Zinc, [1,2,3-propanetriolato(2-)-.kappa.O1,.kappa.O2]-, homopolymer, stereoisomer

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

(1995)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test, 2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

zinc(2+) 3-hydroxypropane-1,2-bis(olate)

EC Number:

700-107-9

Cas Number:

87189-25-1

Molecular formula:

(C3H6O3Zn)n

IUPAC Name:

zinc(2+) 3-hydroxypropane-1,2-bis(olate)

Details on test material:

- Substance type: Organic
- Physical state: Powder
- Storage condition of test material: At room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Strain as stated in the report: Wistar rat; Crl:Wl (Han)
- Source: Males: Charles River Laboratories, Germany ; Females (nulliparous and non-pregnant): Charles River, France
- Age at study initiation: 11-12 weeks
- Weight at study initiation: Males: 329.9 – 332 g, Females: 212.5 – 217.9 g
- Housing: Individually in type-MIII polycarbonate cages supplied by Becker & Co., Castrop-Rauxel, Germany. Pregnant females were provided with nesting material (cellulose wadding) toward the end of pregnancy.
- Diet ad libitum: Ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland.
- Water ad libitum: tap water
- Acclimation period: At least 5 days before start of treatment under laboratory conditions
- Number of animal per dose group: 3

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h artificial fluorescent light and 12h darkness per day

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing solutions were prepared once at the start of the study, divided in daily portions and stored deep frozen until the daily use.
The test substance was applied as a suspension. The test substance was suspended in 1% Carboxymethylcellulose and mixed using a homogenizer. Homogeneity during the administration was achieved using a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 1, 3 and 10 g/100ml at 100, 300 and 1000 mg/kg bw/d, respectively.
- Amount of vehicle (if gavage): 10 ml/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of gestation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses were carried out as a separate study at the test facility Competence Center Analytics Department of BASF SE, Ludwigshafen, Germany (10L00004). The homogeneity and the concentration control analyses were performed in samples of all concentrations at the start and at the end of the administration period. The analytical concentrations ranged from 96.3 to 103.3% of the nominal concentration. The homogeneity of the substance was confirmed. No stability analyses were performed since the test substance hydrolyzes in aqueous solution and zinc concentrations do not reflect the actual state of the molecule.

Duration of treatment / exposure:

Males approx. 5 weeks, females approx. 7 weeks.
The duration of treatment covered a premating period of 2 weeks and a mating period (of max. 2 weeks) in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females.

Frequency of treatment:

daily via gavage

Details on study schedule:

Male and female animals were treated via daily gavage 14 days prior and throughout mating (max. 14 days). The females were allowed to litter and rear their pups until day 4 after parturition. On PND 4, all pups were sacrificed and examined. At the end of the study, the parental animals were sacrificed (males on study day 35, females on study day 49) after a fasting period (withdrawal of food) for at least 16-20 hours.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

MORTALITY AND CLINICAL SYMPTOMS
The animals were checked twice daily for mortality (dead and moribund animals) and clinical symptoms of toxicity on work day and once daily on weekend and public holidays. Particular attention was given to littering and lactation behavior of the dams, but only special findings were documented.

BODY WEIGHT
- Parental animals: generally, body weight was determined once weekly.
- During mating body weight of the F0 females was determined on the day of sperm evidence in the vaginal smear and thereafter on days 7, 14 and 20 of gestation, on day of parturition (PND0), and on day PND4. - Females without litter were weighed weekly.

FOOD CONSUMPTION
- Parental animals: food consumption was determined once weekly, with following exceptions:
The food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on gestation day (GD) 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined on PND 1 and 4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period) and females without litter (during the lactation).

Litter observations:

From all pups delivered by the F0 females the total number of pups, the number of liveborn and stillborn pups in each litter and the sex of the pups were determined as soon as possible on the day of birth. The sex of the pups was finally confirmed at necropsy.

MORTALITY AND CLINICAL SYMPTOMS
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented with the dam concerned. In addition the pups were checked twice daily for mortality (i.e. dead and moribund pups) on workdays (once a day on week ends and public holidays).

BODY WEIGHT
The pups were weighed one day after birth (PND 1) and on day 4 after birth.
Furthermore, the body weights on PND 1 were used for the calculation of "runts" (pups, which weighed less than 25% of the mean weight of the respective control pups).

