Phosphonic acid

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 August - 21 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 August - 21 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

Principles of method if other than guideline:

In addition, the procedures described in the report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks (Groups 1-4) and 12 weeks (additional animals for Group 4).
- Weight at study initiation: mean weight at start of treatment was 324 - 330 gr (males) or 211 - 214 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages. Pups were kept with the dam until termination
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES
From: 31 August to 21 November 2012

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

- Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. No correction was made for the purity/composition of the test substance.
- Storage conditions of formulations: At ambient temperature.
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
- Preventative measures: The pH of the dose preparations for Groups 2-4 was relatively low. To avoid possible local esophageal effects, the following preventative measures were taken:
1 Wiping of the flexible catheter prior to dosing of each animal.
2 From 16 October 2012 onwards, additional rinsing of the flexible catheter with approximately 0.5-0.6 mL water (Elix, Millipore S.A.S., Molsheim, France) after dosing each animal with the test formulations.

One Group 2 female was dosed according to her Day 20 post-coitum body weight, while she had just delivered her litter (which was not completely cleaned yet). The slightly higher dose volume resulted in a dose level of 103 mg/kg instead of 80 mg/kg.
Evaluation: Handling of this animal did not disturb maternal care as all pups survived until planned necropsy and no findings were noted. A slightly higher dose level for just one day of treatment will not affect data of this animal.

From 12 November 2012 onwards, it was not noted in the raw data if the flexible catheter was wiped prior to dosing of each animal.
Evaluation: In the unlikely event that this preventative action was not taken, no local esophageal effects were caused as no esophageal abnormalities were seen at necropsy.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The delegated phase was performed by the Principal Investigator for Formulation Analysis.

Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. Samples remained on dry ice until receipt at TNO Triskelion, The Netherlands.

Formulation samples were not transferred to the weighing room, but were dispatched directly to the test site.
Evaluation: Samples were received in good order by the test site.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41-48 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).Two Group 1 females, three Group 2 females, one Group 3 female and one Group 4 female were not dosed during littering.

Frequency of treatment:

Once daily, 7 d/w

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Remarks:

During the first four days high dose animals were dosed at 750 mg/kg however due to unexpected severe toxicity the dose level was lowered to 500 mg/kg from Day 5 onwards.

No. of animals per sex per dose:

Group 1-3: 10/sex
Group 4: 13 males and 11 females. (one female and three males lost during the first 5 days of treatment were replaced by spare animals)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of a 10-Day dose range finding study (See attached results).

Based on the results of this range finding study, dose levels for the main study were: 0, 80, 250 and 750 mg/kg.
Since no clear peak effect of occurrence of clinical signs was observed in the range finding study, clinical observations in the main study were conducted immediately after dosing.

During the first four days of the treatment period, Group 4 animals were dosed at 750 mg/kg. From Day 5 onwards, they were dosed at 500 mg/kg.

- Selection of animals for selected measurements: 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.

Positive control:

Not required.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstance of any death was recorded in detail.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals, at least immediately after dosing (no clear peak effect of occurence of clinical signs in the dose range finding study). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION
- Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY: yes

WATER CONSUMPTION
No. Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION
No

HAEMATOLOGY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS
No

NEUROBEHAVIOURAL EXAMINATION
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: According to test guidelines

Sacrifice and pathology:

GROSS PATHOLOGY
All animals except for non-selected females were fasted overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- The number of former implantation sites and corpora lutea were recorded for all paired females.
- Selected 5 animals/sex/group and all animals that died spontaneously or were killed in extremis: According to test guidelines
- All remaining animals and females which failed to deliver: According to test guidelines

ORGAN WEIGHTS
- Selected 5 animals/sex/group: According to test guidelines
- All remaining males: Epididymides and testes

Inadvertently, the terminal body weights of two Group 1 females were not determined at necropsy, and inadvertently organ weights were collected from two Group 4 males when they were not required.
Evaluation: Sufficient data was available for evaluation. Moreover, as it concerned two control animals historical control data could be taken into account. The extra organ weights were taken from animals who had been fasted and are thus representative. These data are reported.

HISTOPATHOLOGY
- According to test guidelines

By error, the thyroid of one control Female was not weighed at necropsy and a few tissues were not available for histopathology. Reasons for missing a few tissues included that these tissues were not discernible at necropsy or trimming, or were erroneously not collected at necropsy. Missing tissues are listed in raw data and pathology report.
Evaluation: Sufficient data was available for evaluation.

