Glutaral

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-03-20 until 1990-04-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(1981)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: "Commission Directive 87/302/EEC of 18 November 1987 adapting to technical progress for the ninth time Council Directive 67/548/EEC, pp. 24 - 26" (1988)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: "EPA/FIFRA Pesticide Assessment Guidelines", Subdivision F, § 83-3, pp. 126 -130, NTIS (Nov. 1984)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: "EPA/TSCA New and Revised Health Effects Test Guidelines [Developmental Toxicity Study] ", NTIS (Oct. 1984).

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Glutaral

EC Number:

203-856-5

EC Name:

Glutaral

Cas Number:

111-30-8

Molecular formula:

C5H8O2

IUPAC Name:

glutaraldehyde

Test material form:

liquid

Specific details on test material used for the study:

_ Name of test material (as cited in study report): Glutaraldehyde
- Physical state: colourless-fluid
- Analytical purity: 50.3% in water
- Lot/batch No.: no data
- Stability under test conditions: stability of the test substance (content of active ingredient > 51%) proven by reanalysis at study ending.
- Storage condition of test material: refrigerator, under N2, in the dark

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Karl Thomae, Biberach/Riss, Germany
- Age at study initiation: about 68 to 77 days old at study start (corresponding to day 0, detection of sperms in the vaginal smear)
- Weight at study initiation: ca. 230 g
- Housing: individual housing in type D III stainless steel wire mesh cages (floor area about 800 cm2)
- Diet (e.g. ad libitum): ground Kliba 343 feed rat/mouse/hamster supplied by Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland, ad libitum
- Water (e.g. ad libitum): drinking water was available ad libitum during days 0 to 6 p.c. and 16 to 20 p.c., while doubly distilled water with or without addition of glutaraldehyde was available ad libitum during the treatment period (days 6 to 16 p.c.)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): the animals were kept in fully air-conditioned rooms, not further specified
- Photoperiod (hrs dark / hrs light): 12 hrs/ 12 hrs

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test solutions were prepared in doubly distilled water. The test material was weighed in a beaker depending an the dose group and topped up with doubly distilled water, which also was weighed). Thereafter, the solutions were placed on a magnetic stirrer for about 15 minutes. The solutions were filled into Makrolon@ drinking water bottles using a semi-automatic device.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test material in doubly distilled water were carried out before the beginning of the study.
Furthermore, samples of the test solutions were sent to the analytical laboratories of BASF AG twice during the study period for verification of the concentrations.
The test solutions were analyzed by colorimetrically after reaction with thiobarbituric acid and FeCl3.

Details on mating procedure:

Three to four untreated females were mated with one untreated fertile male rat of the same breed. Mating took place from ca. 16.00 hours to ca. 7.30 hours on the following day; the mating period was therefore about 15 to 16 hours.

Duration of treatment / exposure:

day 6 to day 16 of gestation

Frequency of treatment:

continuously in drinking water

Duration of test:

21 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 ppm (nominal)

Remarks:

in water; 5.2 mg/kg bw/day actual ingested

Doses / concentrations 2

Dose / conc.:

250 ppm (nominal)

Remarks:

in water; 25.7 mg/kg bw/day actual ingested

Doses / concentrations 3

Dose / conc.:

750 ppm (nominal)

Remarks:

in water; 68 mg/kg bw/day actual ingested

No. of animals per sex per dose:

25 females/group

Control animals:

yes, concurrent no treatment

Details on study design:

CHOICE OF TEST DOSES
Dose selection was based on 2 range-finding studies
In the first range-fincling study (BASF AG, 1991, Report No: 13R0599/89035), the test substance was administered to pregnant rats (10/dose) with the drinking water from day 6 to day 16 post coitum at doses of 0, 100 and 500 ppm, corresponding to 0, 10 and 50 mg/kg body weight/day. Some findings assessed as possibly being substance-related were reported for the 500 ppm level, including a diminished food consumption (day 6 to 8 p.c.), a reduced water intake (day 6 to 13 p.c.), and foci in the glandular stomach (2 cases only). The dose level of 100 ppm was defined as the NOAEL for both, the dams and the fetuses.
In the second range-finding study (BASF AG, 1991, Report No: 10R0599/89048), the test substance was administered by gavage to pregnant rats (10/dose) from day 6 to day 15 post coitum) at doses of 0, 10 and 50 mg/kg bw/day. A series of findings assessed as possibly being substance-related was reported for the 50 mg/kg bw/day dose level, including reduced food consumption, reduced mean body weights, diminished body weight gains, reduced corrected body weight gain, reduced nutritional state, labored breathing and piloerection seen in some cases, one case of vaginal hemorrhage on days 15 and 16 p.c. with absence of viable fetuses, lowering of total protein and globulin concentration, and increased relative kidney weights. Moreover, all animals showed thickening of margo plicatus, and in 3 cases, the animals additionally showed lesions of the glandular stomach. A slight increase in post implantation loss due to a marginally increased number of mainly late resorptions also was reported. In the 10 mg/kg bw/day group, one case of margo plicatus was reported.

