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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.
- 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

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

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

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

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: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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.

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'

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.

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

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.

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'

Reproductive effects observed:

not specified

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was conducted under GLP conditions and in accordance with standardised guidelines. It was therefore assigned a reliability score of 1 in line with the criteria of Klimisch et al. The quality of the database is therefore high.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Toxicity to Reproduction

A screening study for reproductive and developmental toxicity was performed with the test material. The study was conducted under GLP conditions and in accordance with the standardised guideline OECD 421.

During the study male and female Wistar Han rats were treated with test material by oral gavage (10 animals per sex per dose) at dose levels of 30, 100 and 350 mg/kg body weight/day.

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.

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.

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.

No reproductive toxicity was observed at any dose level.

Short description of key information:
NOAEL = 350 mg/kg bw/day, rat, OECD 421, van Tuyl (2010)

Justification for selection of Effect on fertility via oral route:
Only one study is available.

Effects on developmental toxicity

Description of key information

NOAEL (prenatal developmental toxicity) = 300 mg/kg bw/day; NOAEL (maternal toxicity) = 30 mg/kg bw/day, OECD 414, EPA OPPTS 870.3700, Schneider et al. (2016)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 July 2014 - 15 December 2015

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 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.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: ca. 10 - 12 weeks
- Weight at study initiation: 148.4 – 191.4 g
- Housing: rats were housed individually in Polycarbonate cages type III (floor area about 800 cm²) furnished with dust-free wooden bedding. For enrichment, wooden gnawing blocks were offered
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
An amount of test material was weighed, topped up with 1% carboxymethylcellulose suspension in drinking water in a calibrated beaker and intensely mixed with a homogeniser. The aqueous test material preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept in a refrigerator.

VEHICLE (1% carboxymethylcellulose suspension in drinking water)
- Concentration of test material in vehicle: 0, 0.30, 1.00 and 3.00 g/100 mL
- Administered volume: 10 mL/kg bw (the calculation of the administration volume was based on the most recent individual body weight)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSES OF THE TEST MATERIAL PREPARATIONS
Analytical verifications of the stability of the test material in drinking water containing 1% carboxymethylcellulose over a period of 8 days in a refrigerator had been verified prior to the start of the study (project No.: 14L00039; see PART III, Supplement).
Samples of the test material preparations were sent to the analytical laboratory once during the study period (at the beginning of administration) for verification of the concentrations. The samples were also used to verify the homogeneity of the low and the high concentrations (30 and 300 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running.

ANALYTICAL METHOD
> ICP-OES (after digestion with mineral acids)
- Apparatus: Inductively coupled plasma atomic emission spectrometer
- Sample preparation: Each sample was evaporated to dryness. After cooling down to room temperature the residue was moistened with 3 mL conc. sulphuric acid, covered with a watchglass and heated slowly on a heating plate. After the test material was cracked completely at a temperature of ca. 320 °C, the residue was cooled down and 5 mL conc. nitric acid was added in portions. The solution was heated slowly again and oxidised. After cooling down 5 mL of mixed acid was added in portions. TThe solution was repeatedly heated and oxidised until all organic components were destroyed. After oxidation the mineral acids were evaporated and the residue was taken up with 5 mL of concentrated hydrochloric acid anjd filled up to a volume of 50 mL with deionised water.
- Test parameters:
Wavelengths: Fe (238.204 nm), Li (670.780 nm), P (213.618 nm)
Calibration: external

Details on mating procedure:

Time-mated animals were paired by the breeder; the day of evidence of mating (detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”.

Duration of treatment / exposure:

14 days (from implantation to one day prior to the expected day of parturition (GD 6 to GD 19)

Frequency of treatment:

Daily

Duration of test:

Surviving dams were sacrificed on GD 20.

