B generation, produced in first and second reproduction cycle, respectively), F1B (F2A and B generation) and F2B generation (F3A and B) after sacrifice on Day 21 post partum. Moreover, a more detailed autopsy was performed with F3B young 21 days post partum with special emphasis on urinary tract changes. Pathological abnormalities were observed over all generations to low incidences and without a consistent relationship to dietary concentrations, including congenital changes (cranioschisis, anophthalmia, tail amputation and discoloration) and effects on the eyes (dry and cloudy appearance or signs of haemorrhage). Moreover, the young of the F2 generation revealed enlarged hearts, which might represent a technical artefact as a similar abnormality was neither observed in the F1 nor F3 generation. Abnormalities observed in the kidney, including pelvic dilatation and cysts were observed occasionally in F1A, F2A and F2B young without a consistent dose-relationship. A dose-related increase in the occurrence of renal pelvic dilatation was observed in young of the F3 generation.

Microscopic examination was restricted to control and high-dose animals of the F3B generation except for the urinary bladder, which was examined from all groups. Moreover, single young from the F1 and F2 generation were included in histopathological examination depending on the result of the macroscopic examination. Anomalies were detected in control and exposed F3B animals in several organs including the liver, lung, spleen, kidney, brain, pituitary, thyroids and lymphoid tissue to low incidences. Alterations in the urinary bladder present in F3B mid- and high-dose animals included minimal papillary hyperplasia of transitional epithelium, epithelial hyperplasia and engorgement of subepithelial blood vessels, dystrophic mineralization in the serosa and minimal hypertrophy of vesicular transition epithelium. As most alterations were associated with crystal deposition in the lumen of the urinary bladder, the observed effects might be related to physical irritation of the surrounding tissues induced by the crystals.

Minor statistically significant but non-dose-related decreases in relative liver and thymus weights were established for the male F3B offspring. F3B females only revealed a reduced relative thymus weight. In addition, decreases in absolute weight of the spleen in the F3B generation were observed in both sexes (not stat sig in females) and showed a dose-response relationship. Although the spleen effects are considered relevant, but in the absence of correlated histopathological findings, this should not be considered as a specific effect of developmental toxicity. Furthermore, relative lung organ weight was increased in high-dose male offspring. Due to 1) the absence of correlating pathological findings, 2) the lack of dose-dependency, 3) low number of examined animals compared to the number of born pups and 4) the marked effect on maternal and pup body weight gain during lactation, biological significance of the observed effects on relative organ weights remains questionable.

Based on the obtained results, the following effect level were defined:

NOAEL (systemic toxicity, P0)≥24000 ppm (equivalent to 1958 mg/kg bw/day (lowest mean)). No treatment-related adverse effects were observed.

NOAEL (fertility, P0): 12000 ppm (equivalent to 954 mg/kg bw/day (lowest mean)). No treatment-related adverse effects were observed.

LOAEL (fertility, P0): 24000 ppm (equivalent to 1958 mg/kg bw/day (lowest mean)). Based on significantly reduced corpora lutea and reduced implantation sites (considered consequent to the lower corpora lutea count).

NOAEL (systemic toxicity, P1 and P2): 6000 ppm (equivalent to 586 mg/kg bw/day (lowest mean)). No treatment-related adverse effects were observed.

LOAEL (systemic toxicity, P1 and P2): 12000 ppm (equivalent to 1186 mg/kg bw/day (lowest mean)). Based on significantly reduced body weight at mid and high dose level, mortaility and associated urinary tract and kidney changes (especially in high-dose males).

NOAEL (fertility, P1 and P2): 12000 ppm (equivalent to 1186 mg/kg bw/day (lowest mean)). No treatment-related adverse effects were observed.

LOAEL (fertility, P1 and P2): 24000 ppm (equivalent to 2999 mg/kg bw/day (lowest mean)). Based on significantly reduced corpora lutea and reduced implantation sites (considered consequent to the lower corpora lutea count).

NOAEL (fetal effects, F1): 12000 ppm (equivalent to 954 mg/kg bw/day (lowest mean)). No treatment-related adverse effects were observed.

LOAEL (fetal effects, F1): 24000 ppm (equivalent to 1958 mg/kg bw/day (lowest mean)). Based on reduced number of live young, reduced mean pup weight at day 21 and reduced litter weight at the highest dose level. It should be noted that a lower number of corpora lutea was evident at the same dose.

LOAEL (fetal effects, F2 and F3): 6000 ppm (equivalent to 586 mg/kg bw/day (lowest mean)). Based on dose-dependent reduced spleen weight (only investigated in F3 fetuses). In the absence of correlated histopathological findings, this should not be considered as a specific effect of developmental toxicity. In addition, at 12000 ppm (F2B second mating) and 24000 ppm (F1 and F2), reduced mean pup weight at day 21 (related to maternal decrease in body weight) and reduced litter weight were observed. In regard to reduced litter weight, it should be noted that a lower corpora lutea count was observed at the same dose. Furthermore, urinary tract changes were observed in animals of the highest dose group.

Conclusion

Taken together, 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. In consequence, and also considering the extreme high doses that led to the effects on female fertility, the data are not sufficient and conclusive for classification in regard to reproductive effects, which is in line with the Guidance on the Application of the CLP criteria (3.7.2.2).

Further, a dose-dependent decrease in spleen weight was observed at all dose levels in F3 fetuses. However, in the absence of correlated histopathological findings, this effect should not be considered as a specific effect of developmental toxicity. Other effects in fetuses observed at higher dose levels, such as the reduced mean body and litter weight and reduced numbers of implantations per dam are considered related to the reduced corpora lutea count and maternal systemic toxicity. Furthermore, observed urinary bladder effects were observed only in fetuses of one generation and were rather mild. As most alterations were associated with crystal deposition in the lumen of the urinary bladder, the observed effects might be related to physical irritation of the surrounding tissues induced by the crystals. Thus, overall observed effects are not considered a classification relevant, specific developmental toxicity effect.

A two-generation reproductive toxicity study was performed with Fosetyl-sodium (DL-1600265), which was conducted equivalent to OECD 416 and in compliance with Grade A pesticide registration toxicology testing agency qualified by China’s Ministry of Agriculture (Certificate No. Toxicology-025). However, since the study was not performed according to GLP and due to major deficiencies, the study is not considered reliable and thus not used as key study. Particularly the lack of individual data, and the fact that key data are not reported including numbers of animals of each sex mated, the time to mate, and gestation length, lack of reporting for each litter in each generation the number of live young, number of deaths, number of resorptions, implantations, corpora lutea, pre- and post-implantation loss, sex ratio of offspring, lack of dose formulation analysis, the study is disregarded for hazard assessment. A brief description of the study is given below for reasons of completeness.