Postmortem examinations (parental animals):

Parental animals were sacrificed by decapitation under isoflurane anesthesia for the purpose of necropsy. Animals that died also were subjected to necropsy as soon as possible after death.
Following organs were weighed: Testes, Epididymides, Ovaries
The following tissues of parental animals were fixed in 4% buffered formaldehyde
or in modified Davidson’s solution:
Adrenal glands, Testes (fixed in modified Davidson’s solution), Epididymides (fixed in modified Davidson’s solution), Pituitary gland, Prostate gland, seminal vesicles, coagulation glands, Ovaries (fixed in modified Davidson’s solution), Uterus, oviducts, vagina, Fore and glandular stomach (male animals/ test groups 0 and 1), Duodenum, ileum, jejunum (male animals/ test groups 0 and 1), Cecum, colon, rectum (male animals/ test groups 0 and 1), all gross lesions.
The ovaries of animals that died or had to be sacrificed intercurrently were fixed in 4% buffered formaldehyde solution.
Following tissues/organs were subjected to light microscopical examination: Testes, Epididymides and Ovaries from all animals of the control and the 1000 mg/kg bw/d group, as well as all gross lesions throughout all dose levels. Particular attention was given to correlate gross lesions with microscopical findings.

Postmortem examinations (offspring):

All surviving pups were examined externally and sacrificed on PND 4. Their organs were assessed macroscopically. Stillborn pups as well as pups that died before PND 4 also were subjected to necropsy.

Statistics:

The statistical assessment of the different data obtained within the present study was based on following methods, depending on the parameters considered: Dunnett test, Fisher´s exact test, Wilcoxon test, Kruskall-Wallis test.

Reproductive indices:

REPRODUCTIVE INDICES FOR THE MALES:
Male mating index (%) = N (males with confirmed mating) x 100 / N (males placed with females)
Male fertility index (%) = N (males with proved fertility) x 100 / N (males placed with females)
Males were defined as “with confirmed mating” by the presence of vaginal sperm in the female, or by the production of a litter, or by the presence implants in the uterus.
Males were defined as “with proved fertility” by female with implants in the uterus.

REPRODUCTIVE INDICES FOR THE FEMALES:
Female mating index (%) = N (females mated) x 100 / N (females placed with males)
Female fertility index (%) = N (pregnant females) x 100 / N (mated females)
Gestation index (%) =N (females with live pups on day of birth) x 100 / N (pregnant females)
Postimplantation loss (%) = N (implantations) – N (pups delivered)x100 / N (implantations)
Females were defined as mated when vaginal sperm was evidenced, or when they had implants in the uterus.
Females were defined as pregnant when they had implants in the uterus.

Offspring viability indices:

Live birth index (%) = N (liveborn pups at birth) x 100 / N (total pups born)
Viability index (%) = N (live pups on day 4 after birth) x 100 / N (total live pups on day of birth)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

MALES
Salivation was seen in 4/10 males after treatment (week 3 and 4) and a poor general state was seen in 2/10 males during week 4 and 5 at the high dose. Except for salivation during week 3 and 4 in males treated with 300 mg/kg bw/d, no treatment related findings were seen in males from the mid and low dose.

FEMALES
No clinical signs of toxicity were evident in any female during premating period. However, clinical signs in females at 1000 mg/kg bw/d during lactation and gestation period consisted of:
- Piloerection in 4/10 animals during gestation (GD 21 onwards) and 3/10 animals during Lactation
- Poor general state in 6/10 animals during gestation (GD 2 onwards) and 3/10 animals during lactation
One female did not nurse its pups properly and the pups died on day of birth. No treatment related findings were seen in females of the low and mid dose group.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

No male died during the study period. Three of 10 females were found dead during gestation period (Gestation Day 17-23). In addition, 1/10 females which was unable to deliver was sacrificed moribundly (palpable fetuses in abdomen) at the high dose.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MALES
Males of the high dose group showed significantly lower body weights during the entire study period. Body weight changes were also significantly lower between study weeks 0 to 1 and 0 to 5. No treatment related findings were seen in animals from the mid and low dose.

FEMALES
In females, no treatment related changes in body weights were observed during the premating period. However, body weight change was significantly decreased from week 0 to 2 at 1000 mg/kg bw/d. The body weights during gestation (GD 7 and GD 20) and lactation (PND 4) were significantly decreased. Body weight gain during entire study period was decreased in females of the high dose group. No changes were observed in females of the low and mid dose groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

MALES
Males of the high dose group showed decreased food consumption during the first study week (mean food consumption of 17.9 g versus 22.7 g for control, respectively).

