Sodium 5-oxo-L-prolinate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 November 2012 to 12 January 2013

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:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD guidance document 43.

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

sodium (2S)-5-oxopyrrolidine-2-carboxylate

Cas Number:

28874-51-3

Molecular formula:

C5H6NO3. Na

IUPAC Name:

sodium (2S)-5-oxopyrrolidine-2-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: White to pale yellow powder.
- Storage condition of test material: Controlled room temperature (15-25 °C, below 70 RH%), protected from humidity.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:WI rats.
- Age at study initiation: (P) Young adult rats, approximately 10 weeks old at starting of dosing and 12 weeks at mating.
- Weight at study initiation: (P) 362-401 g males and 260-291 g females (did not exceed ± 20% of the mean weight for each sex at onset of treatment).
- Housing: Rats were housed in groups of 5/sex/cage, in Type II and III polypropylene/polycarbonate cages. However during mating and gestation/delivery period, they were housed in pairs or individually, respectively.
- Diet: Complete breeding and maintenance diet for rats and mice, provided ad libitum.
- Water: tap water from municipal supply, provided ad libitum.
- Acclimation period: 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 - 24.9 °C
- Humidity (%): 31 – 70 %
- Air changes (per hr): 15-20 air exchanges/hour.
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

(distilled)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
- Method: The test material was formulated in the vehicle at the appropriate concentrations according to the dose level and volume selected. Formulations were prepared according to stability results, within 1 day at room temperature or 7 days when stored refrigerated at 2-8 °C.
- Dosing volume: 20 mL/kg bw/day, the actual volume administered was calculated and adjusted based on each animal’s most recent body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test formulations for concentration and homogeneity was performed on two occasions during the study. Top, middle and bottom duplicate samples were taken from formulations, during the first and last week of treatment, one set to analyse, collected in replicates as practical, and one set as aback-up, which was not required for confirmatory analyses. Similarly, one sample was taken in duplicate from the Group 1 (control) solution for concentration measurements.

IDENTIFICATION AND QUANTIFICATION OF TEST SUBSTANCE/PRODUCT
- Separation method: HPLC-UV method.

Details on mating procedure:

- Initiation of mating: Mating began 2 weeks after the initiation of treatment.
- M/F ratio per cage: 1:1.
- Length of cohabitation: Females remained with the same male until copulation occurred, or up to 14 days.
- Proof of pregnancy: A vaginal smear was prepared daily for each female during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (considered as Day 0 of pregnancy). There was no female during the study that showed no-evidence of copulation (nonmated).
- After successful mating each pregnant female was caged: Sperm positive females were caged individually. Mating pairs were clearly identified in the data, mating of siblings was avoided.

Duration of treatment / exposure:

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post- mating). Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy, 4 days post-partum.

Frequency of treatment:

Daily, on a 7 days/week basis.

No. of animals per sex per dose:

12 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the sponsor in consultation with the Study Director, based on available data and information from previous experimental work, including the results of a 7-day preliminary dose range finding study.
Dose levels were selected with the aim of inducing toxic effects but, if possible, no death or suffering at the highest dose, and a NOAEL.
The oral route was selected as it is a possible route of exposure to the test material in humans.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Signs of morbidity and mortality were recorded twice daily (at the beginning and end of each day). General clinical observations were made once a day, after treatment at approximately the same time each day.
- Cage side observations: Behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were monitored.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Examinations were made once before the first exposure (to allow for within-subject comparisons), and at least once a week thereafter.
- These observations were made outside the home cage in a standard arena, approximately at similar times. Signs evaluated included monitoring of any changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards). Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
On gestation day GD13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).
Females were allowed to litter and rear their offspring and the following observations were recorded: the delivery process, nursing instinct and suckling efficiency.

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed on Day 0, afterwards at least weekly and at termination. Parent females were weighed on gestation Days GD0, 7, 14 and 20 and on post-partum Days PPD0 (within 24 hours after parturition) and 4 (before termination). The females were additionally weighed on GD3, 10 and 17 in order to give accurate treatment volumes.

FOOD CONSUMPTION: Yes, measured at weekly intervals.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day.