Parental generations were exposed continuously throughout the study via the feed to 80, 400, or 2000 mg/kg bw/day (corresponding to a fosetyl intake of 7.87 and 7.44 mg/kg bw/day in the low dose; 37.94 and 36.21 mg/kg bw/day in the mid-dose, and 189.53 and 187.44 mg/kg bw/day in the high dose group for females and males, respectively, as calculated from ingested food quantity per kg of body weight, average for P0 and P1 generation). The concurrent control group received plain diet.

Thirty rats per sex were treated in each test group for the parent generation (P0) and the adult F1 generation (P1). Standardisation of litter to 4 pups/sex and litter occurred on postnatal day (PND) 4. Examinations in parental generations included general condition, food consumption, body weight, reproductive indices, sperm parameters, organ-body weight ratio, and pathology tests for the main reproductive tissues and organs. In F1 and F2 offspring, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain and survival rate were determined.

Overall, no adverse effects on fertility were observed in the conducted 2-generation toxicity study. Furthermore, no signs indicative for systemic toxicity were apparent in the parental generations. Thus, a NOAEL of 2000 mg/kg bw/day corresponding to 189.53 mg/kg bw/day and 187.44 mg/kg bw/day in female and male rats, respectively, was derived for systemic parental and reproductive toxicity. Exposure during pregnancy and lactation was not correlated to adverse effects in the F1/P1 and F2 generation. Due to missing dose-responses and consistency between different generations, observed alterations in relative organ weights were not considered as indicative for treatment-related effects leading to adversity and thus, a NOAEL of 2000 mg/kg bw/day was set for developmental toxicity. As discussed above, the outcome of the study is not considered as reliable due to major deficiencies in reporting and hence, the data are not considered for hazard assessment.

Effects on developmental toxicity

Description of key information

Developmental toxicity, (according to OECD TG 414), rabbit: NOAEL (maternal systemic and developmental effects and fetal developmental effects) >= 300 mg/kg bw/day

(Read-across to Fosetyl-Al, CAS 39148-24-8)

Developmental toxicity, (equivalent to OECD TG 414), rat: NOAEL (maternal systemic and developmental effects and fetal developmental effects) = 1000 mg/kg bw/day

(Read-across to Fosetyl-Al, CAS 39148-24-8)

Three-generation reproduction study, rat:

NOAEL (fetal effects, F1): 12000 ppm (equivalent to 954 mg/kg bw/day (lowest mean).

LOAEL (fetal effects, F1): 24000 ppm (equivalent to 1958 mg/kg bw/day (lowest mean).

LOAEL (fetal effects, F2 and F3): 6000 ppm (equivalent to 586 mg/kg bw/day (lowest mean).

(Read-across to Fosetyl-Al, CAS 39148-24-8)

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:: developmental toxicity
Type of information:: migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:: key study
Study period:: 7 Sept - 8 Oct 1999
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: other: see 'Remark'
Qualifier:: according to guideline
Guideline:: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:: draft document August 1996
Deviations:: no
Qualifier:: according to guideline
Guideline:: EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:: adopted in August 1998
Deviations:: no
GLP compliance:: yes
Limit test:: no
Species:: rabbit
Strain:: other: New Zealand White Rabbits Hra (NZW) SPF
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Covance Research Products, Denver, USA
- Age at study initiation: 5 - 6 months (days 0 - 2 of gestation)
- Weight at study initiation: 3 - 5 kg (pregnant females)
- Fasting period before study: no
- Housing: individually in suspended stainless steel wire mesh cages
- Diet: pelleted diet (Certified Rabbit Diet, No. 5322 (PMI Nutrition International, St .Louis, MO)), 50 g feed on the day of arrival, 125 g on the second day and approx. 200 g thereafter
- Water: facility water, ad libitum
- Acclimation period: 2 - 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 25
- Humidity (%): 40 - 96
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 7 Sept 1999 To: 8 Oct 1999
Route of administration:: oral: gavage
Vehicle:: other: methyl cellulose (Methocel ® (0.5% w/v in water))
Details on exposure:: PREPARATION OF DOSING SOLUTIONS:
The required amount of test substance was added to methyl cellulose and stirred on a magnetic stir plate for at least 30 min and continued to be stirred until completion of dosing.

VEHICLE
- Amount of vehicle (if gavage): 2 mL/kg bw day
- Lot/batch no. (if required): 97H0980
- Purity: 100%
The aqueous solution of methyl cellulose was prepared weekly and stored refrigerated at 2 - 8 °C.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Samples of dosing solutions were tested for homogeneity and dose accuracy via HPLC-UV. Homogeneity analysis revealed a recovery rate of 96.4% for all concentrations thereby demonstrating homogeneity of the solution. Moreover, dose level confirmatory tests determined concentrations of dosing solutions of ±10% of the nominal concentration which demonstrates the presence of acceptable concentrations.
Details on mating procedure:: not applicable (pregnant rats were obtained from the supplier)
Duration of treatment / exposure:: Day 4 - 28 of gestation
Frequency of treatment:: daily, 7 days/week
Duration of test:: 25 days
Dose / conc.:: 50 mg/kg bw/day (actual dose received)
Dose / conc.:: 100 mg/kg bw/day (actual dose received)
Dose / conc.:: 300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:: 25
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale: dose selection was based on a range-finding teratology study performed with 75, 125, 250, 500 and 1000 mg Fosetyl-Al/kg bw/day administered via gavage during Days 4 - 28 of gestation. Maternal toxicity was observed at the two top doses tested identified by reduced body weight gain and food comsumption. At 250 mg/kg bw/day, some evidence of decreased maternal body weight gain was still observed. Furthermore, 250 and 500 mg/kg bw/day induced slight decreases in the number of life fetuses and a slight increase in post-implantation loss. Due to the small animal number included in the study of only 5 dams per dose group, data on alterations in uterine implantation is inconclusive. No adverse effects have been observed either on the dams or fetuses at the two lowest doses applied. Thus, doses ranging from 50 - 300 mg/kg bw/day were chosen as applicable for the prenatal developmental toxicty study including a high-dose which is expected to produce slight maternal toxicity and a low-dose, which should represent the NOEL. The mid-dose was proposed to provide dose-response information.
Maternal examinations:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (including post-dose observations approx. 1.5 to 2 h after dosing)
- Cage side observations included observations for mortality, morbidity and signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
- Observations included general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia and evaluation of respiration.

