Phosphoric acid, iron(2+) lithium salt (1:1:1)

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 September 2009- 30 October 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Pysical state: solid
- Appearance: green-grey lumpy powder
- Stability under storage conditions: stable
- Storage condition of test material: At rrom temperature in the dark

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.
- Age at study initiation: (P) 10 wks
- Weight at study initiation: (P) Males: 276-305g; Females: 176-202 g;
- Fasting period before study: No
- Housing:
Pre-mating Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation Offspring was kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).
General Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico &Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0-21.9°C
- Humidity (%): 32-87%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day

IN-LIFE DATES: From: 3 September 2009 To: 30 October 2009

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations.
- Concentration in vehicle: 0, 6, 20 and 70 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually housed in Macrolon cages (MIII type, height 18 cm).
- Any other deviations from standard protocol:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature for at least 6 hours.

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-52 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Frequency of treatment:

Daily, 7 days a week.

Details on study schedule:

- Age at mating of the mated animals in the study: at least 12 weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 30, 100 and 350 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected after consultation with the Sponsor based on a 28-day study with Lithium Iron Phosphate (NOTOX Project 467955).

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No

MORTALITY/VIABILITY: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily, detailed clinical observations were made in all animals.

BODY WEIGHT: Yes
- 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: YES
- Time schedule for examinations: Weekly, for males and females. Food consumption was not recorded during the breeding period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

WATER CONSUMPTION : YES
- Time schedule for examinations: Subjective appraisal (visual) was maintained until Day 9. From Day 9 of treatment onwards, water consumption was measured. No water consumption was determined during the mating period.

OTHER:
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 and recorded.

Oestrous cyclicity (parental animals):

Not determined

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Not applicable

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

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after at least 28 days of treatment
- Maternal animals: All surviving animals on Day 4 of lactation or shortly thereafter or if not littering Post-coitum Day 25-27.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Identification marks: not processed
Preputial gland
Adrenal glands**
Prostate gland
Cervix
Seminal vesicles
Clitoral gland
Stomach**
Coagulation gland
Testes*
Epididymides*
Uterus
Kidneys**
Vagina
Ovaries
All gross lesions
Pituitary gland

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed on Day 4 of lactation or shortly thereafter
- These animals were subjected to postmortem examinations (gross macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
No histopathology, no organ weights.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (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 (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data.

The following additional methods of statistical analysis were used at the discretion of the Study Director:
The number of corpora lutea was subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal, 1952) to determine intergroup difference. If the results of the ANOVA were significant (p<0.05), the Wilcoxon test (Wilcoxon, 1945) was applied to the data 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.

No statistical analysis was performed on histopathology findings.

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:

Percentage mating males= Number of males mated/Number of males paired x 100

Percentage mating females= Number of females mated/Number of females paired x 100

Fertility index males= Number of males generating a pregnancy/Number of males paired x 100

Fertility index females= Number of pregnant females/Number of females paired x 100

Conception rate = 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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

treatment related clinical signs comprised hunched posture and piloerection in all males for several days and in one female during Days 5 to 16 or 13 post-coitum, respectively at high dose level.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced body weight and food consumption at high dose level

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

reduced body weight and food consumption at high dose level

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

MORTALITY/VIABILITY:
At 350 mg/kg body weight/day, two unscheduled deaths occurred:
- One male treated at 350 mg/kg (no. 36) was found dead on Day 11 of treatment. Hunched posture was noted the day before its death. At necropsy this animal showed beginning autolysis, black contents in the GI-tractus, reddish foci on the glandular mucosa of the stomach, a thickened limiting ridge of the stomach, black foci on the pancreas and reduced size of the spleen and thymus.
- In addition, one Group 4 male (no. 31) was sacrificed moribund after 23 days on test. At necropsy this animal showed reduced size of spleen and seminal vesicles. Clinical signs consisted of hunched posture and piloerection for several days and lean appearance for one day.
In these animals several microscopic findings were noted, but no definitive cause of death could be established. These deaths were considered to be treatment-related.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
treatment related clinical signs comprised hunched posture and piloerection in all males for several days and in one female during Days 5 to 16 or 13 post-coitum, respectively.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
Body weights and body weight gain were reduced during the complete treatment period in males treated at 350 mg/kg. On Day 1, before the start of treatment, body weights of males at this dose level were already slightly lower compared to the control group. As this difference compared to the control group was initially very slight but became worse over time, the reduced body weights and body weight gain were considered to be treatment related. In females treated at 350 mg/kg, body weight was slightly reduced at the end of the post-coitum period and during early lactation. Body weight loss was noted for several animals of the high dose group.
At 350 mg/kg, food consumption before or after allowance for body weight was slightly lower for both sexes during the first two weeks of treatment when compared to control levels. This was however not statistical significant.

WATER CONSUMPTION (PARENTAL ANIMALS):
Water consumption was increased up to 4-fold in males treated at 350 mg/kg during premating Days 9-12 and 14-15 (not measured on the other days of premating). In females treated at 350 mg/kg water consumption was increased up to 2-fold during premating, post-coitum up to lactation Day 2. On the last days of lactation, this effect was no longer observed.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Organ weights and organ:body weight ratios of treated animals were considered to be similar to those of control animals

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No toxicologically significant effects on reproductive parameters were noted. Spermatogenic staging profiles were normal for all Group 1 and Group 4 males. Three males and three females failed to mate, conceive, sire or deliver healthy offspring. The infertility of male no. 10 (Group 1) could be attributed to moderate seminiferous tubular atrophy of the testes. Female no. 79 (Group 4) had a deciduoma present in the uterus, which is presumptive evidence for implantation with subsequent foetal loss. These findings could not be attributed to treatment. No cause of infertility was found for the other animals.
No deficiencies in maternal care were observed. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No signs of difficult or prolonged parturition were noted among the pregnant females