No. of animals per sex per dose:

25 dams per dose

Control animals:

yes

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations included checks for: signs of morbidity, pertinent behavioural changes and signs of overt toxicity. A check for mortality was performed twice a day on working days or once a day on Saturdays, Sundays or on public holidays.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.
The body weight change of the animals was calculated based on the obtained results. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION: Yes
- Time schedule: The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

WATER CONSUMPTION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On GD 20, blood samples were obtained in a randomised order from all females by retrobulbar venous puncture
- How many animals: all dams
- Parameters checked included: leukocyte count (WBC), erythrocyte count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes (RET).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On GD 20, blood samples were obtained in a randomised order from all females by retrobulbar venous puncture
- How many animals: all dams
- Parameters checked included: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), inorganic phosphate (INP), clacium (Ca), urea, creatine (CREA), glucose (GLUC), total bilirubin (TBIL), total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in randomised order.
- Examination: After the dams had been sacrificed, they were necropsied and assessed for gross pathology

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes (and distribution)
- Number of early resorptions: Yes (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)
- Number of late resorptions: Yes (embryonic or fetal tissue in addition to placental tissue visible)
- Live foetuses: Yes
- Dead foetuses: Yes (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

Fetal examinations:

FOETAL EXAMINATIONS
At necropsy each foetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the foetuses and the condition of placentas, umbilical cords, foetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the foetuses were sacrificed by a subcutaneous injection of pentobarbital. Approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half was placed in Harrison’s fluid for fixation.

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live foetuses, proportions of pre-implantation loss, proportions of post-implantation loss, proportions of resorptions, proportion of live foetuses in each litter, litter mean foetal body weight, and litter mean placental weight were all analysed using the DUNNETT-test (two-sided) for the hypothesis of equal means.

Female mortality, females pregnant at terminal sacrifice, and number of litters with foetal findings were analysed using FISHER'S EXACT test (one-sided) for the
hypothesis of equal proportions.

Proportions of foetuses with malformations and variations and/or unclassified observations in each litter were analysed using the WILCOXON- test (one-sided) for the hypothesis of equal medians.

For blood parameters with bidirectional changes non-parametric one-way analysis was performed using the KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians.
For blood parameters with unidirectional changes a pairwise comparison of each dose group was performed with the control group using the WILCOXON-
test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians

In all cases * for p ≤ 0.05; ** for p ≤ 0.01

Indices:

Conception rate (%) = (number of pregnant females / number of fertilised animals) x 100

Pre-implantation loss (%) = [(number of corpora lutea – number of implantations) / number of corpora lutea] x 100

Post-implantation loss (%) [(number of implantations – number of live foetuses) / number of implantations] x 100

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
> IN LIFE EXAMINATIONS
MORTALITY
There were no test material-related or spontaneous mortalities in any females of the test groups (0, 30, 100 or 300 mg/kg bw/day).

CLINICAL FINDINGS
No clinical signs or changes of general behaviour, which may be attributed to the test material, were detected in any female at dose levels of 30, 100 or 300 mg/kg bw/day during the entire study period.

BODY WEIGHTS
The mean body weight and the mean body weight gain of the test material-treated dams in test groups 1-3 (30, 100 or 300 mg/kg bw/day) were generally comparable to the controls throughout the entire study period.
The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) of the test groups 1-3 (30, 100 or 300 mg/kg bw/day) was not influenced by treatment with the test material. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.

FOOD CONSUMPTION
The mean food consumption of the dams in all test groups (30, 100 and 300 mg/kg bw/day) was generally comparable to the concurrent control throughout the study period. If calculated for the entire treatment period (GD 6-19) or study period (GD 0-20), the treated dams consumed a comparable amount of food as the controls.