SACRIFICE
- Maternal animals: All surviving animals were euthanized 4 days post-partum. Females which not delivered were sacrificed 26 days after the last day of mating, as practical.
- Method: Animals were sacrificed under pentobarbital anaesthesia by exsanguination.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cranium, thoracic, the abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues indicated below were weighed, paired organs were weighed individually; absolute organ weights were measured and are reported. Relative organ weights (to body and brain weight) were calculated and are reported.
Uterus (with and without cervix), vagina, testes, brain, ovaries, pituitary.

- Detailed histological examination was performed on the selected list of retained organs in the control and high dose groups, including the brain, pituitary, uterine cervix+body+horn, ovary, vagina and all macroscopic findings (abnormalities) from all animals. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data

Fetal examinations:

- Pups euthanized at post-partum day 4.
- These animals were carefully examined at least externally for gross abnormalities.

Statistics:

The statistical evaluation of appropriate data was performed with a statistical program package. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.

Indices:

Male Mating Index
Female Mating Index
Male Fertility Index
Female Fertility Index
Gestation Index
Survival Index
Post-natal mortality
Pre-implantation mortality
Intrauterine mortality
Total mortality
Sex ratio

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects. Remark: No treatment related effects observed up to 1000 mg/kg bw/day.

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
There was no unscheduled mortality during this study.
No test material related clinical signs were noted during the study, either at the daily clinical observation or at the weekly evaluation.
In one female treated at 1000 mg/kg bw/day (4509, high-dose group) a subcutaneous mass in the urogenital region was observed from Day 30. This observation was regarded as an incidental finding, unrelated to treatment.

BODY WEIGHT AND FOOD CONSUMPTION
No adverse effects on body weight or body weight gain were observed.
Body weights were comparable to the control in all treated groups. Compared to the control mean, slightly higher body weight gain values were recorded in the treated females during the mating period (Day 14-G0; p<0.05 or p<0.01). These differences were regarded as normal biological variation, not associated with the treatment.
When compared to the control, there were no statistically or toxicologically significant differences in food consumption in the female animals administered with the test material up to and including 1000 mg/kg bw/day.

REPRODUCTIVE PERFORMANCE
There were no differences between the control and treated groups with regard to reproductive ability or in the mating, fertility and gestation indices, which showed minor variations within the normal biological ranges, unrelated to treatment.
The administration of the test material was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within up to 5 days of pairing (cohabitation), with the exception of the low-dose female 2503 and the high-dose female 4505, which had a sperm positive vaginal smear after 14 or 13 days of mating. These variations were regarded as individual, normal biological variability, without toxicological significance.
There was no effect of treatment noted during gestation, parturition or the post-partum period.
The mean duration of pregnancy was similar in the control and treated groups and varied between 21 and 23 days.
All the parturitions were normal.
The numbers of corpora lutea and implantation sites were similar in the treated groups up to and including 1000 mg/kg bw/day, compared to the controls. There were no treatment related adverse effects on the pre/post-implantation, post-natal or total mortality values (%) in any of the dose groups.

ORGAN WEIGHTS
There were no statistically significant differences or changes considered to reflect an adverse effect on organ weights in females at any of the dose groups evaluated, up to and including 1000 mg/kg bw/day.
A Statistically significant decreased in absolute and relative weight was observed in the brain and the vagina in the mid dose females. However, there was no consistent dose response or there were no correlated pathological and histopathological findings, thus, these variations were regarded as normal variations unrelated to treatment.

GROSS PATHOLOGY
No treatment related gross changes were observed at dose levels up to 1000 mg/kg bw/day.
Uterine dilatation in 1/12 control female, subcutaneous mass/firm (brown at the cut surface, 2x1x0.5 cm in size) at the skin/subcutis within the mammary gland area in 1/12 high dose female and two placentas present in left uterine horn 1/12 low-dose female were observed at necropsy.

HISTOPATHOLOGY
No treatment related microscopic findings were observed in the experimental animals.
The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were of similar histological structure in both the control and high-dose females.
There was an incidental occurrence of mammary gland hyperplasia (lobular with focal atypia) in one high-dose female (No. 4509). This microscopic lesion correlated with gross change at the skin/subcutis within the mammary gland area in this female. There were no histopathological changes in reproductive organs including the ovary, uterus and vagina in this female rat.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: No treatment related effects observed up to 1000 mg/kg bw/day.