Inadvertently, the adrenals, aorta, clitoral gland, thymus, liver and lungs of one Group 4 female were fixated a few minutes in Modified Davidson instead of 10% buffered formalin.
Evaluation: This did not affect the quality of the slides.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 750/500 mg/kg laboured respiration, rales, gasping, piloerection, chromodacryorrhoea of the snout, lethargy, hunched posture, salivation, hypothermia and lean appearance were noted for the animals that died or were euthanized in extremis.

Animals at 750/500 mg/kg that survived to the end of the treatment period were noted with rales, salivation, hunched posture, laboured respiration, piloerection, lean appearance, lethargy, spasms and flat posture.

Piloerection, rales, chromodacryorrhoea of the snout, hunched posture, laboured respiration and salivation were noted transiently for a few animals at 250 mg/kg. As these signs were transient and noted for a limited number of animals at this dose level, they were not considered to be adverse. Scabs on the right flank was noted for one male at 250 mg/kg, this was incidental.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were three males and one female dosed at 750 mg/kg that were euthanized in extremis or died spontaneously after 2-4 days of treatment. The dose level was subsequently lowered to 500 mg/kg. After that time, there were an additional four females and one male that died or were euthanized in extremis. The females died or were euthanized after 29-36 days and the male was killed in extremis after 9 treatment days.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights and body weight gains were lower for males at 750/500 mg/kg than controls from Day 8 of the premating period through the entire mating period (not always statistically significant). Body weights and gains were lower for females at 750/500 mg/kg than controls from Days 14-20 of the post coitum period (only absolute body weight on Day 14 was significantly lower than controls). The difference for females was mostly attributed to weight loss and reduced gains for two Group 4 females (one Group 4 female also contributed to this on post coitum Day 14; she was killed in extremis after this point).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 750/500 mg/kg, a trend towards slightly lower food consumption (absolute and relative to body weight) was noted for males and females during the premating period (not statistically significant). Absolute and relative food consumption was significantly lower for high dose females from Days 4-17 of the post coitum period.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Absolute food consumption was also significantly lower for females at 250 mg/kg from Days 7-11 post coitum, though the effect was only transient, and in the absence of corresponding effects on body weights, it was not considered to be adverse.

The significantly lower food consumption (absolute and relative) noted for females at 80 mg/kg from Days 0-7 and for females at 250 mg/kg from Days 0-4 of the post coitum period, respectively, were not considered to be toxicologically relevant as they occurred in the absence of a dose-related distribution.

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

no effects observed

Description (incidence and severity):

Based on subjective appraisal.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant effects on haematology parameters up to 750/500 mg/kg.

Haemoglobin and haematocrit values were significantly lower for males at 750/500 mg/kg and significantly higher for females at 750/500 mg/kg than controls. These were not considered to be toxicologically relevant, however, as these changes were opposite for males and females and the values remained within the range of normal biological variation and did not show a dose-dependent distribution.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 750/500 mg/kg significantly lower values for total protein (males) and albumin (females) and a higher concentration of bile acids (males) compared to controls were noted.
The remaining statistically significant changes noted in this study were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. These changes included increased concentrations of sodium (males at 250 mg/kg), chloride (males at 80, 250 and 750/500 mg/kg), and lower concentrations of urea (females at 250 mg/kg), sodium (females at 80 and 750/500 mg/kg) and inorganic phosphates (females at 250 and 750/500 mg/kg).

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 750/500 mg/kg females had lower absolute and relative uterine weights and lower absolute spleen weights. Females at 250 mg/kg had lower absolute spleen weights as well. The lower spleen weights at 750/500 mg/kg parallel the decrease in hemopoietic foci seen in females at the microscopic examination.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic findings noted for the animals at 750/500 mg/kg that died or were euthanized in extremis included isolated or several reddish or dark red foci on the stomach glandular mucosa, irregular surface or isolated dark red or black foci of the forestomach and/or glandular mucosa, perforations of the forestomach, fibrin-like coating on the diaphragm, thickened limiting ridge of the stomach, reduced size of spleen and thymus, enlarged lungs, GI-tract distended with gas, beginning autolysis, greenish discoloration of the lungs, pelvic dilation of the kidney, emaciated appearance, uterus contains live fetuses (one Group 4 female) and many dark red foci on the thymus.

There were no treatment-related macroscopic findings in the animals surviving to their scheduled necropsy dates.