HAEMATOLOGY
At gestation day 20 total white blood cell (WBC) and absolute neutrophil counts were increased in dams of test groups 2 and 3 (100 and 300 mg/kg bw/day). In dams of test group 3 (300 mg/kg bw/day) relative neutrophil counts were increased and relative lymphocyte counts were decreased in addition.
Total white blood cell (WBC) counts were already higher in dams of test group 1 (30 mg/kg bw/day), but the mean was within the historical control range (WBC 4.04-5.84 Giga/L). Relative reticulocyte counts were higher in dams of test groups 1, 2 and 3 (30, 100 and 300 mg/kg bw/day), but the values were within the historical control range (relative reticulocyte counts 1.2-3.1%). Therefore, these alterations were regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY
At gestation day 20 aspartate aminotransferase (AST) activities were decreased and urea and total bilirubin levels were increased in dams of test group 3 (300 mg/kg bw/day). Total bilirubin values were already higher in dams of test group 2 (100 mg/kg bw/day). However, all mentioned values were within historical control ranges (AST 0.87-1.54 μkat/L; urea 4.52-7.14 mmol/L; total bilirubin 1.00-1.65 μmol/L). Therefore, these changes were regarded as incidental and not treatment-related.

> TERMINAL EXAMINATIONS
UTERUS WEIGHT
The mean gravid uterus weights of the animals of test group 1-3 (30, 100 and 300 mg/kg bw/day) were not influenced by the test material. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.

NECROPSY FINDINGS
No test material-related or spontaneous findings occurred at necropsy in any dam.

REPRODUCTION DATA
The conception rate reached 96% in the mid-dose group (100 mg/kg bw/day) and 100% in the control, low- and high-dose groups (0, 30 and 300 mg/kg bw/day). With these rates, a sufficient number of pregnant females were available for the purpose of the study.
There were no test material-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 30, 100 and 300 mg/kg bw/day) with regard to the conception rate, the mean number of corpora lutea and implantation sites or the values calculated for the pre- and the post-implantation losses, the number of resorptions and viable foetuses. All observed differences were considered to reflect the normal range of fluctuations for animals of this strain
and age.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

clinical biochemistry

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Remarks on result:

other:

Remarks:

NOAEL was highest dose tested

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
> EXAMINATION OF THE FOETUSES
SEX DISTRIBUTION
The sex distribution of the foetuses in test groups 1-3 (30, 100 and 300 mg/kg bw/day) was comparable to the control foetuses.

WEIGHT OF THE PLACENTAE
The mean placental weights of test material-treated groups 1-3 (30, 100 and 300 mg/kg bw/day) were comparable to the corresponding control group.

WEIGHT OF THE FOETUSES
The mean foetal weights were similar in all test groups (0, 30, 100 and 300 mg/kg bw/day) and did not show any biologically relevant differences.

> EXTERNAL EXAMINATION OF THE FOETUSES
FOETAL EXTERNAL MALFORMATIONS
One foetus of test group 2 (100 mg/kg bw/day) showed two external malformations and had associated skeletal findings (Table 2). The incidences of these malformations were neither significantly different from control nor dose-dependent and, therefore, not considered biologically relevant (Table 3).

FOETAL EXTERNAL VARIATIONS
No external variations were recorded.

FOETAL EXTERNAL UNCLASSIFIED OBSERVATIONS
No unclassified external observations were recorded.

> SOFT TISSUE EXAMINATION OF THE FOETUSES
FOETAL SOFT TISSUE MALFORMATIONS
No soft tissue malformations were recorded.

FOETAL SOFT TISSUE VARIATIONS
Two soft tissue variations were detected in all test groups including the control (0, 30, 100 or 300 mg/kg bw/day), i.e. dilated renal pelvis and dilated ureter. The incidences of these variations were neither significantly different from control nor dose-dependent and, therefore, not considered biologically relevant. All of them can be found in the historical control data at higher incidences.

FOETAL SOFT TISSUE UNCLASSIFIED OBSERVATIONS
No unclassified soft tissue observations were detected.

> SKELETAL EXAMINATION OF THE FOETUSES
FOETAL SKELETAL MALFORMATIONS
Skeletal malformations were detected in test groups 1-3 (30, 100 or 300 mg/kg bw/day), as shown in Table 5. One foetus had associated external findings. The total incidence of skeletal malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data. None of these individual findings is considered to be related to treatment.