BODY WEIGHT: Yes
- Time schedule for examinations: gestation day 0 and daily thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: uterus, lung, kidney, bladder, ovary
Ovaries and uterine content:: The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:: - External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: [all per litter]
Statistics:: Mean values and standard errors were calculated from the examined parameters. Statistical analysis were performed with ANOVA followed by a Dunnett's test to identify differences between the control and treatment groups (in regard to body weights, body weight change, feed consumption, number of corpura lutea, uterine implantation data, pre- and post-implantation loss, number of male and female fetuses, fetal weight distinguished by sex and as a composite for both sexes). A Kruskal-Wallis test was performed to test equality of means. All statistical tests were conducted at the 5 and 1%, two-sided risk levels. Statistical evaluation of equality of means was made by the appropriate one way analysis of variance (ANOVA) technique, followed by a multiple comparison procedure, if needed. If ANOVA showed no difference, no additional comparisons were made. If ANOVA was significant, Dunnett's test was used to determine which data, if any, differed from the control.

The Kruskal-Wallis test (2Xn) was used to test equality of means. If no differences were seen, no additional analyses were performed. If differences were indicated, a 2X2 Kruskal-Wallis test was used to determine which treatments differed from control.

Further, a Fisher Exact Test with Bonferonni correction was performed to identify differences in mortality rate, pregnancy rates, incidence of females with only resorptions and incidence of litters containing fetuses with malformations. All statistical tests were conducted at the 5 and 1%, two-sided risk levels.
Clinical signs:: effects observed, non-treatment-related
Description (incidence and severity):: No clinical signs of systemic toxicity were observed despite scabs, alopecia and decreased faecal volume. As these effects were determined only at low incidence in the control and dose groups and they represent common laboratory findings, they are interpreted as fortuitous and not substance-specific.
Dermal irritation (if dermal study):: not examined
Mortality:: mortality observed, non-treatment-related
Description (incidence):: 50 mg/kg bw/day: 1/25 animals was euthanized on Gestation Day 27 as a result of an apparent abortion, fetus found under the cage. Since abortions where not seen in the other treatment groups and spontaneous abortions are common among rabbits when stressed, the death is not considered to be related to treatment. Moreover, postmortem examinations revelead a discoloured uterus and a fluid-filled uterine horn on the left side.

100 mg/kg bw/day: 1/25 animals found dead on Day 11 of gestation. Postmortem examination, revealed possible gavage trauma in 5 of 6 dead rabbits since the lungs were discolored and/or the lungs and trachea were fluid filled. This theory is supported as these animals did not exhibit any overt clinical observations prior to death (non-treatment-related).

300 mg/kg bw/day: 6/25 animals were found dead during the study on Days 6 (two unscheduled death), 9, 12, 18 and 27. Postmortem examination, revealed possible gavage trauma in 5 of 6 dead rabbits since the lungs were discolored and/or the lungs and trachea were fluid filled. This theory is supported as these animals did not exhibit any overt clinical observations prior to death (non-treatment-related).
The remaining animal of this dose group, died on Gestation Day 27 postmortem examination was unable to determine the cause of death, however findings included discolored lung foci and hemorrhagic kidneys (due to the single apperance, the death is rather considered unrelated to treatment).
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: 100 and 300 mg/kg bw/day: Despite a significant increase in food consumption from Day 27 - 29 of gestation in mid- and high-dose females, food consumption was unremarkable in all animals. As body weight gain appeared normal in all animals, the biological significance of the altered food consumption remains questionable (non-adverse).
Food efficiency:: no effects 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
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
Gross pathological findings:: no effects observed
Description (incidence and severity):: No effect of treatment was evident from the macroscopic postmortem examination for the female rabbits.
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: not examined
Histopathological findings: neoplastic:: not examined
Other effects:: not examined
Number of abortions:: effects observed, non-treatment-related
Description (incidence and severity):: 50 mg/kg bw/day: 1/25 animals was euthanized on Gestation Day 27 as a result of an apparent abortion, fetus found under the cage.
Pre- and post-implantation loss:: no effects observed
Total litter losses by resorption:: not examined
Early or late resorptions:: no effects observed
Dead fetuses:: no effects observed
Changes in pregnancy duration:: not examined
Changes in number of pregnant:: no effects observed
Other effects:: no effects observed
Description (incidence and severity):: Gravid uterine weight: gravide uterine weight were comparable among the groups (please refer to Table 1).

Corpora Lutea: no effects observed
Details on maternal toxic effects:: Pregnancy rates and gravide uterine weight were comparable among the groups (please refer to Table 1). Uterine implantation data, number of corpora lutea and pre-and postimplantation loss and mean number of live fetuses were unremarkable.
: Key result
Dose descriptor:: NOEL
Remarks:: systmic toxicity
Effect level:: >= 300 mg/kg bw/day (actual dose received)
Based on:: test mat.
Basis for effect level:: other: maternal systemic toxicity
: Key result
Dose descriptor:: NOEL
Remarks:: maternal developmental toxicity
Effect level:: >= 300 mg/kg bw/day (actual dose received)
Based on:: test mat.
Basis for effect level:: other: maternal developmental effects
: Key result
Abnormalities:: no effects observed
Fetal body weight changes:: no effects observed
Description (incidence and severity):: Fetal body weights were not affected by treatment when compared to the control group.
Reduction in number of live offspring:: no effects observed
Description (incidence and severity):: No effect of treatment at any dose level was observed on mean number of live fetuses per female rabbit compared to control group.
Changes in sex ratio:: no effects observed
Changes in litter size and weights:: no effects observed
Anogenital distance of all rodent fetuses:: not examined
Changes in postnatal survival:: not examined
External malformations:: effects observed, non-treatment-related
Description (incidence and severity):: There was one dead fetus in the 50 mg/kg/day treated group which exhibited external malformations of gastroschisis, hindlimbs hyperflexion, and agenesis of the tail. The malformations in the dead fetus are not considered a teratogenic treatment-related effect since they occurred at a low incidence and were not observed in the other treatment groups.
Skeletal malformations:: effects observed, non-treatment-related
Description (incidence and severity):: One commonly observed skeletal malformation (fused stemebrae) was present in all treatment groups, but at a single incidence in each group. This skeletal malformation has been observed previously in this strain of rabbit, are considered to be of spontaneous origin, and are not attributed to treatment. Skeletal variations were observed in the treatment groups, but the variations appeared at an incidence which were comparable to that of the control group, and thus were considered to be of spontaneous origin, and not attributed to treatment.
Visceral malformations:: effects observed, non-treatment-related
Description (incidence and severity):: Visceral variations were observed in the treatment groups, but the variations appeared at an incidence which were comparable to that of the control group, and thus were considered to be of spontaneous origin, and not attributed to treatment.
Other effects:: not examined
Details on embryotoxic / teratogenic effects:: Details on embryotoxic / teratogenic effects:
Fetal body weights, fetal sex ratio and the number of live fetuses per female rabbit were comparable among the groups (please refer to Table 2). External, visceral and skeletal examinations did not reveal treatment-related effects and malformations were observed to a low incidence of 0.5 and 2% without being dose-related (please refer to Table 3). Fused sternebrae observed as skeletal malformation was present in all test groups in only 1 fetus. Due to the low incidence at any applied dose and the fact that fused sternebrae has been previously observed in this strain, the appearance of this skeletal malformation is considered as incidental and not treatment-related. Moreover, the death of 1 low-dose fetus which exhibited several external malformations including gastroschisis, hindlimbs hyperflexion and agenesis of the tail is not interpreted as treatment-related due to the low incidence and a missing dose-response relationship. External malformations were only present in control and low-dose fetuses. Moreover, visceral malformations were detected in 1 fetus each of control and high-dose animals (please refer to Table 3).
: Key result
Dose descriptor:: NOAEL
Effect level:: >= 300 mg/kg bw/day (nominal)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: no substance-related effects were observed on fetuses on the evaluated endpoints
: Key result
Abnormalities:: no effects observed
: Key result
Developmental effects observed:: no
Conclusions:: Based on the results obtained in this prenatal developmental toxicity study in rabbits, the NOAEL for maternal and developmental toxicity can be set at => 300 mg/kg bw/day.