Incidental findings noted for control and/or treated animals included enlarged or reddish discolored mesenteric lymph node, enlarged mandibular lymph node, isolated reddish foci on the thymus, reduced size of the left seminal vesicle, scabbing on the right flank, isolated tan or dark red focus/foci on one or both clitoral glands, several yellowish, soft nodules on the clitoral glands, yellowish, soft nodule on the epididymis, pelvic dilation of the kidneys and yellowish, soft nodule on the liver. These findings remained within the background range encountered among rats of this age and strain, did not show a dose-related incidence trend and were thus not considered to be treatment related or toxicologically relevant.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were two Group 4 males and one Group 4 female found dead. The two males had macroscopic perforation of the forestomach, which for one Group 4 male correlated to ulceration of the forestomach. For another Group 4 male the autolysis of the stomach was too advanced for a thorough microscopic evaluation, though the presence of a few mucosal erosions still could be noted. For one Group 4 female complete ulceration of the trachea epithelium was the cause of death.
In addition, there were two Group 4 males and four Group 4 females sacrificed moribund. For one Group 4 male the cause of moribundity was marked necrosis of the trachea epithelium, for another Group 4 male no definite cause of moribundity could be established. For one Group 4 female ulceration of the glandular stomach and for another Group 4 female moderate ulceration of the trachea and bronchial epithelium were the cause of moribundity. For two Group 4 females no definite cause of moribundity could be established. These deaths are considered test item related.

There were treatment-related microscopic findings present in rats surviving to their scheduled necropsy dates in the:

Stomach
- Glandular inflammation, lymphogranulocytic was present in male rats treated at 750/500 mg/kg (4/5 rats) up to a slight degree.
- Glandular stomach erosion was present in male rats treated at 750/500 mg/kg (1/5) at a minimal degree.
- Hyperplasia of the forestomach squamous epithelium was present in male rats treated at 750/500 mg/kg (2/5) up to slight degree.
- Vacuolation of the limiting ridge was present at increased severity in male rats treated at 750/500 mg/kg (2/5) at slight degree. Minimal degrees were present in control, 80 and 250 mg/kg treated rats (all 1/5 rats) and this was considered to be the background level.
- Thickened limiting ridge was present in male rats treated at 750/500 mg/kg (2/5) up to moderate degree.

Spleen
- Hemopoietic foci, primarily erythropoiesis showed a slight decrease in 750/500 mg/kg treated female rats.

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals.

The variation in motor activity did not indicate a relation with treatment.
All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

Key result

Dose descriptor:

NOAEL

Remarks:

Parental generation

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Remark

Critical effects observed:

not specified

No test substance was detected in the Group 1 formulations. The concentrations analyzed in the formulations of Group 2, 3, 4 and 4/5 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

 

The formulations of Group 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

 

Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours (i.e. relative difference ≤ 10%).

Conclusions:

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 250 mg/kg (both for local and systemic toxicity).
Reproduction NOAEL: at least 500 mg/kg. As 750 mg/kg was only dosed for four days, this NOAEL could only be set at 500 mg/kg.
Developmental NOAEL: at least 250 mg/kg.
The likelihood that this NOAEL is actually higher cannot be excluded. However, the number of litters (n=6) available at the higher dose level of 750/500 mg/kg was too low to ensure a thorough evaluation of possible developmental effects.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

Principles of method if other than guideline:

In addition, the procedures described in the report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Phosphonic acid (Phosphorous acid)

IUPAC Name:

Phosphonic acid (Phosphorous acid)

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): Phosphonic acid (Phosphorous acid)
- Substance type: White crystalline solid
- Physical state: Solid
- Purity: See Certificate of Analysis
- Lot/batch No.: PAFK07F002
- Expiration date of the lot/batch: 31 July 2013 (allocated by WIL Research Europe B.V., 1 year after receipt of the test substance)
- Storage condition of test material: At room temperature in the dark desiccated
- pH: <1 at concentration of 1%
- Purity/composition correction factor required: No
- Hygroscopic: Yes, store desiccated
- Volatile: No
- Stability at higher temperatures: Yes, maximum temperature: <150°C
- Solubility in vehicle (water): >1067 g/L

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks (Groups 1-4) and 12 weeks (Group 5).
- Weight at study initiation: mean weight at start of treatment was 324 - 330 gr (males) or 211 - 214 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages. Pups were kept with the dam until termination
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES
From: 31 August to 21 November 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

- Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. No correction was made for the purity/composition of the test substance.
- Storage conditions of formulations: At ambient temperature.
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
- Preventative measures: The pH of the dose preparations for Groups 2-4 was relatively low. To avoid possible local esophageal effects, the following preventative measures were taken:
1 Wiping of the flexible catheter prior to dosing of each animal.
2 From 16 October 2012 onwards, additional rinsing of the flexible catheter with approximately 0.5-0.6 mL water (Elix, Millipore S.A.S., Molsheim, France) after dosing each animal with the test formulations.