FEMALES
The food consumption during the entire study period was decreased for females at 1000 mg/kg bw/d (11.3, 13.7 and 12.5 compared to 15.8, 15.7 and 15.7 g at 0 mg/kg bw/d for weeks 0 to 1, 1 to 2, and 0 to 2, respectively). No treatment related findings were seen at the mid dose a group, while the food consumption of females treated with 100 mg/kg bw/d was decreased between week 1 and 2.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Forestomach: The macroscopically thickened margo plicatus correlated with a minimal or slight focal squamous hyperplasia that was observed in 8 males and all females of test group 2 (300 mg/kg bw/d) as well as in 8 males and 8 females of test group 3 (1000 mg/kg bw/d). In the same region, a minimal or slight multifocal epithelial degeneration occurred in 4 males and all females of test group 2 (300 mg/kg bw/d) as well as in 8 males and 2 females of test group 3 (1000 mg/kg bw/d). One male of test group 3 (1000 mg/kg bw/d) showed an erosion/ ulcer.
Glandular stomach: In most cases, the red or light red discoloration turned out to be multifocal hyperemia or erosion/ ulcer. A multifocal hyperemia was observed in the glandular epithelium of 6 males and 3 females of test group 2 (300 mg/kg bw/d) as well as of 9 males and 6 females of test group 3 (1000 mg/kg bw/d). Erosions or ulcers occurred in 1 male and 3 females of test group 2 (300 mg/kg bw/d) as well as in 5 males and 7 females of test group 3 (1000 mg/kg bw/d). The macroscopically diagnosed erosions or ulcers in 3 females of test group 3 (1000 mg/kg bw/d) could be confirmed histopathologically. The thickening of wall corresponded with a submucosal inflammation that was observed in 9 males and all females of test group 2 (300 mg/kg bw/d) as well as in all males and 6 females
of test group 3 (1000 mg/kg bw/d).
Duodenum: In most animals there were no histopathological correlates for the grossly thickened duodenal wall: Only 1 female of test group 3 (1000 mg/kg bw/d) showed a focal inflammation. Therefore, a test substance-related effect seemed rather unlikely.
Jejunum: Because there was no histopathological correlate for the macroscopically observed thickening of wall in three females of test group 3 (1000 mg/kg bw/d) the finding was considered to be incidental.
Adrenal cortex: One of the females with grossly enlarged adrenal cortex showed a focal congestion, another one a diffuse congestion, and in the third female there was no correlate. These findings were considered to be incidental.
All other findings noted were single observations. All of them were considered to be incidental and spontaneous in origin and without any relation to treatment.

Decedents: Three females of test group 3 (1000 mg/kg bw/d) died prematurely. Two of them showed erosions or ulcers in the glandular stomach; in the third female a multifocal hyperemia was observed in the epithelium of the glandular stomach. One female with erosions or ulcers in the glandular stomach was sacrificed in a moribund state. These findings were considered to be treatment-related. The erosions or ulcers in the glandular stomach might have caused the earlier deaths.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

MALES
The male mating index and the male fertility index was 100% in all treatment groups.

FEMALES
The post implantation loss was increased (31.3%) and the live birth index (72% with 28% stillborn) as well as the gestation index (50%) was decreased in females treated with 1000 mg/kg bw/d.
Three dams of this dose group had a complete litter loss and 1/6 females did not nurse its pups properly. The pups died on day of birth. The female mating index and the female fertility index were 100%.
No effects on the reproductive performance were seen in females treated with the low and mid dose.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

sytemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Pathological findings in the GI tract like erosions or ulcers, squamous hyperplasia, submucosal inflammation and epithel degeneration were seen (males and females) at 300 mg/kg bw/d.

2

Dose descriptor:

NOAEL

Remarks:

Reproduction/fertility

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: The post implantation loss was increased (31.3%) and the live birth index (72% with 28% stillborn) as well as the gestation index (50%) in females was decreased at 1000 mg/kg bw/d.

3

Dose descriptor:

NOAEL

Remarks:

Reproduction/fertility

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No effects on reproductive performance and on fertility were seen even at the highest dose.

