2-methylpropane-2-thiol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-methylpropane-2-thiol (CAS #75-66-1)

IUPAC Name:

2-methylpropane-2-thiol (CAS #75-66-1)

Details on test material:

TS: t-butyl mercaptan product TBM (2-methylpropane-2-thiol)
Source: NGO Chemical, Baytown, Texas
Batch: ODB
Purity: > 99.0%

Test animals

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The Charles River Breeding Laboratories Inc., Portage, Michigan, USA
- Age at study initiation: approximately 12 weeks old
- Weight at study initiation: 25-36 grams at the time of mating
- Housing: individually housed, except during mating, in suspended wire-mesh cages
- Diet (e.g. ad libitum): Purina® Certified Rodent Chow #5002
- Water (e.g. ad libitum): tap water
- Acclimation period: 26 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

Animal Exposure Methods:
Exposures were conducted in one cubic meter glass and stainless steel exposure chambers. Air for the chamber ventilation was supplied from a HVAC system separate from the general laboratory systems. This air was particulate filtered (99.9% + 0.3 µ) and controlled for temperature and humidity. Chamber airflow rate varied between 200 and 260 L/min depending on desired exposure concentrations.
Exposure chamber temperatures and relative humidity were recorded each day alter three and six hours of exposure. Table 1 presents the minimum and maximum and the mean temperature and relative humidity at the six hour measurement time for each group over the course of the study.

Exposure Atmosphere Generation Methods:
A vapor atmosphere of the test material was generated utilizing a counter-current vaporization system. This system operated as follows: The test material was pumped at a known and constant rate to the top of the bead column by a FMI® fluid metering pump or Sagee syringe drive.
Dry-compressed air passed up the bead column in a countercurrent manner relative to the liquid. Vaporization occurred on the bead column. The concentrated vapors were piped to the exposure chamber air inlet where dilution with chamber ventilation air reduced the concentration to the desired level. Table 2 summarizes the vapor generation system operating conditions.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Nominal exposure concentrations were calculated for all exposures. Actual exposure concentrations were measured by non-dispersive Infrared spectrophotometry utilizing a Wilks (MIRAN®) lA analyzer. The analyzer was calibrated by volumetric dilution of pure (99%) t-Butyl Mercaptan in saran gas bags. The calibration was checked once daily.

Details on mating procedure:

One female and one male animal of the same species and strain were placed together for mating. The occurrence of copulation was determined by daily inspection for a copulatory plug. The day evidence of mating was detected was designated day 0 of gestation and the female was returned to an individual cage.

Duration of treatment / exposure:

gestation days 6 - 16

Frequency of treatment:

6 hour/day

Duration of test:

until GD17

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
10, 100 and 200 ppm
Basis:
other: desired conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
11, 99 and 195 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

25

Control animals:

yes, sham-exposed

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
Prior to initiation of the treatment period all animals were observed twice daily for mortality and overt changes in appearance and behavior. All animals were observed daily for mortality and clinical signs of toxicity from gestation day 6 through sacrifice.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
Individual maternal body weights were recorded on gestation days 0, 6, 9, 12, 15 and 17 .

FOOD CONSUMPTION: No

WATER CONSUMPTION : No

POST-MORTEM EXAMINATIONS: Yes
On gestation day 17, all surviving dams were sacrificed by carbon dioxide inhalation. The abdominal and thoracic cavities and organs of the dams were examined for grossly evident morphological changes and the carcasses discarded. Uteri from females that appeared nongravid were placed in 10% ammonium sulfide solution for confirmation of pregnancy.

Ovaries and uterine content:

The ovaries and uterine content were examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes
All fetuses were individually weighed and examined for external malformations and variations, including the palate and eyes. Each fetus was externally sexed and individually numbered and tagged for identification

- Soft tissue examinations: Yes
Approximately one-half of the fetuses were placed in Bouin's fixative for subsequent visceral examination by razor-blade sectioning as described by Wilson.

- Skeletal examinations: Yes
The remaining one-half of the fetuses were fixed in alcohol, macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson2 for subsequent skeletal examination

- Head examinations: No

Statistics:

The male to female fetal sex distribution and the numbers of fetuses and litters with malformations were compared using the X2 test criterion with Yate's correction for 2X2 contingency tables and/or Fisher's exact probability test. The numbers of early and late resorptions, nonviable fetuses, and postimplantation loss were compared by the Mann-Whitney U test. The mean numbers of viable fetuses, total implantations, and corpora lutea, and mean fetal body weights were compared by analysis of variance (one way classification). Bartlett's test for homogeneity of variances, and the appropriate t test using Dunnett's multiple comparison tables were used to judge significance of differences. All statistical analyses compared the treatment group to the control group with the level of significance at p<0.05.