Details on embryotoxic / teratogenic effects:
VIABILITY AND CLINICAL SIGNS
There were no adverse effects on pups’ viability or any clinical observations recorded in the F1 generation that could be considered toxicologically relevant, or related to test material administration. The mean and total number of viable pups as well as pups survival indices showed no statistically or toxicologically significant differences to control.
There were no pups found dead and intact. A low number of pups were found cannibalized, 2/150 (Day 2 or day 4), 3/176 (Day 0 or Day 1), 2/182 (Day 2) and 1/145 (Day 2) in the control, low-, mid- and high-dose groups, respectively, and 1/182 mid-dose pup was noted to be gray on Day 0; these minor variations were regarded as incidental and ascribed to normal biological variability, unrelated to treatment.

BODY WEIGHT
There was no adverse effect of treatment on the offspring body weight or body weight gain.
Mean body weight and body weight gain values evaluated for all pups or on a litter basis were similar in all treated groups.

INDICES
The sex ratios were similar in the control and treated groups.

GROSS PATHOLOGY
There were no abnormalities at the external examination of all pups surviving to PND4.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at the maximum dose tested.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

ANALYSIS OF DOSE FORMULATIONS
No test material was detected in the Control solution samples. All formulations were shown to be homogeneous. All formulations were found to be in the range of 100 to 107 % of nominal concentrations. Based on these results, the formulations were considered suitable for the study purposes. The analytical results are summarized in the Table 1.

Table 1: Dose Formulation Analysis

Sampling Time	Nominal Concentration (mg/mL)	Measured Concentrations ± SD (mg/mL)	Measured Concentration as % of Nominal
First Week	3.125	3.19 ± 0.03	102
	12.5	12.5 ± 0.25	100
	50	51.5 ± 1.00	103
Last Week	3.125	3.24 ± 0.08	104
	12.5	13.4 ± 0.17	107
	50	53.1 ± 1.40	106

Table 2: Offspring Generation

Females	Dose (mg/kg bw/day)
	Control	62.5	250	1000
Number of liveborns (total) on PND0	150	174	182	145
Number of viable pups (mean) on PND0	13.64	14.50	15.17	14.50
Number of viable pups (mean) on PND4	13.45	14.42	15.00	14.40
Number of viable pups (total) on PND0	150	174	182	145
Number of viable pups (total) on PND4	148	173	180	144
Survival index PND0	100.00	98.99	100.00	100.00
Survival index PND0-4	98.69	98.23	98.91	99.29
Sex ratio (%) on PND0	47.32	54.45	54.53	56.25
Sex ratio (%) on PND4	46.71	54.22	54.97	56.58

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test, no adverse effects were observed as a direct result of treatment up to and including 1000 mg/kg bw/day. Therefore the NOAEL for maternal, and developmental, toxicity was determined to be 1000 mg/kg bw/day.

Executive summary:

The developmental toxicity of the test material was determined in a reproduction/developmental toxicity screening study performed using Wistar rats. The study was conducted under GLP conditions and in line with the standardised guidelines OECD 421 and OECD guidance document 43.

During the study groups of 12 male and 12 female rats were exposed to the test material, formulated in distilled water, at concentrations of 0.0, 62.5, 250 and 1000 mg/kg bw/day via oral gavage. Animals were dosed daily at 20 mL/kg bw/day, 7 days per week. Males were dosed for 28 days (14 days pre-mating and 14 days mating/post- mating). Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy, 4 days post-partum.

Analytical verification confirmed that all formulations within the range of 100 to 107 % of nominal concentrations, and were thus suitable for the purpose of the study.

Based on the detailed maternal observations no adverse effects were recorded as a direct result of treatment up to and including 1000 mg/kg bw/day. Offspring indices and post-mortem examination of the pups revealed no treatment related effects.

Therefore the NOAEL for maternal, and developmental, toxicity was determined to be 1000 mg/kg bw/day.