Taking into consideration the results above, it became obvious that the administration of glutaraldehyde with the drinking water was the more appropriate route of administration, because the dams showed only minimal signs of maternal toxicity in concentrations which produced frank toxicity in the dams when given by gavage; however, a high dose of 500 ppm (i.e., ca. 50 mg/kg bw/day) seemed to be too low to fulfill the recommendations af the test guidelines, since the highest dose level to be selected should induce some overt maternal toxicity such as slight weight loss. Thus the dose levels for the present study were fixed as follows: 0, 50, 250, 750 ppm.

Examinations

Maternal examinations:

MORTALITY AND CLINICAL SYMPTOMS
the animals were checked daily, at least once for clinical symptoms and twice for mortality on working days. On Saturdays, Sundays or public holidays, the animals were only checked once a day.

BODY WEIGHT
Body weight was determined on day on day 0, 1, 3, 6, 8, 10, 13, 15, 17 and 20 p.c., and body weight change was calculated on the basis of the values obtained.

FOOD CONSUMPTION
Except for day 0 p.c., food consumption was determined on the same days as body weight, and only pregnant dams were considered for calculation.

WATER CONSUMPTION
Except for day 0 p.c., water consumption was determined on the same days as body weight and additionally on day 16 p.c., and only pregnant dams were considered for calculation.

TEST SUBSTANCE INTAKE
The intake of test substance (IT, in mg/kg bw/day) was calculated according to following formula:
ITx = WC x D /BWx

D = dose in ppm
WC = mean daily water consumption on day x + y, y = 1, 2 or 3; in g
BWx = body weight on day x; in g
Only pregnant dams were considered for calculation

POST-MORTEM EXAMINATIONS
On day 20 p.c. all dams were sacrificed for the purpose of necropsy. The sacrificed dams were subjected to gross pathology and the uterus and ovaries were removed.
Only pregnant dams, which were sacrificed at the end of the study period, were considered for the evaluations of following parameters: gravid uterine weight, mean net maternal body weight gain, reproduction data.
Following sacrifice, the corrected body weight gain (i.e. the net maternal body weight change) was calculated from the terminal body weight by subtraction of (1) the uterus weight and (2) the body weight measured on day 6 p.c.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination; the examinations included:
- Gravid uterus weight;
- Number of corpora lutea;
- Number of distribution of implantations sites classified as live fetuses and dead implantations, with dead implantations comprising early resorptions, late resorptions and dead fetuses.

The conception rate (CR) as well as the pre- and post- implantation losses (Pre-I¸ Post-I) were calculated according to following formula:
- CR = Number of pregnant animals x 100 / Number of fertilized animals
- Pre-I = (Number of corpora lutea - Number of implantations) x 100 / Number of implantations
- Post-I = (Number of implantations - Number of live fetuses) x 100 / Number of implantations

Fetal examinations:

GENERAL EXAMINATION
The fetuses were extracted from the uterus and were examined for following parameters:
Fetal weight, sex (measurement of the anogenital distance, later confirmed by internal examination), external abnormalities, viability, placentae, umbilical cords, fetal membranes, and fetal liquids. Individual placental weights were recorded.
One half of the fetuses per dam were placed in ethyl alcohol whereas the other half was fixed in Bouin´s solution for further evaluation.

SKELET
The skeletons of the fetuses fixed in ethyl alcohol were stained according to the method of Dawson (Stain Technol. 1: 123, 1926) for examination under a stereomicroscope.

SOFT TISSUES
The fetuses fixed in Bouin´s solution were examined for effects in the organs according to the method of Barrow and Taylor (J. Morph. 127: 291-306, 1969).

Statistics:

Food and water consumption, body weight and body weight change, corrected maternal body weight gain, gravid uterine weight, weight of the fetuses, weight of the placentae, corpora lutea, implantations, pre- and post implantation losses, resorptions and live fetuses, were assessed by means of Dunnett´s Test.
Conception rate, maternal mortality and all fetal findings were assessed by means of Fisher´s Exact Test.