Reference 2

Endpoint:: developmental toxicity
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 1977
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:: No guideline was in place when the study was conducted. However, the study design is similar to OECD 414 (please refer to details provided in the respective fields below). Briefly, the following deviations exist:
treatment period during organogenesis from GD6 -15; high dose tested was four times higher than the limit dose of 1000 mg/kg bw/day and missing examinations, incl hormone measurements and anogenital distance.
GLP compliance:: no
Remarks:: The study was conducted prior to GLP.
Limit test:: no
Species:: rat
Strain:: other: CFY
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Anglic laboratory animals, Alconbury, Huntingdon, Cambrigeshire
- Age at study initiation: sexually mature, not further specified
- Weight at study initiation: 180 - 250 g
- Housing: 5 animals per cage
- Diet: Spratt's laboratory diet No.1, ad libitum
- Water: tap water, ad libitum
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not specified
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified

IN-LIFE DATES: not specified
Route of administration:: oral: gavage
Vehicle:: water
Remarks:: sterile
Details on exposure:: PREPARATION OF DOSING SOLUTIONS: The test material was prepared freshly on a daily basis in sterile water. The dosage volume was calculated for individual animals on day 6 and adjusted on day 10 and 14.

VEHICLE
- Concentration in vehicle: 2.5, 5 and 20%
Analytical verification of doses or concentrations:: no
Details on mating procedure:: - Impregnation procedure: purchased timed mated
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:: Day 6 to 15 of pregnancy
Frequency of treatment:: daily
Duration of test:: 15 days
Dose / conc.:: 500 mg/kg bw/day
Remarks:: Concentration of solution 2.5 % (w/v)
Dose / conc.:: 1 000 mg/kg bw/day
Remarks:: Concentration of solution 5 % (w/v)
Dose / conc.:: 4 000 mg/kg bw/day
Remarks:: Concentration of solution 20 % (w/v)
No. of animals per sex per dose:: 20
Control animals:: yes, concurrent vehicle
Maternal examinations:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (obvious changes)

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Days 1, 3, 6, 10, 14, 17 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: congenital abnormalities and macroscopic pathological changes in maternal organs; the ovaries and uteri
Ovaries and uterine content:: The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Yes
- Number and distribution of live young
- Litter weight from which the mean pup weight was calculated
Blood sampling:: - Plasma: No
- Serum: No
Fetal examinations:: - External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: No
- Anogenital distance of all live rodent pups: No
Statistics:: Group mean values were calculated from individual observations in two ways:
Mean A: Includes all surviving animals that provided evidence of pregnancy including those showing total resorption.
Mean B: Includes all animals with live young at termination.
Results were statistically analysed on a litter basis using non-parametric methods
Indices:: Pre-implantation loss: (Number of corpora lutea - Number of implantations) x100 / Number of corpora lutea