ORGAN WEIGHTS (PARENTAL ANIMALS)
The body weights of males and females treated at 350 mg/kg were reduced on the day of necropsy. However, organ weights and organ:body weight ratios of treated animals were considered to be similar to those of control animals.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related microscopic findings in the reproductive organs.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related microscopic findings in the reproductive organs.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

Parental

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

>= 350 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproductive toxicity was observed at any dose level.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
This consisted of a slightly decreased number of living pups at first litter check (not statistical significant), increased postnatal loss and decreased viability index, which was due to 2 litters of this group. One female (no. 73) had 5 pups found dead at the first litter check and the two remaining pups were sacrificed due to bad health (small, no milk, cold, dehydrated) on Day 1 of lactation. Another female (No. 80) had 6 missing pups on Day 3 of lactation (assumed to be cannibalised), the other 6 pups survived until necropsy.

CLINICAL SIGNS (OFFSPRING)
No effects.

BODY WEIGHT (OFFSPRING)
No effects.

GROSS PATHOLOGY (OFFSPRING)
No effects.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Treatment with the test material by oral gavage in male and female Wistar Han rats at dose levels of 30, 100 and 350 mg/kg bw/day revealed parental and developmental toxicity at 350 mg/kg body weight/day. No reproductive toxicity was observed for treatment up to 350 mg/kg bw/day.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 100 mg/kg/day
Reproductive NOAEL: at least 350 mg/kg/day
Developmental NOAEL: 100 mg/kg/day

Executive summary:

In a reproductive screening study according to OECD 421 in rats with oral gavage of several dose levels (0- 30 - 100 - 350 mg/kg bw/d) the following results were obtained:

MORTALITY/VIABILITY: At 350 mg/kg body weight/day, two unscheduled deaths occurred: - One male treated at 350 mg/kg (no. 36) was found dead on Day 11 of treatment. Hunched posture was noted the day before its death. At necropsy this animal showed beginning autolysis, black contents in the GI-tractus, reddish foci on the glandular mucosa of the stomach, a thickened limiting ridge of the stomach, black foci on the pancreas and reduced size of the spleen and thymus. - In addition, one Group 4 male (no. 31) was sacrificed moribund after 23 days on test. At necropsy this animal showed reduced size of spleen and seminal vesicles. Clinical signs consisted of hunched posture and piloerection for several days and lean appearance for one day. In these animals several microscopic findings were noted, but no definitive cause of death could be established. These deaths were considered to be treatment-related.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): treatment related clinical signs comprised hunched posture and piloerection in all males for several days and in one female during Days 5 to 16 or 13 post-coitum, respectively.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): Body weights and body weight gain were reduced during the complete treatment period in males treated at 350 mg/kg. On Day 1, before the start of treatment, body weights of males at this dose level were already slightly lower compared to the control group. As this difference compared to the control group was initially very slight but became worse over time, the reduced body weights and body weight gain were considered to be treatment related. In females treated at 350 mg/kg, body weight was slightly reduced at the end of the post-coitum period and during early lactation. Body weight loss was noted for several animals of the high dose group. At 350 mg/kg, food consumption before or after allowance for body weight was slightly lower for both sexes during the first two weeks of treatment when compared to control levels. This was however not statistical significant.

WATER CONSUMPTION (PARENTAL ANIMALS): Water consumption was increased up to 4-fold in males treated at 350 mg/kg during premating Days 9-12 and 14-15 (not measured on the other days of premating). In females treated at 350 mg/kg water consumption was increased up to 2-fold during premating, post-coitum up to lactation Day 2. On the last days of lactation, this effect was no longer observed.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS) Organ weights and organ:body weight ratios of treated animals were considered to be similar to those of control animals

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS) No toxicologically significant effects on reproductive parameters were noted. Spermatogenic staging profiles were normal for all Group 1 and Group 4 males. Three males and three females failed to mate, conceive, sire or deliver healthy offspring. The infertility of male no. 10 (Group 1) could be attributed to moderate seminiferous tubular atrophy of the testes. Female no. 79 (Group 4) had a deciduoma present in the uterus, which is presumptive evidence for implantation with subsequent foetal loss. These findings could not be attributed to treatment. No cause of infertility was found for the other animals. No deficiencies in maternal care were observed. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No signs of difficult or prolonged parturition were noted among the pregnant females

ORGAN WEIGHTS (PARENTAL ANIMALS) The body weights of males and females treated at 350 mg/kg were reduced on the day of necropsy. However, organ weights and organ:body weight ratios of treated animals were considered to be similar to those of control animals.

GROSS PATHOLOGY (PARENTAL ANIMALS) There were no treatment-related microscopic findings in the reproductive organs.

HISTOPATHOLOGY (PARENTAL ANIMALS) There were no treatment-related microscopic findings in the reproductive organs.

NOAEL:

Systemic: At 350 mg/kg body weight/day, parental toxicity consisted of mortality (two males), clinical signs (hunched posture and piloerection in all males and one female), reduced body weight gain, slightly reduced food consumption during the first two weeks of treatment and increased water consumption. No toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (i.e. macroscopic examination, organ weights, and microscopic examination). No parental toxicity was observed at 30 and 100 mg/kg/day.

Fertility: No reproductive toxicity was observed at any dose level.

Development: At 350 mg/kg/day, developmental toxicity consisted of a slightly decreased number of living pups at first litter check, increased postnatal loss and decreased viability index.

Gestation index, duration of gestation, number of dead pups at first litter check, sex ratio, and early postnatal pup development (clinical signs, body weight and external macroscopy) were unaffected.

No developmental toxicity was observed at 30 and 100 mg/kg body weight/day.