One Group 2 female was dosed according to her Day 20 post-coitum body weight, while she had just delivered her litter (which was not completely cleaned yet). The slightly higher dose volume resulted in a dose level of 103 mg/kg instead of 80 mg/kg.
Evaluation: Handling of this animal did not disturb maternal care as all pups survived until planned necropsy and no findings were noted. A slightly higher dose level for just one day of treatment will not affect data of this animal.

From 12 November 2012 onwards, it was not noted in the raw data if the flexible catheter was wiped prior to dosing of each animal.
Evaluation: In the unlikely event that this preventative action was not taken, no local esophageal effects were caused as no esophageal abnormalities were seen at necropsy.

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The delegated phase was performed by the Principal Investigator for Formulation Analysis.

Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. Samples remained on dry ice until receipt at TNO Triskelion, The Netherlands.

Formulation samples were not transferred to the weighing room, but were dispatched directly to the test site.
Evaluation: Samples were received in good order by the test site.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41-48 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).Two Group 1 females, three Group 2 females, one Group 3 female and one Group 4 female were not dosed during littering.

Pups were not dosed directly but could have potentially been exposed to the test substance in utero, via maternal milk or from exposure to maternal urine/faeces.

Frequency of treatment:

Once daily for 7 d/w.

Details on study schedule:

- Age at mating of the animals in the study: Approximately 13 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

Remarks:

During the first four days high dose animals were dosed at 750 mg/kg. However, due to unexpected severe toxicity the dose level was lowered to 500 mg/kg from Day 5 onwards.

No. of animals per sex per dose:

Group 1-3: 10/sex
Group 4: 13 males and 11 females (one female and three males lost during the first 5 days of treatment were replaced by spare animals).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of a 10-Day dose range finding study (See attached results).

Based on the results of this range finding study, dose levels for the main study were: 0, 80, 250 and 750 mg/kg.
Since no clear peak effect of occurrence of clinical signs was observed in the range finding study, clinical observations in the main study were conducted immediately after dosing.

During the first four days of the treatment period, Group 4 animals were dosed at 750 mg/kg. From Day 5 onwards, they were dosed at 500 mg/kg.

- Selection of animals for selected measurements: 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.
- Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.
- Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.

Positive control:

Not required.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstance of any death was recorded in detail.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals, at least immediately after dosing (no clear peak effect of occurence of clinical signs in the dose range finding study). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION
- Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY: yes

WATER CONSUMPTION
No. Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION
No

HAEMATOLOGY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS
No

NEUROBEHAVIOURAL EXAMINATION
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: According to test guidelines

GENERAL REPRODUCTION DATA
- Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.
- Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Oestrous cyclicity (parental animals):

Not determined.

Sperm parameters (parental animals):

Slides of the testes were prepared for histopathological staging of spermatogenesis (selected 5 males of the control and high dose group and animals suspected to be infertile).

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

No clinical observations were registered in the computer for all pups of one Group 3 litter on Day 3 of lactation.
Evaluation: Sufficient information available. Moreover, no findings were noted on the other days.

On Day 3 of lactation, one extra pup was found in a Group 3 litter. No body weight is available for Day 1 and no clinical observations for Day 1 and 2. The body weight of this pup on Day 3 of lactation was 7.90 gram.
Evaluation: Sufficient information available.

From one Group 4 Female (killed in extremis on Day 19 post-coitum), the fetuses found in the uterus during necropsy were not sexed.
valuation: Sufficient information available for a thorough evaluation.

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

GROSS PATHOLOGY
All animals except for non-selected females were fasted overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- The number of former implantation sites and corpora lutea were recorded for all paired females.
- Selected 5 animals/sex/group and all animals that died spontaneously or were killed in extremis: According to test guidelines
- All remaining animals and females which failed to deliver: According to test guidelines

ORGAN WEIGHTS
- Selected 5 animals/sex/group: According to test guidelines
- All remaining males: Epididymides and testes

HISTOPATHOLOGY
- According to test guidelines

By error, the thyroid of one control Female was not weighed at necropsy and a few tissues were not available for histopathology. Reasons for missing a few tissues included that these tissues were not discernible at necropsy or trimming, or were erroneously not collected at necropsy. Missing tissues are listed in raw data and pathology report.
Evaluation: Sufficient data was available for evaluation.