Post-implantation loss: (Number of implantations - Number of live young) x100 / Number of implantations
Clinical signs:: effects observed, treatment-related
Description (incidence and severity):: At 4000 mg/kg bw/day: 5/20 rats that died or were sacrificed showed marked gastric dilatation and fluid retention was found at post mortem examination. Prior to death all animals lost weight and three showed chromodacryorrhea
Dermal irritation (if dermal study):: not examined
Mortality:: mortality observed, treatment-related
Description (incidence):: Occasional mortalities at 500 and 1000mg/kg bw/day were attributable to dosing errors (non-treatment related).
At 4000mg/kg bw/day 5/20 rats died, or were sacrificed during days 9, 10 and 11 (treatment-related, adverse). (Please refer to table 1 in the "Any other information on results including tables section")
No deaths occured amongst control rats.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: There was a dosage related retardation of weight gain during the first four days of dosing : at 500 and 1000 mg/kg bw/day, but the divergence from control values was not marked (treatment-related, non-adverse).
At 4000 mg/kg bw/day weight gain was almost completely suppressed among surviving animals (treatment-related, adverse). (Please refer to table 2 and 3 in the "Any other information on results including tables section")
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
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
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: At 4000 mg/kg bw/day: 5/20 rats that died or were sacrificed showed marked gastric dilatation and fluid retention was found at post mortem examination.
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: not examined
Histopathological findings: neoplastic:: not examined
Other effects:: not examined
Number of abortions:: not specified
Pre- and post-implantation loss:: no effects observed
Description (incidence and severity):: There were no statistically significant (P> 0.05) intergroup variations in respect of post-implantation loss. (Please refer to table 4 in the "Any other information on results including tables section")
Total litter losses by resorption:: effects observed, treatment-related
Description (incidence and severity):: One animal (No. 74) of the 4000 mg/kg bw/day dose group showed total litter loss. (Please refer to table 1 and 4 in the "Any other information on results including tables section")
Total litter loss occured in the presence of marked body weight loss during pregnancy compared to control animals. In detail, from start of the experiment till GD6, the dam´s body weight was comparable to the mean body weight of the control group. Starting on GD 10, a marked reduction in body weight was observed about 21, 24, 26 and 32% (determined on GD 10, 14, 17 and 20, respectively) compared to the mean body weight of the control group.
Early or late resorptions:: effects observed, treatment-related
Description (incidence and severity):: One animal (No. 74) of the 4000 mg/kg bw/day dose group showed total litter loss. (Please refer to table 1 and 4 in the "Any other information on results including tables section")
As described above, total litter loss was accompanied by marked systemic toxicity.
Dead fetuses:: no effects observed
Description (incidence and severity):: No signinificant differences in the number of embryonic deaths was determined between treatment and control groups. (Please refer to table 4 in the "Any other information on results including tables section")
Changes in pregnancy duration:: not specified
Changes in number of pregnant:: no effects observed
Description (incidence and severity):: Pregnancy rate was comparable for all groups. (Please refer to table 4 in the "Any other information on results including tables section")
: Key result
Dose descriptor:: NOAEL
Remarks:: systemic toxicity
Effect level:: 1 000 mg/kg bw/day
Based on:: test mat.
Basis for effect level:: other: no effects observed at this dose level
: Key result
Dose descriptor:: LOAEL
Remarks:: systemic toxicity
Effect level:: 4 000 mg/kg bw/day
Based on:: test mat.
Basis for effect level:: body weight and weight gain; clinical signs; mortality
: Key result
Dose descriptor:: NOAEL
Remarks:: developmental toxicity
Effect level:: 1 000 mg/kg bw/day
Based on:: test mat.
Basis for effect level:: other: no adverse effect observed at this dose level
: Key result
Dose descriptor:: LOAEL
Remarks:: developmental toxicity
Effect level:: 4 000 mg/kg bw/day
Based on:: test mat.
Basis for effect level:: total litter losses by resorption
Remarks on result:: other: Total litter loss at occured in the presence of severe systemic toxicity
: Key result
Abnormalities:: effects observed, treatment-related
Localisation:: uterus
Description (incidence and severity):: Total litter loss was observed in 1/15 dams, which occured in the presence of marked systemic toxicity.
Fetal body weight changes:: effects observed, treatment-related
Description (incidence and severity):: At 4000 mg/kg bw/day: mean fetal body weight significantly reduced.
Fetal body weight was comparable to the control group up to and including 1000 mg/kg bw/day. (Please refer to table 4 in the "Any other information on results including tables section")
Reduction in number of live offspring:: no effects observed
Description (incidence and severity):: (Please refer to table 4 in the "Any other information on results including tables section")
Changes in sex ratio:: no effects observed
Description (incidence and severity):: (Please refer to table 4 in the "Any other information on results including tables section")
Changes in litter size and weights:: effects observed, treatment-related
Description (incidence and severity):: At 4000 mg/kg a combination of slightly low litter size and slightly low foetal weight led to a low litter weight which was significantly (P< 0.05) different from the control value. Litter size and weight appeared unaffected up to a concentration of 1000 mg/kg bw/day. (Please refer to table 4 in the "Any other information on results including tables section")
Anogenital distance of all rodent fetuses:: not examined
Changes in postnatal survival:: not examined
External malformations:: effects observed, treatment-related
Description (incidence and severity):: At 4000 mg/kg slightly higher incidences of major malformations were observed. The results in the lower dose groups were comparable to the control group. (Please refer to table 5 in the "Any other information on results including tables section")
Skeletal malformations:: effects observed, treatment-related
Description (incidence and severity):: At 4000 mg/kg slightly higher incidences of major malformations and minor anomalies were observed. The minor anomalies particularly of the skeleton, were mainly forms of relative immaturity (eg retarded ossification), correlating with the lower mean foetal weight at this dosage. These effects are considered more likely to be secondary consequences of maternal toxicity rather than to a direct effect on the embryo.

At 4000 mg/kg there was an increased proportion of foetuses with variant sternebrae the difference from controls attaining statistical significance (P< 0.05)for normal ribbed foetuses. Since the variants were mainly forms of reduced ossification the increase would correlate with the lower mean foetal weight of this group. These effects are considered more likely to be secondary consequences of maternal toxicity rather than to a direct effect on the embryo.

At 500 and 1000mg/kg bw incidences of skeletal variants did not differ significantly (P>0.05) from control values.

(Please refer to table 5 and 6 in the "Any other information on results including tables section")
Visceral malformations:: no effects observed
Description (incidence and severity):: No signinificant differences between treatment and control groups.
Other effects:: not examined
: Key result
Dose descriptor:: NOAEL
Effect level:: 1 000 mg/kg bw/day
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: no adverse effect observed at this dose level
: Key result
Dose descriptor:: LOAEL
Effect level:: 4 000 mg/kg bw/day
Based on:: test mat.
Sex:: male/female
Basis for effect level:: fetal/pup body weight changes; changes in litter size and weights; external malformations; skeletal malformations
Remarks on result:: other:
Remarks:: All effects were observed in the presence of marked systemic toxicity in maternal animals.
: Key result
Abnormalities:: effects observed, treatment-related
Localisation:: skeletal: sternum; skeletal: rib
Description (incidence and severity):: At 4000 mg/kg bw/day there was an increased proportion of foetuses with variant sternebrae attaining statistical significance (P< 0.05) for normal ribbed foetuses. Since the variants were mainly forms of reduced ossification the increase would correlate with the lower mean foetal weight of this group.
: Key result
Developmental effects observed:: yes
Lowest effective dose / conc.:: 4 000 mg/kg bw/day
Treatment related:: yes
Relation to maternal toxicity:: developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:: yes
Relevant for humans:: not specified
Conclusions:: Based on the results obtained in this prenatal developmental toxicity study in rats, the NOAEL for maternal and developmental toxicity can be set at 1000 mg/kg bw/day.
There is no evidence of teratogenicity .

Effect on developmental toxicity: via oral route

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 300 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: The available information comprises an adequate and reliable study (Klimisch score 1) from an analogue substance. Read across is justified based on (bio)transformation to common compounds. The selected study is thus sufficient to fulfill the standard information requirements set out in Annex IX, 8.7, of Regulation (EC) No 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:: no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:: no study available

Additional information

No reliable data on Fosetyl-sodium are available. However, hazard assessment is conducted by means of a read-across to reliable data on the analogue substance Fosetyl-Al. The selected study is an adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall quality assessment (refer to the endpoint discussion for further details). (for further details on the read-across approach please refer to chapter 13 in the technical dossier).