FOETAL SKELETAL VARIATIONS
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of foetal skeletons and appeared without a relation to dosing (Table 7). The overall incidences of skeletal variations were comparable to the historical control data. For a better overview, all skeletal variations with statistically significant differences between control and the treated groups were compiled in the table below (Table 8). All incidences were expressed on a foetus per litter basis and any statistically significant differences were shown.
The mid-dose finding ‘supernumerary thoracic vertebra’ and the low-dose finding ‘supernumerary rib (14th) (cartilage present)’ were not considered to be treatment-related, because there is no dose-response. Also, as can be seen from Table 6, the increased incidences of ‘incomplete ossification of skull; unchanged cartilage’ and ‘wavy rib’ were well inside the historical control range. Thus, despite of an apparent dose response, these findings were not considered as adverse events.

FOETAL SKELETAL UNCLASSIFIED CARTILAGE OBSERVATIONS
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups (Table 9). The observed unclassified cartilage findings were related to the skull, the vertebral column, the ribs and the sternum and did not show any relation to dosing. The incidence of notched cartilage between basisphenoid and basioccipital was significantly increased in the low-dose group (30 mg/kg bw/day). Since there is no dose-response relationship and other test material-treated groups were unaffected, an association with the treatment as well as a toxicological relevance were not assumed.

Dose descriptor:

NOAEL

Remarks:

prenatal development

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: no developmental effects observed

Remarks:

NOAEL was highest tested dose

Abnormalities:

no effects observed

Developmental effects observed:

not specified

Analyses

The concentration of the test material in the samples were calculated by means of its iron, lithium and phosphorus contents.

For the samples 3 - 9 the recoveries found are in good compliance with the expected concentrations (91 - 98 %). The test material was found to be homogeneously distributed in the vehicle (related to the samples 3 - 5 and 7 - 9).

Table 1: Analytical Results

Sample no.	Values found (g/100 g)	Value of test material found (g/100 mL)	Expected concs. of test material (g/100 mL)	Recovery found/expected values (%)
1 (test material)	Fe: 35.0 LI: 4.4 P: 19.7	-	-	-
2 (vehicle)	Fe: < 0.001 Li: < 0.001 P: < 0.001	-	-	-
3	Fe: 0.099 Li: 0.012 P: 0.054	Fe: 0.283 Li: 0.273 P: 0.274	0.3	Fe: 94 Li: 91 P: 91
4	Fe: 0.100 Li: 0.012 P: 0.055	Fe: 0.286 Li: 0.273 P: 0.279	0.3	Fe: 95 Li: 91 P: 93
5	Fe: 0.098 Li: 0.012 P: 0.054	Fe: 0.280 Li: 0.273 P: 0.274	0.3	Fe: 93 Li: 91 P: 91
6	Fe: 0.330 Li: 0.13 P: 0.180	Fe: 0.943 LI: 0.932 P: 0.914	1.0	Fe: 94 Li: 93 P: 91
7	Fe: 1.00 Li: 0.13 P: 0.57	Fe: 2.857 Li: 2.955 P: 2.893	3.0	Fe: 95 Li: 98 P: 96
8	Fe: 1.00 Li: 0.13 P: 0.58	Fe: 2.857 Li: 2.955 P: 2.944	3.0	Fe: 95 Li: 98 P: 98
9	Fe: 1.00 Li: 0.13 P: 0.57	Fe: 2.857 Li: 2.955 P: 2.893	3.0	Fe: 95 Li: 98 P: 96

 

Table 2: Individual Foetal External Malformations

Test group	Dam no. - Foetus no., Sex	Finding
0 (0 mg/kg bw/day)	none
1 (30 mg/kg bw/day)	none
2 (100 mg/kg bw/day)	61-03 F^†	mandibular micrognathia, aglossia
3 (300 mg/kg bw/day)	none