Inadvertently, the terminal body weights of two Group 1 females were not determined at necropsy, and inadvertently organ weights were collected from two Group 4 males when they were not required.
Evaluation: Sufficient data was available for evaluation. Moreover, as it concerned two control animals historical control data could be taken into account. The extra organ weights were taken from animals who had been fasted and are thus representative. These data are reported.

Inadvertently, the adrenals, aorta, clitoral gland, thymus, liver and lungs of one Group 4 female were fixated a few minutes in Modified Davidson instead of 10% buffered formalin.
Evaluation: This did not affect the quality of the slides.

Postmortem examinations (offspring):

SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation Days 5-7.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

HISTOPATHOLOGY / ORGAN WEIGHTS
No.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100
- Pre-implantation loss (%): (number of corpora lutea - number of implantation sites)/number of corpora lutea x 100
- Post-implantation loss (%): (number of implantation sites - number of live fetuses) / number of implantation sites x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 750/500 mg/kg laboured respiration, rales, gasping, piloerection, chromodacryorrhoea of the snout, lethargy, hunched posture, salivation, hypothermia and lean appearance were noted for the animals that died or were euthanized in extremis.

Animals at 750/500 mg/kg that survived to the end of the treatment period were noted with rales, salivation, hunched posture, laboured respiration, piloerection, lean appearance, lethargy, spasms and flat posture.

Piloerection, rales, chromodacryorrhoea of the snout, hunched posture, laboured respiration and salivation were noted transiently for a few animals at 250 mg/kg. As these signs were transient and noted for a limited number of animals at this dose level, they were not considered to be adverse. Scabs on the right flank was noted for one male at 250 mg/kg, this was incidental.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were three males and one female dosed at 750 mg/kg that were euthanized in extremis or died spontaneously after 2-4 days of treatment. The dose level was subsequently lowered to 500 mg/kg. After that time, there were an additional four females and one male that died or were euthanized in extremis. The females died or were euthanized after 29-36 days and the male was killed in extremis after 9 treatment days.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights and body weight gains were lower for males at 750/500 mg/kg than controls from Day 8 of the premating period through the entire mating period (not always statistically significant). Body weights and gains were lower for females at 750/500 mg/kg than controls from Days 14-20 of the post coitum period (only absolute body weight on Day 14 was significantly lower than controls). The difference for females was mostly attributed to weight loss and reduced gains for two Group 4 females (one Group 4 female also contributed to this on post coitum Day 14; she was killed in extremis after this point).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 750/500 mg/kg, a trend towards slightly lower food consumption (absolute and relative to body weight) was noted for males and females during the premating period (not statistically significant). Absolute and relative food consumption was significantly lower for high dose females from Days 4-17 of the post coitum period.

Absolute food consumption was also significantly lower for females at 250 mg/kg from Days 7-11 post coitum, though the effect was only transient, and in the absence of corresponding effects on body weights, it was not considered to be adverse.

The significantly lower food consumption (absolute and relative) noted for females at 80 mg/kg from Days 0-7 and for females at 250 mg/kg from Days 0-4 of the post coitum period, respectively, were not considered to be toxicologically relevant as they occurred in the absence of a dose-related distribution.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant effects on haematology parameters up to 750/500 mg/kg.

Haemoglobin and haematocrit values were significantly lower for males at 750/500 mg/kg and significantly higher for females at 750/500 mg/kg than controls. These were not considered to be toxicologically relevant, however, as these changes were opposite for males and females and the values remained within the range of normal biological variation and did not show a dose-dependent distribution.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 750/500 mg/kg significantly lower values for total protein (males) and albumin (females) and a higher concentration of bile acids (males) compared to controls were noted.

The remaining statistically significant changes noted in this study were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. These changes included increased concentrations of sodium (males at 250 mg/kg), chloride (males at 80, 250 and 750/500 mg/kg), and lower concentrations of urea (females at 250 mg/kg), sodium (females at 80 and 750/500 mg/kg) and inorganic phosphates (females at 250 and 750/500 mg/kg).