A GLP-compliant study according to OECD Guideline 414 was performed to characterize developmental toxicity of Fosetyl-aluminium (Fosetyl-Al, M-205472 -01 -1). Groups of 25 New Zealand White rabbits were exposed daily to 50, 100 and 300 mg Fosetyl-Al/kg bw via gavage from Day 4 – 28 of gestation. Dose selection was based on the results of a range-finding teratology study performed with 75, 125, 250, 500 and 1000 mg Fosetyl-Al /kg bw/day administered via gavage during Days 4 - 28 of gestation. Control animals received the respective vehicle (0.5% (w/v) methyl cellulose). Dams were sacrificed on Day 29 of gestation. Unscheduled deaths were recorded at any dose level: 1 low-dose dam was elective sacrificed on Day 27 of gestation as a result of an abortion. As spontaneous abortions related to stress are common among rabbits and no abortion occurred in the other dose groups, the abortion and subsequent sacrifice was considered as not substance-related. Further, 1 mid-dose female was found dead on Day 11 of gestation which was most probable due to gavage trauma identified through discoloured lungs and fluid-filled lungs and trachea. In the high-dose group, 6/25 rabbits were found dead on Days 6 (two unscheduled death), 9, 12, 18 and 27 of gestation. As fluid-filled lungs and/or discoloured lungs were observed post-mortem in 5/6 rabbits, mortality due to gavage trauma seems to be the cause of death for at least 5 high-dose rabbits. The cause of death of the remaining rabbit, which died on Day 27 of gestation, remained unclear. No clinical signs of systemic toxicity were observed in test and control animals despite scabs, alopecia and decreased faecal volume. As these effects were determined only at low incidence in the control and dose groups and they represent common laboratory findings, they are interpreted as fortuitous and not substance-specific. Furthermore, body weight gain was comparable among the groups. Despite a significant increase in food consumption from Day 27 - 29 of gestation in mid- and high-dose females, food consumption was unremarkable in all animals. As body weight gain appeared normal in all animals, the biological significance of the altered food consumption remains questionable. Furthermore, pregnancy rates and gravide uterine weight were comparable among the groups. Uterine implantation data, number of corpura lutea and pre-and postimplantation loss were unremarkable. Fetal body weights, sex ratio and the number of live fetuses per dam were comparable among the groups. Furthermore, external, visceral and skeletal examinations did not reveal treatment-related effects. Moreover, the death of 1 low-dose fetus which exhibited several external malformations including gastroschisis, hindlimbs hyperflexion and agenesis of the tail is not interpreted as treatment-related due to the low incidence. Based on the reported findings, no maternal toxic or developmental effects were observed after administration of Fosetyl-Al. Thus, a NOAEL of ≥ 300 mg/kg bw/day was defined for maternal and developmental toxicity.

In addition, an oral pre-natal developmental toxicity study in rats (similar to OECD TG 414, conducted prior to GLP) was performed with Fosetyl-Al (M-158819 -01 -1). Groups of 20 CFY females rats were administered 0, 500, 1 000 or 4 000 mg/kg bw/day of Fosetyl-Al by gavage, once daily from Day 6-15 of gestation. Control animals received the respective vehicle (water). All rats were sacrificed on Day 20 of gestation.

Occasional deaths seen in the 500 and 1000 mg/kg bw/day dose groups were attributable to dosing errors. At the highest dosel level, 4000 mg/kg bw/day, 5/20 rats died or were sacrificed on Day 9; 10 and 11. All rats exhibited body weight loss and 3/5 rats showed chromodacryorrhea prior to death. In addition, post mortem examinations revealed marked gastric dilatation and fluid retention.

Pregnancy rate was comparable among all dose groups. Litter parameters, including litter size, post implantation loss, litter and mean foetal weights were not affected in the 500 and 1000 mg/kg bw/day dose groups. One animal of the highest dose group showed total litter loss. It should be noted that total litter loss occured in the presence of marked body weight loss during pregnancy. At 4000 mg/kg bw/day slightly higher post implantation loss and slightly lower values for litter size and mean foetal weight led to a lower litter weight which was significantly different from the control value.

Embryonic and foetal development, as assessed by the incidences of major malformations, minor vis ceral and skeletal anomalies and the distribution of skeletal variants, was not significantly affected by treatment at 500 or 1000 mg/kg bw/day. Foetuses from the 4000 mg/kg bw/day group exhibited a slightly higher incidence of major abnormalities (thoracic asymmetry, displaced kidney and testes, hydrocephaly, vein/artery transposition, intra - abdominal and subcutaneous haemorrhage) and minor abnormalities (subcutaneous oedema, medial displacement of the testis). A higher incidence of skeletal (sternebrae) variants was only seen in the 4000 mg/kg bw/day group which correlates with the lower mean foetal weight. The incidences of mild to moderate subcutaneous oedema and small interparietal and occipital bone were significantly increased in the high-dose group. Frequently, several abnormalities occurred in a single pup, so that more abnormalities appeared to occur in the high dose group, whereas the proportion of pups with abnormalities was not different from controls.

Overall, no adverse effects were observed in maternal animals and fetuses up to a dose of 1000 mg/kg bw/day. Thus, based on the obtained results, a NOAEL of 1000 mg/kg bw/day was defined for maternal systemic and developmental toxicity. Likewise, a NOAEL of 1000 mg/kg bw/day was set for fetal developmental toxicity. The LOAEL for maternal systemic and developmental effects was determined at 4000 mg/kg bw/day, due to reduced body weight, mortality and a higher implantation loss and lower litter weight in the highest dose group. For fetal developmental toxicity, the LOAEL was also set at 4000 mg/kg bw/day, based on the increased incidence of major and minor abnormalities and skeletal variants in the highest dose group. Fetal effects were clearly related to maternal toxicity and thus, it is concluded that the observed developmental effects are a secondary non-specific consequence of maternal toxicity effects.