Historical control data:

See the attached file

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The livers of all female mice, preserved in 10% formalin as specified in the protocol, were externally normal. At 100 and 200 ppm, the mean absolute and relative liver weights were increased when compared to the control group (Table 6), but were not statistically significant and likely an adaptive response. The values et 10 ppm were comparable to the control group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

no effects observed

Description (incidence and severity):

corpora lutea

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Maternal Observations:
Survival was 100% in all groups. The mice in the treated groups were similar in appearance and behavior to the control group mice. No treatment-related trend in necropsy findings was observed. The livers of all female mice, preserved in 10% formalin as specified in the protocol, were externally normal. At 100 and 200 ppm, the mean absolute and relative liver weights were increased when compared to the control group (Table 6), but were not statistically significant and likely an adaptive response. The values et 10 ppm were comparable to the control group.
There were no biologically meaningful differences in mean maternal body weight (Table 4) during the treatment period (gestation days 6-17) or over the entire gestation period (gestation day 0-17) in the t-butyl Mercaptan treated mice when compared to the control group mice. In addition, the adjusted (dam weight on gestation day 17 minus the gravid uterus weight) mean maternal body weight change (Table 4) from gestation days 0-17 in the treated groups in this study segment was comparable to the control group.

Caesarean Section Observations:
There were no biologically meaningful or statistically significant differences in the mean number of viable fetuses, postimplantation loss, total implantations, corpora lutea, fetal body weight or the fetal sex distribution in the treated groups when compared to the control group (Table 5) and the historical control data.

Effect levels (maternal animals)

Dose descriptor:

NOAEC

Effect level:

>= 195 ppm (analytical)

Basis for effect level:

other: no observed effects

Remarks on result:

other:

Remarks:

Equivalent to 721 mg/m³

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

not specified

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not specified

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

An increase in the total litters with malformations, due primarily to the increased incidence of the malformation vertebral anomaly, was noted in the t-butyl Mercaptan treated groups when compared to the control group. However, this incidence did not occur in a dose-related pattern (47.8% and 28.6% of the litters in the 100 and 200 ppm groups, respectively, had malformed fetuses) and was not statistically significant (p>0.05).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Vertebral anomalies were present in 16.7% of the litters in both the control group and the 10 ppm group, in 43.5% of the litters in the group 100 ppm and in 23.8% of the litters in the 200 ppm. The only other malformation present in the 200 ppm group was a single instance of rib anomalies. In the 10 and 100 ppm groups, the remaining malformations did not occur in a dose-related pattern and/or were within the range of the historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

An increase in the percent of fetuses per group with the variations misaligned sternebrae and/or 14th rudimentary ribs was noted in the treated groups when compared to the control group. The incidence of misaligned sternebrae in the control and treated groups was greater than the highest value in the historical control data, while the occurrence of 14th rudimentary ribs in the control and treated groups was within the range of the historical control data. There were no other trends in the incidence of genetic or developmental variations in the treated groups when compared to the control group.

Effect levels (fetuses)

Dose descriptor:

NOAEC

Effect level:

>= 195 ppm (analytical)

Sex:

male/female

Basis for effect level:

other: no observed effects

Remarks on result:

other: Equivalent to 721 mg/m³

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

TABLE 4: Summary of Group Mean Maternal Body Weights

	Control	10 ppm	100 ppm	200 ppm
Day of gestation	Mean±S.D.	Mean±S.D.	Mean±S.D.	Mean±S.D.
0	29±2.39	29±2.1	29±2.6	29±2.0
6	31±2.6	31±2.3	32±2.7	30±4.0
9	33±2.6	33±2.7	34±2.6	33±3.2
12	38±3.0	38±2.9	39±3.6	38±3.0
15	44±3.5	45±3.3	46±4.4	46±3.4
17	51±4.0	51±3.6	52±5.6	52±4.2
17 (adjusted)^a	35±3.9	35±2.4	35±3.7	36±2.4

^aDam body weight on gestation day 17 minus gravid uterus weight

TABLE 5: Summary of Group Mean Maternal and Fetal Observations at Cesarean Section