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The variation in motor activity did not indicate a relation with treatment.
All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were two Group 4 males and one Group 4 female found dead. The two males had macroscopic perforation of the forestomach, which for one Group 4 male correlated to ulceration of the forestomach. For another Group 4 male the autolysis of the stomach was too advanced for a thorough microscopic evaluation, though the presence of a few mucosal erosions still could be noted. For one Group 4 female complete ulceration of the trachea epithelium was the cause of death.
In addition, there were two Group 4 males and four Group 4 females sacrificed moribund. For one Group 4 male the cause of moribundity was marked necrosis of the trachea epithelium, for another Group 4 male no definite cause of moribundity could be established. For one Group 4 female ulceration of the glandular stomach and for another Group 4 female moderate ulceration of the trachea and bronchial epithelium were the cause of moribundity. For two Group 4 females no definite cause of moribundity could be established. These deaths are considered test item related.

There were treatment-related microscopic findings present in rats surviving to their scheduled necropsy dates in the:

Stomach
- Glandular inflammation, lymphogranulocytic was present in male rats treated at 750/500 mg/kg (4/5 rats) up to a slight degree.
- Glandular stomach erosion was present in male rats treated at 750/500 mg/kg (1/5) at a minimal degree.
- Hyperplasia of the forestomach squamous epithelium was present in male rats treated at 750/500 mg/kg (2/5) up to slight degree.
- Vacuolation of the limiting ridge was present at increased severity in male rats treated at 750/500 mg/kg (2/5) at slight degree. Minimal degrees were present in control, 80 and 250 mg/kg treated rats (all 1/5 rats) and this was considered to be the background level.
- Thickened limiting ridge was present in male rats treated at 750/500 mg/kg (2/5) up to moderate degree.

Spleen
- Hemopoietic foci, primarily erythropoiesis showed a slight decrease in 750/500 mg/kg treated female rats.

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

No toxicologically relevant effects on reproductive parameters were noted.
 
The mating, fertility and conception indices, precoital time, and the number of corpora lutea and implantation sites were unaffected by treatment.
 
For two Group 4 females dosed at 750/500 mg/kg, the number of implantation sites was relatively low compared to the number of corpora lutea, resulting in a pre-implantation loss of 53 and 44%, respectively. This finding was not considered to be toxicologically relevant as based on the in-life data both females were in good physical condition during the critical period when implantation of the conceptus takes place (Days 4-5 post-coitum) and all present implantations developed normally, Moreover, all other females of the high dose group carried 10-15 implantations which was within the normal range of biological variation.

For two Group 3 females (250 mg/kg) and 2 Group 4 females (750/500 mg/kg), the number of pups were slightly higher than the number of implantations. This was attributable to normal resorption of these areas as these enumerations were performed on Days 5-7 of lactation.

The gestation index was lower for females at 750/500 mg/kg (67% compared to 100% for controls). All females in Groups 1, 2 and 3 were pregnant and had live pups. There were nine pregnant females in Group 4 (750/500 mg/kg), but ultimately only six females had litters with viable pups on Day 1 of lactation. Of the nine pregnant females at this dose level, one was found dead and two were euthanized in extremis during the post coitum period. The lower gestation index was secondary to maternal toxicity, and was not reflective of actual developmental toxicity.

No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.No deficiencies in maternal care were observed.

Details on results (P0)

MORTALITY
There were three males and one female dosed at 750 mg/kg that were euthanized in extremis or died spontaneously after 2-4 days of treatment. The dose level was subsequently lowered to 500 mg/kg. After that time, there were an additional four females and one male that died or were euthanized in extremis. The females died or were euthanized after 29-36 days and the male was killed in extremis after 9 treatment days.

CLINICAL SIGNS
At 750/500 mg/kg laboured respiration, rales, gasping, piloerection, chromodacryorrhoea of the snout, lethargy, hunched posture, salivation, hypothermia and lean appearance were noted for the animals that died or were euthanized in extremis.

Animals at 750/500 mg/kg that survived to the end of the treatment period were noted with rales, salivation, hunched posture, laboured respiration, piloerection, lean appearance, lethargy, spasms and flat posture.

Piloerection, rales, chromodacryorrhoea of the snout, hunched posture, laboured respiration and salivation were noted transiently for a few animals at 250 mg/kg. As these signs were transient and noted for a limited number of animals at this dose level, they were not considered to be adverse. Scabs on the right flank was noted for one male at 250 mg/kg, this was incidental.

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals.

The variation in motor activity did not indicate a relation with treatment.
All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

BODY WEIGHTS
Body weights and body weight gains were lower for males at 750/500 mg/kg than controls from Day 8 of the premating period through the entire mating period (not always statistically significant). Body weights and gains were lower for females at 750/500 mg/kg than controls from Days 14-20 of the post coitum period (only absolute body weight on Day 14 was significantly lower than controls). The difference for females was mostly attributed to weight loss and reduced gains for two Group 4 females (one Group 4 female also contributed to this on post coitum Day 14; she was killed in extremis after this point).