  ^† foetus with additional skeletal malformation

Table 3: Total External Malformations

		Test group 0 (0 mg/kg bw/day)	Test group 1 (30 mg/kg bw/day)	Test group 2 (100 mg/kg bw/d)	Test group 3 (300 mg/kg bw/d)
Litter Foetuses	N N	25 258	25 258	24 251	25 261
Foetal incidence	N (%)	0.0	0.0	1 (0.4)	0.0
Litter incidence	N (%)	0.0	0.0	1 (4.2)	0.0
Affected foetuses/litter	Mean (%)	0.0	0.0	0.5	0.0

Table 4: Total Soft Tissue Variations

		Test group 0 (0 mg/kg bw/day)	Test group 1 (30 mg/kg bw/day)	Test group 2 (100 mg/kg bw/day)	Test group 3 (300 mg/kg bw/day)
Litter Foetuses	N N	25 123	25 125	24 120	25 124
Foetal incidence	N (%)	5 (4.1)	5 (4.0)	8 (6.7)	5 (4.0)
Litter incidence	N (%)	4 (17)	3 (12)	5 (21)	4 (16)
Affected foetuses/litter	Mean (%)	4.5	4.7	6.7	4.2

Table 5: Individual Foetal Skeletal Malformations

Test group	Dam no. - Foetus no., Sex	Finding
0 (0 mg/kg bw/day)	none
1 (30 mg/kg bw/day)	44-06 M	cleft sternum
2 (100 mg/kg bw/day)	61-03 F^†	severely malformed skull bones
3 (300 mg/kg bw/day)	81-05 M	exoccipital fused with 1st cervical arch, misshapen basisphenoid, branched rib

^† foetus with additional external malformation

Table 6: Total Skeletal Malformations

		Test group 0 (0 mg/kg bw/day)	Test group 1 (30 mg/kg bw/day)	Test group 2 (100 mg/kg bw/d)	Test group 3 (300 mg/kg bw/d)
Litter Foetuses	N N	25 135	25 133	24 131	25 137
Foetal incidence	N (%)	0.0	1 (0.8)	1 (0.8)	1 (0.7)
Litter incidence	N (%)	0.0	1 (4.0)	1 (4.2)	1 (4.0)
Affected foetuses/litter	Mean (%)	0.0	0.7	0.7	0.6

Table 7: Total Foetal Skeletal Variations

		Test group 0 (0 mg/kg bw/day)	Test group 1 (30 mg/kg bw/day)	Test group 2 (100 mg/kg bw/d)	Test group 3 (300 mg/kg bw/d)
Litter Foetuses	N N	25 135	25 133	24 131	25 137
Foetal incidence	N (%)	134 (99)	130 (98)	129 (98)	136 (99)
Litter incidence	N (%)	25 (100)	25 (100)	24 (100)	25 (100)
Affected foetuses/litter	Mean (%)	99.0	98.0	98.3	99.3

Table 8: Occurrence of Statistically Significantly Increased Skeletal Variations (expressed as mean percentage of affected foetuses/litter)

Finding	Test group 0 (0 mg/kg bw/day)	Test group 1 (30 mg/kg bw/day)	Test group 2 (100 mg/kg bw/day)	Test group 3 (300 mg/kg bw/day)	HCD Mean % (range)
Incomplete ossification of skull; unchanged cartilage	3.1	9.0	12.7**	23.9**	7.6 (0.0 - 25.9)
Supernumerary thoracic vertebra	2.4	3.8	6.3*	6.3	4.1 (0.0 - 19.1)
Supernumerary rib (14th); cartilage present	5.8	13.6*	9.3	5.6	5.7 (0.0 - 27.3)
Wavy rib	3.1	11.5*	12.6**	16.3**	4.9 (0.0 - 17.0)

