Methyl isothiocyanate

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:

other: GLP Guideline study (EPA 870.3700)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methyl isothiocyanate

EC Number:

209-132-5

EC Name:

Methyl isothiocyanate

Cas Number:

556-61-6

Molecular formula:

C2H3NS

IUPAC Name:

isothiocyanatomethane

Details on test material:

MITC was purchased from Osmose Wood Preserving (Buffalo, New york).
Purity = 99.6%

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Portage, Michigan)
- Age at study initiation: 236-343g
- Weight at study initiation: 98 days (14 weeks)
- Fasting period before study: no data
- Housing: individually housed in clean wire-mesh cages suspended above cage-board.
- Diet (e.g. ad libitum): Certified Rodent labDiet 5002 (pMI Nutrition International), ad libitum
- Water (e.g. ad libitum): Municipal water, ad libitum
- Acclimation period: 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72.2-72.7°F
- Humidity (%): 32-50%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Dose volume = 5 ml/kg/dose.
Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On December 15, 1997 (prior to the initiation of dosing), a 10-ml aliquot was taken from the middle stratum of the control group formulation and 10-ml aliquots were collected from the top, middle and bottom strata of each treated group formulation. These samples were analyzed for homogeneity. An additional 10-ml aliquot was taken from the middle stratum of the 0.3 mg/ml formulation and stored for 8-day stability verification. Eight-day stability of the test article in the vehicle at concentrations of 0.5 and 3 mg/mi was established in a previous study (WIL-316001'). For the December 22 and 29, 1997, and January 5, 1998, weekly dosing preparations, a 10-ml aliquot was collected from the middle stratum of each dosing formulation, including the control, and analyzed for concentration. The dosing formulations were homogeneous, stable for 8 days and contained the amounts of the test article specified in the protocol.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1M/1F
- Length of cohabitation: no data (in the home cage of the male)
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug OR sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

on gestation days 6 through 19

Frequency of treatment:

Twice daily (approximately four hours apart).
The twice daily treatment regimen was used because of the irritative properties of MITC.

Duration of test:

on gestation day 20.

Doses / concentrations

Remarks:

Doses / Concentrations:
3, 10, 30 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

22-25 females / dose

Control animals:

yes, concurrent vehicle

Details on study design:

no

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes, twice daily (morning and afternoon)
DETAILED CLINICAL OBSERVATIONS: Yes, one hour following each dosing
BODY WEIGHT: Yes, daily on gestation days 0 and 6-20.
FOOD CONSUMPTION : Yes, daily on gestation days 0 and 6-20.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
WATER CONSUMPTION : No
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20

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: Yes
- Number of late resorptions: Yes

Fetal examinations:

The fetuses were weighed, wexed and examined.
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

Statistics:

All analyses were conducted using two-tailed tests for a minimum significance level of 5%, comparing each treated group to the vehicle control group. Means were presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. The following statistical tests were performed by a Digital MicroVAX 3400 computer (with appropriate programming).
One-way ANOVA with Dunnett's test for Corpora Lutea, Total Implantations, Viable Fetuses, Fetal Body Weights, Maternal Body Weights and Weight Changes, Maternal Net Body Weight Changes and Gravid Uterine Weights, Food Consumption.
Kruskal-Wallis test with Mann-Whitney U test for Litter Proportions of Intrauterine Data (Considering the Litter, Rather than the Fetus, as the Experimental Unit), Litter Proportions of Malformations and Developmental Variations.

Indices:

Group mean litter basis, proportional litter basis.

Historical control data:

yes

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS : All animals survived to the scheduled necropsy on gestation day 20. Test article-related clinical signs were observed primarily at a dose level of 30 mg/kg/day. The predominant findings in this group included salivation and clear matting on the neck, forelimbs and around the mouth; these signs were observed primarily at the time of both daily dosings. Tan matting on the neck, forelimbs, and around the mouth was noted in the 30 mg/kg/day group one hour following both daily dosings. Yellow matting on the urogenital area was noted in this group during the daily examinations and at the post-dosing observations. Salivation at the time of dosing was also noted occasionally in the 10 mg/kg/day group. All of the findings were generally noted during gestation days 8 to 19. Other clinical findings in the treated groups were noted sporadically or for single animals and not considered to be related to treatment.

BODY WEIGHT AND GRAVID UTERINE WEIGHT :
At 30 mg/kg/d, a reduced mean body weight gain were observed on GD 6-9, 9-12, 12/20 (p<0.01), 19-20 (p<0.01) and 6-20 (p<0.01). Mean net bw in the 30 mg/kg/d was slightly lower than the control group value but not statistically significant. Mean gravid uterine weight and net body weight gain in this group were reduced and statistically significant (p <0.01) when compared to the control group values.
Mean body weights, body weight gains, net body weights, net body weight gains and gravid uterine weights in the 3 and 10 mg/kg/day groups were unaffected by MITC. Mean gravid uterine weights in the 3 and 10 mg/kg/day groups were reduced and statistically significant (p < 0.05) when compared to the control group values. These reductions were attributed to the reduced numbers of implantation sites which resulted in fewer viable fetuses. Since implantation occurs prior to gestation day 6 (the first day of dosing with the test article) and a similar decrease in viable lifter size was not observed at the higher dose level of 30 mg/kg/day, the reduced number of viable fetuses and the resulting reduced gravid uterine weights in the 3 and 10 mg/kg/day groups were not attributed to treatment with the test article.

FOOD CONSUMPTION : Food consumption in the 30 mg/kg/day group was reduced statistically significant when compared to the control group (GD 6-9, 12-20, 6-20). Food consumption was unaffected by MITC administration at dose levels of 3 and 10 mg/kg/day.