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Most F1 pups did not show adverse clinical signs up to scheduled sacrifice on PND 4. In one litter (1000 mg/kg bw/d) 2/2 liveborn pups were not properly nursed and died on PND 0. Upon necropsy both pups showed an empty stomach. No further clinical signs were seen in any dose group.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The mean number of delivered pups in dams treated with 1000 mg/kg bw/d was significantly decreased (47+/-8 vs. 135 +/-14 at control). In addition, the rate of stillborn pups was significantly increased to 28% compared to control. These changes were assessed as being related to the severe signs of maternal toxicity seen in several animals of this test group during gestation, which were also consistent during lactation.
No differences in the mean number of delivered pups per dam and the rate of liveborn and stillborn pups were seen in the other treatment groups when compared to control.
A significant increase of pups that died during lactation was observed at the high dose group and was assumed to be related to maternal toxicity. The viability index as indicator for pup mortality between PND 0-4 was 62% for the highest dose group and between 98% and 100% for control, low and mid dose.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean pup body weights and the pup body weight change values were significantly decreased in male and female pups of the high dose group (1000 mg/kg bw/d), being statistically significant for males only. This decrease was assumed to be the consequence of maternal toxicity. In addition, one male runt was seen in this dose group.
Mean pup body weights/pup body weight changes of all pups in the low and high dose were comparable to the concurrent control values.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between all test groups.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Upon necropsy 2 stillborn pups of the control group and 10 stillborn pups of the high dose group (1000 mg/kg bw/d) showed post mortem autolysis. In two pups of test group 3 (1000 mg/kg bw/d) that died on PND 0 the stomach was found empty.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The viability index for both sexes as well as the pup weights were decreased at 1000 mg/kg bw/d.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Mean Parental Body Weights in grams
	MALES
	Dose (mg/kg bw/d)
week	0	100	300	1000
0	332+/-9.5	330.2+/-9.1	331.8+/-14.6	329.9+/-10.4
1	357.6+/-14.9	346.1+/-11	355.5+/-23.7	335.4+/-24.3*
2	358.6+/-15.7	350.2+/-14.2	256.5+/-33.4	329.8+/-24.3*
3	380.4+/-14.8	358.9+/-14.4	379.2+/-38.4	346.7+/-22,0*
4	395+/-21.5	373.4+/-19.5	390.6+/-43.6	358.8+/-19.9*
5	404.5+/-21.6	384.8+/-20.2	403.4+/-46.7	368.4+/-22.5*
	FEMALES - PREMATING
Week	0	100	300	1000
0	217.9+/-7.1	215.8+/-7.2	212.5+/-9.6	215.2+/-8.5
1	224.5+/-.10.2	222.1+/-7.5	217.7+/-11.1	215+/-10.9
2	231.3+/-11.8	228.5+/-9.3	225.3+/-13.7	219.4+/-11.7
	FEMALES - GESTATION
Day	0	100	300	1000
0	232.1+/-10.1	228.2+/-9	224.3+/-14.3	222.1+/-11
7	264.8+/-12.9	257.9+/-7.3	259.8+/-18.5	247.6+/-17.7*
14	296.7+/-16.3	288.2+/-7.1	289+/-19.3	282.5+/-12.9
20	365.6+/-21.7	354.2+/-11.6	355.9+/-28.1	322.6+/-18.4*
	FEMALES - LACTATION
Day	0	100	300	1000
0	269.7+/-16.2	266.6+/-9.8	270.1+/-24.8	253.3+/-19.8
4	275.7+/-17.3	277.4+/-9.1	282.3+/-23.3	251.9+/-21.0*
* p<0.05 (two-sided Dunnett-test)

Table 2: Summary of litter data
LITTER AND PUP OBSERVATIONS
Dose (mg/kg bw/d)		0	100	300	1000
Litters	N	10	10	10	6
Litters with liveborn pups	N	10	10	10	5
Litters with stillborn pups	N	4	2	0	4
Pups delivered	N	135	137	119	47**
Pups liveborn	N	130	135	119	34**
	%	96	99	100	72
Pups stillborn	N	5	2	0	13**
	%	3.7	1.5	0	28
Pups died	N	1	0	0	6**
	%	0.7	0	0	13
Pups sacrificed moribund	N	1	0	0	0
Pups cannibalized	N	1	0	0	7**
	%	0.7	0	0	15
**, p<0,01 (Dunnett-test and Fishers exact test, repectively)