 HCD = Historical control data

* = p ≤ 0.05 (Wilcoxon-test [one-sided])

** = p ≤ 0.01 (Wilcoxon-test [one-sided])

Table 9: Total Unclassified Cartilage Observations

		Test group 0 (0 mg/kg bw/day)	Test group 1 (30 mg/kg bw/day)	Test group 2 (100 mg/kg bw/day)	Test group 3 (300 mg/kg bw/day)
Litter Foetuses	N N	25 135	25 133	24 131	25 137
Foetal incidence	N (%)	101 (75)	101 (76)	108 (82)	106 (77)
Litter incidence	N (%)	24 (96)	24 (96)	24 (100)	25 (100)
Affected foetuses/litter	Mean (%)	72.7	76.1	82.5	77.6

 

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test material to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused signs of maternal toxicity at dose levels of 100 and 300 mg/kg bw/day taking clinical pathology parameters into account. However, there were no toxicologically relevant adverse foetal findings evident.
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity was 30 mg/kg bw/day.
The NOAEL for prenatal developmental toxicity was 300 mg/kg bw/day, the highest dose tested.

Executive summary:

The prenatal developmental toxicity of the test material was assessed in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 414 and EPA OPPTS 870.3700.

During the study the test material was administered as an aqueous suspension to groups of 25 time-mated female Wistar rats by gavage at dose levels of 0, 30, 100 and 300 mg/kg bw/day on gestation days (GD) 6 through 19. The control group was dosed with the vehicle in parallel (1% carboxymethylcellulose suspension in drinking water). A standard dose volume of 10 mL/kg bw was used for each test group. At terminal sacrifice on GD 20, 24-25 females per group had implantation sites.

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

On GD 20, blood samples were obtained from all females by retrobulbar venous puncture following isoflurane anesthesia. After blood sampling all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead foetuses) were determined. The foetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the foetuses of each litter were examined for soft tissue findings and the remaining foetuses for skeletal (inclusive cartilage) findings.

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test material to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused signs of maternal toxicity at dose levels of 100 and 300 mg/kg bw/day taking clinical pathology parameters into account. However, there were no toxicologically relevant adverse foetal findings evident.

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity was 30 mg/kg bw/day.

The NOAEL for prenatal developmental toxicity was 300 mg/kg bw/day, the highest dose tested.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was conducted under GLP conditions and in accordance with standardised guidelines. It was therefore assigned a reliability score of 1 in line with the criteria of Klimisch et al. The quality of the database is therefore high.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental Toxicity

The prenatal developmental toxicity of the test material was assessed in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 414 and EPA OPPTS 870.3700.

During the study the test material was administered as an aqueous suspension to groups of 25 time-mated female Wistar rats by gavage at dose levels of 0, 30, 100 and 300 mg/kg bw/day on gestation days (GD) 6 through 19. The control group was dosed with the vehicle in parallel (1% carboxymethylcellulose suspension in drinking water). A standard dose volume of 10 mL/kg bw was used for each test group. At terminal sacrifice on GD 20, 24-25 females per group had implantation sites.

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

On GD 20, blood samples were obtained from all females by retrobulbar venous puncture following isoflurane anesthesia. After blood sampling all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead foetuses) were determined. The foetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the foetuses of each litter were examined for soft tissue findings and the remaining foetuses for skeletal (inclusive cartilage) findings.

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test material to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused signs of maternal toxicity at dose levels of 100 and 300 mg/kg bw/day taking clinical pathology parameters into account. However, there were no toxicologically relevant adverse foetal findings evident.

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity was 30 mg/kg bw/day.

The NOAEL for prenatal developmental toxicity was 300 mg/kg bw/day, the highest dose tested.

Justification for selection of Effect on developmental toxicity: via oral route:
Only one study is available.

Justification for classification or non-classification

The substance does not fulfil the classification criteria for reproductive or developmenetal toxicity according to European Regulation (EC) No 1272/2008.