Historical control data:

Historical control data were added to the study report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related signs of toxicity were observed.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The maternal body weights and body weight changes were quite similar for the treated and the control group. The values were within the range of biological variation and differences between the groups were without biological relevance.

Net maternal body weight change from day 6 p.c.: No significant differences between treated and control groups were seen. The values for all groups ranged between 43.9 +/- 8.45 g ( 50 ppm group) and 48.5 +/- 9.32 g (750 ppm group).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The maternal food consumption was quite similar for the treated and the control group. The values were within the range of biological variation and differences between the groups showed no treatment-relationship.

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption was inconspicuous in the 50 ppm group whereas a slight decrease in water consumption (up to 12%) was reported for the 250 ppm group from day 10 to day 15 p.c. In the 750 ppm group, water consumption was clearly reduced to about 19% below control during the period ranging from day 6 to day 16 p.c.

The approximate test substance intake (mg/kg bw/day) was 0, 5.2, 25.7 and 68.0 mg/kg bw/day for 0, 50, 250 and 750 ppm, respectively.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean gravid uterine weight was quite similar for all treated and the control groups; the values for all groups ranged between 74.9 +/- 25.13 g (control) and 82.2 +/- 16.14 g (250 ppm group).

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy revealed no treatment-related abnormalities.

Description (incidence and severity):

Females excluded because of non pregnancy:
Following females were partly or totally excluded from data evaluation/calculation because of non pregnancy: 5 females of the control group, 3 females of the 50 ppm group, 2 females of the 250 ppm group, and 2 females of the 750 ppm group.

Maternal developmental toxicity

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pre- and post implantation losses were inconspicuous.

Early or late resorptions:

no effects observed

Description (incidence and severity):

Number of resorptions was inconspicuous.

Dead fetuses:

no effects observed

Description (incidence and severity):

Number of live fetuses was inconspicuous.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

A conception rate of 92% respectively for the 250 and 750 ppm groups was reported, versus 80% for control. Conception rate was inconspicuous.
Conception rate, number of corpora lutea, number of implantation sites were inconspicuous.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Remarks:

maternal toxicity

Effect level:

50 ppm

Basis for effect level:

other: No adverse effetcs observed at this dose.

2

Dose descriptor:

LOEL

Remarks:

maternal toxicity

Effect level:

250 ppm

Basis for effect level:

water consumption and compound intake

Results (fetuses)

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on sex ratio, placental weight, fetal weight were reported. No treatment-related external malformations were seen. In fact, a single case of malformation (aglossotomia) was reported for a fetus of the 750 ppm group; this type of malformation is known to occur in the historical control at a low frequency and was therefore considered to be a spontaneous finding. One case of fused placenta was reported for one fetus of the 50 ppm group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations of the skull, the sternum (e.g. dislocated ossification centers), the vertebral column and/or the ribs were reported for 9 fetuses of the control group, 8 fetuses of the 50 ppm group, 2 fetuses of the 250 ppm group and 9 fetuses of the 750 ppm group. The only statistically significant difference was the lower number of fetuses affected of the 250 ppm group. Variations concerning the ribs (e.g. shortened 13th ribs or accessory 14th ribs), the sternum and the vertebral column). Signs of retardations (e.g. incomplete or missing ossification of vertebral bodies) were seen in all groups including control. A statistically significantly increased number of 50 ppm fetuses with incomplete ossification of the sternebrae, as well as an increased litter incidence of fetuses with incomplete ossification of the sternebrae were reported, which indeed were considered to be of spontaneous nature.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Organ examination revealed no treatment-related effects. In fact, one case of organ malformation (situs inversus) was reported for one control fetus. Variations were seen, including dilated renal pelvis and hydroureter, which showed no dose-response relationship and were within the normal range of biological variation for the used strain.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Remarks:

embryotoxicity

Effect level:

750 ppm

Sex:

male/female

Basis for effect level:

other: No indication for an embryo-/fetotoxic potential up to the highest tested dose.

Remarks on result:

other: 68 mg/kg bw/day

2

Dose descriptor:

NOAEL

Remarks:

68 mg/kg body weight/day

Effect level:

750 ppm

Sex:

male/female

Basis for effect level:

other: No indication for a teratogenic potential at the highest tested dose.

Remarks on result:

other: 68 mg/kg bw/day

Fetal abnormalities

Abnormalities:

effects observed, non-treatment-related

Overall developmental toxicity

Developmental effects observed:

no