NECROPSY DATA : Stomach adhesions were observed on females treated with 30 mg MITC/kg/d. No other internal findings that could be related to the test article were observed at any dose level.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

3 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

LOAEL

Effect level:

10 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

3

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

4

Dose descriptor:

LOAEL

Effect level:

30 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Mean fetal body weight in the 30 mg/kg/day group (3.4 g) was reduced when compared to the control group value (3.7 g). The difference was statistically significant (p< 0.01). The 30 mg/kg/day group value was within the range of the WIL historical control data (3.3-3.9g). However, four of 22 litters in this group (18%) had mean fetal body weights that were lower than the minimum value observed in the WIL historical control data. Therefore, the reduced fetal body weight in this group was attributed to the test article. Postimplantation loss, viable litter size, fetal sex ratio and the mean numbers of corpora lutea and implantation sites in the 30 mg/kg/day group were similar to the control group values. Intrauterine growth and survival in the 3 and 10 mg/kg/day groups were unaffected by test article administration.

FETAL MORPHOLOGICAL DATA : The numbers of fetuses (litters) available for morphological evaluation were 359(22), 371(25), 337(23) and 345(22) in the control, 3, 10 and 30 mg/kg/day groups, respectively. Malformations were observed in 3(3), 0(0), 0(0) and 0(0) fetuses (litters) in these same respective groups.
An increased incidence of two skeletal variants, unossified sternebrae nos. 1, 2, 3 and/or 4 and unossified sternebrae nos. 5 and/or 6 was observed in the 30 mg/kg/day group. The difference from the control group was statistically significant for unossified sternebrae nos. 5 and/or 6. The increased incidence of these variants was attributed to the retarded fetal growth (reduced fetal body weights) observed in the 30 mg/kg/day group. Other fetal developmental variations in the treated groups occurred similarly in the control group, occurred at a low incidence or were within the range of the WIL historical control data and were not considered to be related to treatment with the test article

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

> 30 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Maternal toxicity was exhibited at a dose level of 30 mg/kg/day by reductions in body weight gain, reduced food consumption and stomach lesions. Developmental toxicity was expressed at this dose level by reduced fetal body weights and an increase in the incidence of skeletal developmental variants (unossified sternebrae). No teratogenic effects were observed for fetuses in this study. No maternal or developmental effects were exhibited at dose levels of 3 and 10 mg/kg/day. Based on the results of this study, a dose level of 10 mg/kg/day was considered to be the NOAEL (no-observed-adverse-effect level) for maternal toxicity or fetal (developmental) toxicity.

Executive summary:

In a developmental toxicity study performed following OECD TG # 414 and according to GLP

, methyl isothiocyanate (99.6% a.i.) was administered in corn oil by gavage twice daily from gestation day (GDs) 6 through 19 to Crl:CD®(SD)BR rats (25/dose level) at dose levels of 0, 3, 10, or 30 mg/kg/day. It was stated that the twice-daily treatment regimen was used because of the irritative properties of the test substance. Dams were sacrificed on GD 20.

No treatment-related findings were noted at 3 mg/kg/day. No premature deaths occurred during the study interval. Many treatment-related clinical signs of toxicity were observed in the 30 mg/kg group at daily examination, at the time of the first and second daily doses, and 1 hour post-dose of the first and second doses. Treatment related salivation was also noted at 10 mg/kg. At 30 mg/kg/day, decreased (p<0.05 or 0.01) body weight gains were observed as follows: during daily measurements on GDs 15-17 (↓22-33%) and GDs 18-20 (↓36 - 47%); for the GDs 12-20 interval (↓29%); the overall treatment interval (↓27%, GDs 6 - 20); and the overall study interval (↓20%, GDs 0-20). At 10 mg/kg/day, body weight gains were statistically decreased during GD 15-16 and 18-19 (↓20% and ↓32%, respectively) compared to controls. At 30 mg/kg/day, a decrease in gravid uterine weight was observed (↓13%), along with a reduction in corrected (for gravid uterine weight) body weight gain (↓31%, p<0.01). Absolute (g/animal/day) and/or relative (g/kg/day) food consumption was reduced in the 30 mg/kg/day group (p<0.05 or 0.01) during the

daily measurements on GDs 15-20 (↓11-24%), during the GDs 6-9 (↓12-15%) and 12-20 (↓10-11%) intervals, and for the overall treatment interval (↓6-12%, GDs 6-20). Females of the highest dose group (8/25 treated) exhibited stomach adhesions and one of

these eight also had a stomach abscess; neither of these observations were noted in control animals. The number of implantations/dam, number of resorptions/dam, percent male, and the extent of pre-and post-implantation losses were similar between control and treated groups.

The maternal LOAEL is 10 mg/kg/day, based on salivation and decreased body weight gain. The maternal NOAEL is 3 mg/kg/day.

At 30 mg/kg/day the mean fetal weight was decreased by 8% compared to controls (p< 0.01). The reduced fetal weight is associated with decreased body weight gain of the maternal animals at the same dose level. Upon skeletal examination, increased incidence of unossified sternebra(e) #1, #2, #3, and/or #4 was observed at 30 mg/kg [2.6 (27.3)] vs controls [0.28 (4.5)]; the litter incidence of this finding was beyond the historical control range (0.0-22.73). No treatment related effects were observed in the 3 or 10 mg/kg/day groups.

The developmental LOAEL is 30 mg/kg/day, based on reduced fetal weight and an increased incidence of the skeletal variation of unossified sternebra(e). The developmental NOAEL is 10 mg/kg/day.