Moreover, the three-generation study performed in rats with Fosetyl-Al provided information on developmental toxicity (M-203019-01-1). Briefly, for developmental analyses, parental generations were exposed to 6000, 12000 and 24000 ppm test substance (corresponding to mean nominal doses of 479, 933 and 1890 mg/kg bw/day (P), 546, 1134 and 2813 mg/kg bw/day (F1) for approx. 23 weeks prior to mating and were kept on the respective diets during the mating (approx. 3 weeks) and gestation period (for further details on study design, please refer to the endpoint summary described above under “Reproductive toxicity”). On Day 20 of gestation, 5 P0 and 10 F1 dams were sacrificed. The number of corpora lutea was slightly reduced in mid and high dose dams, but significance was only reached at high-dose for both generations. Consequent to the lower corpora lutea count the numbers of implantations per dam and litter weight values were lower than those of controls at day 20 of pregnancy. At 24 000 ppm but not at 12 000 ppm differences from control values frequently attained statistical significance. However, only the P0 generation showed an association between the reduced number of corpora lutea and a decreased number of live young (statistically significant), increased post-implantation loss and increased embryonic deaths (both not statistically significant). The litter weight was significantly reduced in F0, F1B and F2B pups of the high dose group. The amount of pre-implantation loss and sex ratio and mean foetal weight at birth (but not day 21) were comparable among the groups in all generations. Body weight of the offspring was comparable for all groups and generations until Day 4 post partum. Afterwards, during mid- and late-lactation, body weight gain was reduced in high-dose offspring, which is most probably related to the reduced body weight gain in maternal animals during lactation. A similar but less marked effect on body weight gain was evident in the mid-dose group. Body weight gain of the low-dose group did not show a consistent effect.

Furthermore, a dose-dependent decrease in spleen weight was observed at all dose levels in F3 fetuses. However, in the absence of correlated histopathological findings, this effect should not be considered as a specific effect of developmental toxicity.

Urinary bladder effects were observed only in F3 fetuses and were rather mild. As most alterations were associated with crystal deposition in the lumen of the urinary bladder, the observed effects might be related to physical irritation of the surrounding tissues induced by the crystals.

Based on the above described findings, the following effect levels were determined for developmental toxicity:

NOAEL (fetal effects, F1): 1200 ppm (equivalent to 954 mg/kg bw/day (lowest mean). No treatment-related adverse effects were observed.

LOAEL (fetal effects, F1): 24000 ppm (equivalent to 1958 mg/kg bw/day (lowest mean). Based on, reduced number of live young, reduced mean pup weight at day 21 and reduced litter weight at the highest dose level. It should be noted that a lower number of corpora lutea was evident at the same dose.

LOAEL (fetal effects, F2 and F3): 6000 ppm (equivalent to 586 mg/kg bw/day (lowest mean). Based on dose-dependent reduced spleen weight (only investigated in F3 fetuses). In the absence of correlated histopathological findings, this should not be considered as a specific effect of developmental toxicity. In addition, at 12000 ppm (F2B second mating) and 24000 ppm (F1 and F2), reduced mean pup weight at day 21 (related to maternal decrease in body weight) and reduced litter weight were observed. In regard to reduced litter weight, it should be noted that a lower corpora lutea count was observed at the same dose. Furthermore, urinary tract changes were observed in animals of the highest dose group.

The observed effects on the offspring are not considered to meet the classification criteria for developmental toxicity as they occur at extreme high doses and in the presence of marked maternal toxicity, which is in line with the Guidance on the Application of the CLP criteria (Annex I: 3.7.4).

Briefly, reduced mean body and litter weight, and reduced numbers of implantations per dam are considered related to the reduced corpora lutea count and maternal systemic toxicity indicated by marked reductions in body weight at specific time intervals. Moreover, altered spleen weights without histopathological correlates and effects on urinary bladder of mild severity not occurring in all fetal generations are not considered to warrant classification. Thus criteria for classification as reproductive/developmental toxicant, as described in the Guidance on the Application of the CLP criteria (3.7.2.2) are not fulfilled.

Conclusion

In conclusion, the available data on the read-across analogue Fosetyl-Al did not indicate adverse effects relevant for classification on the developing offspring. Thus, Fosetyl-sodium is not considered to exhibit developmental toxicity.

In addition, a non-reliable developmental toxicity study on Fosetyl-sodium is available (B-WY-150057), which was performed equivalent to OECD 414 (B-WY-150057, 2015) in rats. Due to the lack of toxicological endpoints and reporting inconsistencies, the study is not considered as reliable key study. Particularly the lack of individual data, the lack of dose formulation analysis and inconsistency of reported dose levels, the study is disregarded (RL3) for hazard assessment. A summary of the study is described below for reasons of completeness.

Young, adult, and nulliparous females were mated with males. Day 0 of pregnancy was defined as the day a vaginal plug or sperm were found. Successfully copulated females were assigned to the different test groups including 22, 22, 21, and 24 females for control, low-, mid- and and high-dose groups, respectively. Starting on gestational day (GD) 6, females were exposed orally via gavage to 100, 400, or 1600 mg/kg bw/day until GD 15. Females were sacrificed on GD20. The uteri were removed and weighted. The uterine contents were examined for the number of implantations, corpora lutea, resorptions, dead and live fetuses. The sex, body weight, body length and tail length of each fetus were determined. Each fetus was examined for external anomalies. Approximately one third of each litter was immersed in Bouin's solution and examined for viscera anomalies. The remainder was immersed in 90% ethanol and examined for skeletal alterations.

No signs of overt toxicity were observed in all dose groups (including period of exposure and the observation period). Body weight and net gain during pregnancy was comparable among groups. In high-dose females, an increased placenta weight was observed. As fetal body weight and body length was unaffected by treatment, only limited toxicological significance was considered for the observed elevated placenta weight and the effect was not considered adverse. The number of corpora lutea, the percentage of live births, absorbed fetuses, stillbirths, uterine weight including fetuses, the average fetal body weight and body length, and tail length were not affected by treatment. No abnormalities were observed in fetal appearance, fetal skeleton or internal organs. Furthermore, no deformities were observed in fetuses of any dose group.

Overall, the test substance did not exhibit developmental toxicity after in utero exposure. Thus, a NOAEL for maternal and developmental toxicity of 1600 mg/kg bw/day was derived in this study. As discussed above, the study was not considered for hazard assessment due to major deficiencies.

Conclusion

In conclusion, based on the available data on developmental toxicity, in utero exposure towards the test substance did not lead to adverse effects sufficiently for classification in the offspring.

Justification for classification or non-classification

The available data on reproductive toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.