Group	Control			10 ppm			100 ppm			200 ppm
	No.	%	S.D.	No.	%	S.D.	No.	%	S.D.	No.	%	S.D
Animals on study:	25	-	-	24	-	-	25	-	-	25	-	-
Animals that were gravid:	24	96.0	-	24	96.0	-	23	92.0	-	21	84.0	-
Animals that died:	0	0.0	-	0	0.0	-	0	0.0	-	0	0.0	-
Nongravid:	-	-	-	-	-	-	-	-	-	-	-	-
Gravid:	-	-	-	-	-	-	-	-	-	-	-	-
Animals that aborted/delivered:	0	0.0	-	0	0.0	-	0	0.0	-	0	0.0	-
Animals examined at Cesarean section:	25	100.0	-	25	100.0	-	25	100.0	-	25	100.0	-
Nongravid:	1	4.0	-	1	4.0	-	2	8.0	-	4	16.0	-
Gravid:	24	96.0	-	24	96.0	-	23	92.0	-	21	84.0	-
Dams with resorptions only:	0	0.0	-	0	0.0	-	0	0.0	-	0	0.0	-
Dams with viable fetuses:	24	100.0	-	24	100.0	-	23	100.0	-	21	100.0	-
Viable fetuses/dam:	11.5	-	2.34	11.1	-	1.65	11.4	-	2.19	11.3	-	1.65
Postimplantation loss/dam:	0.6	-	0.88	0.9	-	1.08	0.7	-	0.76	0.7	-	1.15
Total implantations/dam:	12.1	-	1.04	12.0	-	2.00	12.1	-	2.02	12.0	-	1.80
Corpora lutea/dam:	14.0	-	2.10	13.9	-	2.46	14.5	-	3.51	14.6	-	3.44
Fetal sex distribution                       Male:	130	47.3	-	128	47.9	-	135	51.5	-	110	46.2	-
Female	145	52.7	-	139	52.1	-	127	48.5	-	128	53.8	-
Mean fetal body weight (grams)	0.88	-	0.079	0.92	-	0.094	0.92	-	0.112	0.94	-	0.092
Group mean preimplantation (%)	-	13.4	-	-	13.5	-	-	16.5	-	-	17.3	-
Group mean postimplantation loss (%)^b:	-	5.2	-	-	7.3	-	-	5.8	-	-	5.9	-

^aGroup mean preimplantation loss (%) =Total No. Corpora Lutea - Total No. Implantationsx 100

Total No. Corpora Lutea

^bGroup mean postimplantation loss (%) =Total No. Implantations-Total No. Viable Fetuses x 100

                                                                    Total No. Implantations

^cValue does not include dams with regressing corpora lutea

*Significantly different from control group mean, p<0.05.

**Significantly different from control group mean, p<0.01.

- Not applicable

S.D.- Standard deviation

Table 6: Individual Maternal Liver Weight

Group	Terminal bw (g)	Liver weight (g)	Relative liver weight (%)
Control	51±4.0	2.89±0.287	5.7±0.48
10 ppm	51±3.6	2.97±0.249	5.9±0.46
100 ppm	52±5.6	3.22±0.466	6.2±0.70
200 ppm	52±4.2	3.58±0.385	6.9±0.57

Applicant's summary and conclusion

Conclusions:

No teratogenic effects occurred in mice when administered 2-methylpropane-2-thiol by whole body inhalation at or below the 195 ppm actual exposure level.

Executive summary:

Pregnant female mice (CD-1; 25/group) were repeatedly exposed (inhalation, whole body) to 2-methylpropane-2-thiol for 6 hrs/day during GD 6-16. Exposure conditions consisted of measured concentrations of 0, 11, 99, and 195 ppm (nominal concentrations of 0, 10, 100 and 200 ppm). The study design was similar to OECD Test Guideline No. 414. All animals survived to scheduled termination. there were no statistically significant differences between the dosed animals and controls with respect to maternal endpoints. Fetal malformations were observed in mice exposed to 99 ppm (litter incidence, 47.8 %) and 195 ppm (litter incidence, 28.6%) when compared to the control group (litter incidence, 16.7%). The particular malformation noted (i.e., vertebral anomaly), however, did not increase in a dose-related pattern and was in the range of historical control data. There were no additional statistically significant differences in fetuses exposed to 2 -methylpropane-2-thiol when compared to controls. There were no signs of maternal toxicity or biologically relevant teratogenic effects when t-butyl mercaptan was administered by whole body inhalation at or below the 195 ppm actual exposure; therefore, the maternal and fetal NOAEC was equal or higher than 195 ppm.