FOOD CONSUMPTION
At 750/500 mg/kg, a trend towards slightly lower food consumption (absolute and relative to body weight) was noted for males and females during the premating period (not statistically significant). Absolute and relative food consumption was significantly lower for high dose females from Days 4-17 of the post coitum period.

Absolute food consumption was also significantly lower for females at 250 mg/kg from Days 7-11 post coitum, though the effect was only transient, and in the absence of corresponding effects on body weights, it was not considered to be adverse.

The significantly lower food consumption (absolute and relative) noted for females at 80 mg/kg from Days 0-7 and for females at 250 mg/kg from Days 0-4 of the post coitum period, respectively, were not considered to be toxicologically relevant as they occurred in the absence of a dose-related distribution.

HAEMATOLOGY
There were no toxicologically relevant effects on haematology parameters up to 750/500 mg/kg.

Haemoglobin and haematocrit values were significantly lower for males at 750/500 mg/kg and significantly higher for females at 750/500 mg/kg than controls. These were not considered to be toxicologically relevant, however, as these changes were opposite for males and females and the values remained within the range of normal biological variation and did not show a dose-dependent distribution.

CLINICAL BIOCHEMISTRY
At 750/500 mg/kg significantly lower values for total protein (males) and albumin (females) and a higher concentration of bile acids (males) compared to controls were noted.

The remaining statistically significant changes noted in this study were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. These changes included increased concentrations of sodium (males at 250 mg/kg), chloride (males at 80, 250 and 750/500 mg/kg), and lower concentrations of urea (females at 250 mg/kg), sodium (females at 80 and 750/500 mg/kg) and inorganic phosphates (females at 250 and 750/500 mg/kg).

MACROSCOPIC EXAMINATION
Macroscopic findings noted for the animals at 750/500 mg/kg that died or were euthanized in extremis included isolated or several reddish or dark red foci on the stomach glandular mucosa, irregular surface or isolated dark red or black foci of the forestomach and/or glandular mucosa, perforations of the forestomach, fibrin-like coating on the diaphragm, thickened limiting ridge of the stomach, reduced size of spleen and thymus, enlarged lungs, GI-tract distended with gas, beginning autolysis, greenish discoloration of the lungs, pelvic dilation of the kidney, emaciated appearance, uterus contains live fetuses (one Group 4 female) and many dark red foci on the thymus.

There were no treatment-related macroscopic findings in the animals surviving to their scheduled necropsy dates.

Incidental findings noted for control and/or treated animals included enlarged or reddish discolored mesenteric lymph node, enlarged mandibular lymph node, isolated reddish foci on the thymus, reduced size of the left seminal vesicle, scabbing on the right flank, isolated tan or dark red focus/foci on one or both clitoral glands, several yellowish, soft nodules on the clitoral glands, yellowish, soft nodule on the epididymis, pelvic dilation of the kidneys and yellowish, soft nodule on the liver. These findings remained within the background range encountered among rats of this age and strain, did not show a dose-related incidence trend and were thus not considered to be treatment related or toxicologically relevant.

ORGAN WEIGHTS
At 750/500 mg/kg females had lower absolute and relative uterine weights and lower absolute spleen weights. Females at 250 mg/kg had lower absolute spleen weights as well. The lower spleen weights at 750/500 mg/kg parallel the decrease in hemopoietic foci seen in females at the microscopic examination.

MICROSCOPIC EXAMINATION
There were two Group 4 males and one Group 4 female found dead. The two males had macroscopic perforation of the forestomach, which for one Group 4 male correlated to ulceration of the forestomach. For another Group 4 male the autolysis of the stomach was too advanced for a thorough microscopic evaluation, though the presence of a few mucosal erosions still could be noted. For one Group 4 female complete ulceration of the trachea epithelium was the cause of death.
In addition, there were two Group 4 males and four Group 4 females sacrificed moribund. For one Group 4 male the cause of moribundity was marked necrosis of the trachea epithelium, for another Group 4 male no definite cause of moribundity could be established. For one Group 4 female ulceration of the glandular stomach and for another Group 4 female moderate ulceration of the trachea and bronchial epithelium were the cause of moribundity. For two Group 4 females no definite cause of moribundity could be established. These deaths are considered test item related.

