Benzenethiol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzenethiol

EC Number:

203-635-3

EC Name:

Benzenethiol

Cas Number:

108-98-5

Molecular formula:

C6H6S

IUPAC Name:

benzenethiol

Details on test material:

Thiophenol (CAS No.1 08-98-5), obtained from Aldrich Chemical Company, Milwaukee, Wisconsin, was determined to be >99% pure by high performance liquid. chromatography. Specific impurities were not identified. The molecular formula for thiophenol (also known as phenyl mercaptan) is CeHsSH.

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- sex : Male (breeders) and female
- Source: Hazleton Research Products, Inc.
- Age at study initiation: approximately 5-6 months of age
- Water (e.g. ad libitum):deionized/filtered water were available ad libitum
- Acclimation period: 14days
- Weight at study initiation: 2750 - 4130 g (gestational day 0)-Maternal body weights ,mean body weights/group ranged from 3424 - 3744 g
- The number of animal per group : 24-26 animals per group , 15 animals (40 mg/kg/day)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 67°F (average )
- Humidity (%): 49% (average )
- Photoperiod (hrs dark / hrs light): 12:12 hour light:dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

- Concentration in vehicle: 0, 10, 30, or 40 mglkglday THIO in corn oil , The initial
study design included 50 mglkglday THIO as the high dose. However, during the first
replicate, 50 mglkglday THIO caused 46% maternal mortality during the first week of
dosing. That group was humanely terminated before the end of the first replicate and
was replaced in the second replicate by 40 mglkglday THIO.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Pre-treatment analysis of the THIO formulations indicated that the concentrations of
THIO were within 98-107% of target concentrations (Table 2). The gavage formulation
provided to the control animals was determined to contain less than the detectable limit
of THIO, which was not specifically quantified. Treatment and examination of animals
was performed without knowledge of exposure levels with the exception of the vehicle
control group.

TABLE 2
Analysis of Thiophenol Dose Formulations Prior to Use

Dose (mglkglday) Target Concentration (mglml) Replicate 1 Concentration (% of Target) Replicate 2 Concentration (% of Target)
-----------------------------------------------------------------------------------------------------------------------------------
0 0 - a - a
10 10 107 98
30 30 101 100
40 40 - c 102
50 50 107 b -
------------------------------------------------------------------------------------------------------------------------------------

a = Below the detectable limit of the assay standard curve.
b = This dose was dropped from the second replicate because 6/13 does died during the firs1 week of dosing and
several of the remaining does appeared unhealthy. Based on this information, the 50 mglkglday group was
terminated to prevent further dis1ress. A 40 mglkglday group was added in its place for the second replicate.
c = The 40 mglkglday dose was not included in the fim replicate.

Details on mating procedure:

- mating procedure :
After a fourteen-day quarantine period, the females received
an intravenous injection of Pregnyl (chorionic gonadotropin, 0.1 ml/kg; Organon, Inc., West
Orange, NJ) prior to insemination. Semen was collected from the untreated males using an
artificial vagina (Bredderman et al., 1964) in conjunction with a teaser female.
Prior to insemination, ejaculate collected from the males was evaluated for number of motile sperm.
Females were inseminated with undiluted ejaculate on a day designated as gestational day
(GD) 0.
- Impregnation procedure: artificial insemination

Duration of treatment / exposure:

From gestational day (gd) 6 through gd 19

Frequency of treatment:

single administraton per day

Duration of test:

gestational day (gd) 30 day

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 30, 40, 50(mglkglday)
Basis:

Control animals:

yes

Examinations

Maternal examinations:

Inseminated females were weighed on the mornings of GD 0, 3, 6 through 19
(prior to dosing), 25, and 30 (prior to sacrifice).
Animals were observed for clinical signs of
toxicity at least once daily before, during, and after the treatment period. Maternal food
consumption was recorded every three days from GD 0 through 18 and also on GD 19, 22,
25,28 and 30. All inseminated rabbits were killed on GD 30 by lethal injection.
The maternal body, liver, right kidney were weighed .