Additional information

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Endpoint		Control	50 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day
Number of dams mated		25	25	25	25
Number of pregnant dams		22	24	23 (pregnancy undetermined in 1 animals)	19 (pregnancy undetermined in 5 animals)
Number of nonpregnant dams		3	1	1	1
Pregnancy rate (%)		88	96	96^#	95^#
Number of dams with viable fetuses		22 (88%)	23 (95.8%)	23 (95.8%)	18 (94.7%)
Mortality of dams		0	1 (elective sacrifice, GD 27)	1 (GD 11)	6 (GD 6 (2 animals), 9, 12, 18 and 27)
Examined at caesarean section		25	24	24	19
Clinical findings(frequency/animals)
General appearance	Red exudate from extremities	1/1	0/0	1/1	0/0
General appearance	Swollen vulva	1/1	4/1	0/0	3/2
Dermal (general)	Scabs	37/2	13/1	1/1	21/1
	Alopecia – extremities/snout	66/6	1/1	8/2	21/4
	Black/brown anogenital staining	0/0	0/0	7/3	7/1
	Yellow anogenital staining	7/2	1/1	0/0	0/0
	Ulceration	2/1	0/0	1/1	0/0
	Alopecia (general)	22/3	17/2	25/5	9/4
Miscellaneous	Decreased faecal volume	24/10	18/7	12/6	22/10
	Watery stool	0/0	1/1	2/2	1/1
	One fetus found in the cage	0/0	1/1	0/0	0/0
Post-dose	Excessive salivation (immed post dose)	0/0	0/0	0/0	1/1
Post-dose	Palpable masses	0/0	0/0	2/1	20/1
Body weight gain (g) [GD 0 – 29]		501 ± 229.4	531 ± 189.9	552 ± 209.6	476± 208.6
Food consumption (g/kg bw/day) [GD 27 – 29]		21± 8.2	26± 10.6	31± 9.1**	29± 7.5*
Food consumption (g/kg bw/day) [GD 0 – 29]		39± 7.5	40± 7.6	41± 7.0	38± 6.1
Gravid uterus weight (g)		561±119.2	593±113.6	543±145.6	527± 135.8

Endpoint	Control	50 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day
Corpora lutea (total number)	240	271	246	208
Corpora lutea (no. per aimal [mean])	10.9 ± 1.85	11.8 ± 2.24	10.7 ± 2.95	11.6 ± 2.20
Implantations (total)	192	219	195	157
Implantations (no. per animal [mean])	8.7 ± 2.07	9.5 ± 1.83	8.5 ± 2.87	8.7 ± 2.27
Total number of live foetuses	183	200	190	148
Number of live fetuses per animal [mean]	8.3 ± 2.01	8.7 ± 1.84	8.3 ± 2.68	8.2 ± 2.51
Total number of dead foetuses	0	1	0	0
Pre-implantation loss (% per animal)	20.2 ± 14.97	18.8 ± 8.93	22.1 ± 12.76	24.6 ± 17.75
Post-implantation loss (no per animal)	0.4 ± 0.59	0.8 ± 1.23	0.2 ± 0.42	0.5 ± 0.71
total number of litters	22	23	23	18
Foetus weight (mean) [g]	45.3 ± 3.91	44.7 ± 3.44	45.6 ± 4.33	43.8 ± 4.77
Foetal sex (ratio (% male)	52.1 ± 18.56	55.1 ± 13.39	44.6 ± 15.69	52 ± 12.46

Group mg/kg bw/day	Bodyweight (g) at Day
Group mg/kg bw/day	1	3	6	10	14	17	20
Control	212	229	246	269	294	322	371
500	219	238	257	278	303	331	375
1000	220	239	259	277	302	333	384
4000	218	237	257	260	297	316	365

Group mg/kg bw/day	Percentage of Young* with
	Extra rib(s)	Variant sternebra€
	Extra rib(s)	Normal ribs	Extra rib(s)
Control	24.6	25.9	23.8
500	29.0	26.9	38.2
1000	42.1	18.0	8.9
4000	27.7	58.3+	56.1

Category	Group (mg/kg bw/day)
	control	500	1000	4000
Mated	20	20	20	20
Dead or killed	-	1a	2aa	5
Non-pregnant	1	1	1	-
Total resorption	-	-	-	1
With viable Young	19	18	17	14

Group mg/kg bw/day	Change from Bodyweight (g) at Day 6
Group mg/kg bw/day	1	3	6	10	14	17	20
Control	-34	-17	0	23	48	76	125
500	-38	-19	0	21	46	74	118
1000	-39	-20	0	18	43	74	125
4000	-39	-20	0	3	40	59	108

Group mg/kg bw/day	No. of animals	Pregnancy rate%^a	Live young			Embryonic Deaths			Implantations	Corporalutea	Pre-implantation loss %	Post-implantation loss %	Litter WT.(g)	Mean foetal WT. (g)
Group mg/kg bw/day	No. of animals	Pregnancy rate%^a	m	f	Total	Early	Late	Total	Implantations	Corporalutea	Pre-implantation loss %	Post-implantation loss %	Litter WT.(g)	Mean foetal WT. (g)
Control	19	95	6.4	6.4	12.7	0.4	0.1	0.5	13.2	14.7	10.3	3.5	48.05	3.79
500	18	95	5.8	5.5	11.3	0.6	0.4	0.9	12.3	14.2	12.7	8.9	42.07	3.74
1000	17	95	6.1	6.9	13.1	0.4	0.0	0.4	13.4	15.3	12.1	2.6	48.50	3.72
4000	14^b	95	5.3	6.2	11.5	0.5	0.8	1.3	12.8	14.9	12.1	10.5	39.71*+	3.46*(+)

Group mg/kg bw/day	Number of Young
	Major malformations			Minor malformations (-)
	Major malformations			Visceral			Skeletal
	Examined	Total	Mean %	Examined	Total	Mean %	Examined	Total	Mean %
Control	242	2	0.8	119	8	6.9	121	9	7
500	204	1	0.8	103	7	6.6	100	9	12.6
1000	222	2	0.8	108	8	7.4	112	14	11.8
4000	161	5	3.8	78	8	13.0	78	17	22.4

Registration Dossier

Registration Dossier

Data platform availability banner - registered substances factsheets

Diss Factsheets

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

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Effect on fertility: via oral route

Effect on fertility: via inhalation route

Effect on fertility: via dermal route

Additional information

Effects on developmental toxicity

Description of key information

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Effect on developmental toxicity: via oral route

Effect on developmental toxicity: via inhalation route

Effect on developmental toxicity: via dermal route

Additional information

Justification for classification or non-classification

Additional information

Referenceopen all close all

Referenceopen all close all