There were treatment-related microscopic findings present in rats surviving to their scheduled necropsy dates in the:

Stomach
- Glandular inflammation, lymphogranulocytic was present in male rats treated at 750/500 mg/kg (4/5 rats) up to a slight degree.
- Glandular stomach erosion was present in male rats treated at 750/500 mg/kg (1/5) at a minimal degree.
- Hyperplasia of the forestomach squamous epithelium was present in male rats treated at 750/500 mg/kg (2/5) up to slight degree.
- Vacuolation of the limiting ridge was present at increased severity in male rats treated at 750/500 mg/kg (2/5) at slight degree. Minimal degrees were present in control, 80 and 250 mg/kg treated rats (all 1/5 rats) and this was considered to be the background level.
- Thickened limiting ridge was present in male rats treated at 750/500 mg/kg (2/5) up to moderate degree.

Spleen
- Hemopoietic foci, primarily erythropoiesis showed a slight decrease in 750/500 mg/kg treated female rats.

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item.

REPRODUCTIVE DATA
No toxicologically relevant effects onreproductive parameterswere noted.
 
The mating, fertility and conception indices, precoital time, and the number of corpora lutea and implantation sites were unaffected by treatment.
 
For two Group 4 females dosed at 750/500 mg/kg, the number of implantation sites was relatively low compared to the number of corpora lutea, resulting in a pre-implantation loss of 53 and 44%, respectively. This finding was not considered to be toxicologically relevant as based on the in-life data both females were in good physical condition during the critical period when implantation of the conceptus takes place (Days 4-5 post-coitum) and all present implantations developed normally, Moreover, all other females of the high dose group carried 10-15 implantations which was within the normal range of biological variation.

For two Group 3 females (250 mg/kg) and 2 Group 4 females (750/500 mg/kg), the number of pups were slightly higher than the number of implantations. This was attributable to normal resorption of these areas as these enumerations were performed on Days 5-7 of lactation.

GESTATION
The gestation index was lower for females at 750/500 mg/kg (67% compared to 100% for controls). All females in Groups 1, 2 and 3 were pregnant and had live pups. There were nine pregnant females in Group 4 (750/500 mg/kg), but ultimately only six females had litters with viable pups on Day 1 of lactation. Of the nine pregnant females at this dose level, one was found dead and two were euthanized in extremis during the post coitum period. The lower gestation index was secondary to maternal toxicity, and was not reflective of actual developmental toxicity.

PARTURITION/MATERNAL CARE
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.No deficiencies in maternal care were observed.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproductive toxicity was observed up to the highest dose level tested (750/500 mg/kg). As 750 mg/kg was only dosed for four days, this NOAEL could only be set at 500 mg/kg.

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Incidental clinical symptoms of pups consisted of missing tail apex, blue spot or scab on the abdomen and pale appearance. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Three pups at 80 mg/kg, six pups at 250 mg/kg and one pup at 750/500 mg/kg were found dead or went missing during the first days of lactation. Missing pups were most likely cannibalized. No pups died or went missing in the control group. At 250 mg/kg, the six dead pups contributed to the significantly lower viability index than controls. Five of the dead pups were from a single litter. As this was limited to a single litter and the female was noted with clinical signs indicative of poor physical condition (hunched posture, laboured respiration and piloerection), the increased postnatal loss could be secondary to her poor physical health. This female did have nine pups that survived to the scheduled necropsy date. Taken together, the increased mortality and lower viability index at 250 mg/kg was considered to be a chance occurrence and not reflective of treatment related toxicity. No toxicological relevance was attributed to any other dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were unaffected by treatment.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Incidental macroscopic findings of pups that were found dead included no milk in the stomach. A missing tail apex was the only macroscopic finding noted for surviving pups, which was incidental. These findings are considered normal for pups of this age, and were not toxicologically relevant.

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

EARLY POSTNATAL PUP DEVELOPMENT
The number of dead and living pups at first litter check and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

No test substance was detected in the Group 1 formulations. The concentrations analyzed in the formulations of Group 2, 3, 4 and 4/5 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

 

The formulations of Group 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

 

Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours (i.e. relative difference ≤ 10%).

Applicant's summary and conclusion

Conclusions:

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 250 mg/kg (both for local and systemic toxicity).
Reproduction NOAEL: at least 500 mg/kg. As 750 mg/kg was only dosed for four days, this NOAEL could only be set at 500 mg/kg.
Developmental NOAEL: at least 250 mg/kg.
The likelihood that this NOAEL is actually higher cannot be excluded. However, the number of litters (n=6) available at the higher dose level of 750/500 mg/kg was too low to ensure a thorough evaluation of possible developmental effects.