Ovaries and uterine content:

The intact uterus were weighed and ovarian corpora lutea were
counted. In the uterus, the number of implantation sites were counted - those uteri which
had no visible implantation sites were stained with ammonium sulfide (10%) to detect very
early resorptions (Salewski, 1964).

Fetal examinations:

Live fetuses were dissected from the uterus and
sacrificed by intraperitoneal injection of euthanasia solution (Euthanasia Solution C-\I,
Page 15
Anthony Products, Acadia, CAl. They were weighed, examined for external morphological
abnormalities and dissected for visceral examination by a fresh tissue dissection technique
(Staples, 1974; Stuckhardt and Poppe, 1984). Half of the fetuses were decapitated prior to
dissection; the heads were fixed in Bouin's solution and then examined by a free-hand sectioning
technique (Wilson, 1965). All fetal carcasses (one-half without heads) were stained with Alcian
Blue/Alizarin Red S and examined for skeletal malfonnations (Marr et al., 1988).

Statistics:

General Linear Models (GLM) procedures were applied for the analyses of variance
(ANOVA) of maternal and fetal parameters (SAS Institute, 1989a; 1989b; 1990a; 1990b; 1990c).
Prior to GLM-ANOVA analysis, an arcsine-square root transformation was performed on all
litter-derived percentage data to nonnalize the means (Snedecor and Cochran, 1967) and
Bartlett's test for homogeneity of variance was perfonned on all data to be analyzed by ANOVA
(Winer, 1962). GLM-ANOVA analysis detenninecl the significance of dose-response relationships
and the significance of dose effects, replicate effects and dose x replicate interactions. When
ANOVA revealed a significant dose effect (p < 0.05), Dunnett's Test (Dunnett, 1955; 1964) and
I Williams' Test (Williams, 1971; 1972) were used to compare treated to control groups. One-tailec;t
tests were used for all pair-wise comparisons except maternal food consumption, maternal body
and organ weights, maternal weight gains, fetal body weight, and percent male fetusesllitter.
Nonsignificant (p>0.05) dose x replicate interactions on selected fetal parametric measures were
considered justification for pooling data across replicates for nonparametric analysis on related
measures. When a dose x replicate interaction was significant (p < 0.05), the data for that
endpoint and any related nominal scale data were analyzed .separately for dose effects wHhin
each replicate in the study, as well as for all replicates combined. Nominal scale measures were
analyzed by a Chi-Square Test for Independence and by the Cochran-Armitage Test for linear
trend on proportional data (Agresti, 1990; Annitage, 1955; Cochran, 1954; SAS Institute, 1992).
When a Chi-Square test showed significant experiment-wise differences, a one-tailed Fisher's
exact probability test was used for pair-wise comparisons of treatment and control groups.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
no THIO-related effects on maternal body weight were observed

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

>= 40 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

2

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Examination of the uterine contents revealed that the number of corpora lutealdoe, the
number of implantations/litter, and the percent pre-implantation loss/litter were comparable
across all groups. THIO administration during the period of major organogenesis did not
alter the percent resorptionsllitter, percent late fetal deathsllitter or precent nonlive
implants/litter . The percent litters with early fetal deaths (resorptions) ,late fetal deaths or nonlive implants (early + late fetal deaths) were also unaffected by
THIO-treatment.
Examination of the live litters indicated that the number of live fetuseS/litter, average male
and female body weight/litter, and the sex ratios of the offspring were not adversely affected'
by THIO administration . Morphological examination of the fetuses
determined that THIO did not increase the incidence of external. visceral, or skeletal
malformations or variations except for an increase in the percent females with variations per
litter at 40 mg/kg/day THIO for the study as a whole, or at 30 and 40
mg/kglday for Replicate,1I alone. The increase in variations was mainly
confined to the presence of extra or rudimentary lumbar ribs.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified