Methyl isothiocyanate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

no data available.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April/May 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 422)

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles river Japan Co.
- Age at study initiation: 10 week-old
- Weight at study initiation: 337.9-426.0 g (males), 210.2-279.2 g (females)
- Fasting period before study: no data
- Housing: in metal cages with screen floors (220w x 270d x 190h mm), one animal per cage.
Females after day 18 of gestation were housed into plastic rat breeding cages (350w x 400d x 180h mm)
- Diet (e.g. ad libitum): solid food (CE-2, CLEA Japan), ad libitum
- Water (e.g. ad libitum): Hatano water supply, ad libitum
- Acclimation period: 15 days (including the quarantine and acclimatization period)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25°C
- Humidity (%): 40-75%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (lighed from 7:00 am -7:00 pm)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
After dissolving the test substance in 37°C water, the measured solvent was added to produce 0.16 w/v%. This was gradually diluted with the solvent to produce solutions of 0.04 and 0.01 w/v%.
Administration volume = 5 mL/kg

VEHICLE = Corn oil
- Justification for use and choice of vehicle (if other than water): because MITC was insoluble in water
- Concentration in vehicle: 0.01, 0.04 and 0.16 w/v%
- Amount of vehicle (if gavage): no data
- Lot/batch no. (if required): V2E7069, manufacturer : Nacalai Tesque Inc.
- Purity: no data

Details on mating procedure:

- M/F ratio per cage: 1M/1F
- Length of cohabitation: two weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 or vaginal plugof pregnancy
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For measurement of the test substance concentration in the prepared sample, 1mL each of the prepared substance was collected with the prescribed amount of dichloromethane and then the same solution was prepared by diluting appropriately in dichloromethane. Alternatively, the required amount of test substance was measured and a standard solution (1, 2, 5 ug/mL) dissolved in dichloromethane was prepared. The sample solution and standard solution was measured using the gas chromatography (GC) method and the concentration was determined using a graph created based on the standard solution.

Duration of treatment / exposure:

For males : for a continuous period of 42 days.
For females : from two weeks prior to copulation throughout the maximum of two weeks of the mating period until the day prior to necropsy.
For the satellite group : from two weeks prior to mating, the mating period until copulation, the gestation period and until day 4 of lactation, or for a continuous period of 42 days

Frequency of treatment:

daily

Details on study schedule:

no

Remarks:

Doses / Concentrations:
0.5, 2 and 8 mg/kg bw/ d (0.01 w/v%)
Basis:
actual ingested

No. of animals per sex per dose:

12 animals/sexe/dose
(+ 2 satellite groupe : 12 females/dose with 0 and 8 mg/kg bw/d).

Control animals:

yes, concurrent vehicle

Details on study design:

Based on the results of the preliminary study of combined repeated oral administration toxicity and reproductive/development test of this test substance conducted at the Hatano Research Institute (Test Plan Number: R-02-005), where administration continued from day 14 of gestation to delivery, and there were two mortalities of the three animals in the ITCM 50 mg/kg group, with the final animal moribund. The results of the necropsy on the mortalities confirmed adhesion between the serosa and the organs in the abdominal cavity, separation and thinning of the proventriculus and ascites accumulation, and substantial shrinkage of the thymus was noted. Based on these findings, the cause of the toxicity noted in the 50 mg/kg group is believed to be based on irritation of the stomach by this test substance. In these same findings, all three animals in the 25 mg/kg group and one of the three in the 10 mg/kg group experienced this to a milder degree. From these results, if the administration period for this study is taken into consideration, we believe that administration of 10 mg/kg of this test substance slightly exceeds the maximum dose. Administration of 8 mg/kg was established for the high dose group in this study, and then using a ratio of 4, a moderate dose of 2 mg/kg and a low dose of 0.5 mg/kg were set.

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
All of the animals were subject to observations daily during their care and the recovery period, and twice daily before and after administration during the administration period.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed observations on symptoms were conducted on all of the males during the quarantine period (all animals delivered), as well as days 7, 14, 21, 28, 35 and 42 of administration and days 7 and 14 of the recovery period, using a scoring method. All of the females were observed during the quarantine period (all of the animals delivered), as well as days 7, 14, 21, 28, 35 and 42 of administration. Animals that were in delivery on the observation day were observed on day 0 of lactation, and other animals delivering were observed once between day 0 and day 4 of lactation. Furthermore, the satellite group was observed on days 7 and 14 of recovery.

BODY WEIGHT: Yes
Body weight measurements were conducted on the males and all of the satellite group on days 1 of administration as well as days 7, 14, 21, 28, 35 and 42, and on days 1, 7 and 14 of recovery and the day of necropsy. Mother animals were measured on days 1, 7 and 14 of administration, and then after copulation was verified, days 0, 7, 14 and 20 of gestation, and after delivery, days 0 and 4 of lactation as well as the day of necropsy.

FOOD CONSUMPTION :
Measurements were performed on the males and the entire satellite group on days 1~2, 7~8, 14~15, 29~30, 35~36 and 41~42 of administration and on days 6~7 and 13~14 of recovery. Measurements were conducted on the mother animals on days 1~2, 7~8, 14~15 of administration, then after copulation was verified, days 0~1, 7~8, 14~15 and 20~21 of gestation, and after delivery, day 3~4 of lactation.

WATER CONSUMPTION : No
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy (males = day 42 of administration, females = day 4 of lactation)
- Anaesthetic used for blood collection: Yes (sodium pentobarbital)
- Animals fasted: Yes (18-24 hours prior to necropsy)
- How many animals: 5 males + 5 females
- Parameters examined : red blood cells, white blood cells, white blood cell classification, hemoglobin, mean corpuscular volume, platelets, hematocrit, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, reticulocyte ratio, prothrombin time, activated partial thromboplastin.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy (males = day 42 of administration, females = day 4 of lactation)
- Animals fasted: Yes (18-24 hours prior to necropsy)
- How many animals: 5 males + 5 females
- Parameters examined : total protin, albumin, total cholesterol, glucose, blood urea nitrogene concentration, creatinine, alkaline phosphate activation, AST activation, ALT activation, gamma-glutamyl transpeptidase activation, triglyceride concentration, inorganic phosphorus concentration, total bilirubin, calcium concentration, A/G ratio, sodium concentration, potassium concentration, chlorine concentration.

URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Examination of functions (pupillary reflection, visual orientation, startle response, rear limb reflex, blinking reflex, and righting reflex).

Oestrous cyclicity (parental animals):

not observed

Sperm parameters (parental animals):

not observed

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
General symptoms, number of live and dead pups, body weight, gender ratio

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The weights (actual weights) of the brain, heart, thymus, liver, kidney, spleen, adrenals, testes and epididymis were measured, and the relative weights calculated. Additionally, the brain, pituitary gland, spinal cord, heart, trachea, lungs (including the airway), liver, kidneys, thymus, spleen, adrenals, thyroid gland and glandura parathyroid, stomach, duodenum, jejune, ileum, appendix, colon, rectum, testes, epididymis, prostate, seminal vesicle containing coagulation, ovaries, uterus, vagina, bladder, submandibular lymph nodes, mesentery lymph nodes, sciatic nerve, femur and bone marrow, and areas of pathological change of all animals were extracted and stored.
Out of all of the animals, the only areas exhibiting pathological changes during visual observation were the ovaries, testes and epididymis. Histopathological examination was conducted on other organs and tissues for each of the five males that underwent necropsy at the end of administration and five females that delivered and were subject to hematological and blood chemical examinations from the control and high dose groups.

Postmortem examinations (offspring):

All of the living pups were euthanized with ether on day 4 of lactation and subject to necropsy.

Statistics:

Fisher direct probability was performed on the results from the examination of functions, for the frequency of animals experiencing changes to the sexual cycle, copulation rate, fertilization rate and frequency of abnormalities in live pups (standard of significance: 5%).
Significant differences with the control group were determined using the Mann-Whitney U test (standard of significance: 5%) for data in the histopathological examination findings for the test substance administration groups divided by grade, and using single Fisher direct probability (standard of significance: 5%) for total values for negative grades.
Other data was compared to the satellite group and the other groups using the values obtained for each individual or the mean value for each litter as one sample. In this case, if there were two groups subject to analysis, first an F-test was performed, and if a significant difference was not noted, a Student’s t-test was performed. If a significant difference was noted during the F-test, an Aspin-Welch test was performed. If there were 3 or more groups subject to analysis, first the Bartlett method was employed to test for uniform distribution of each group (standard of significance: 5%). If the distribution was uniform, distribution analysis (standard of significance: 5%) was conducted for uniform arrangement, while if there was significance between the groups, multiple comparisons were conducted using the Dunnett method (standard of significance: 5%). On the other hand, if the distribution for any of the groups was 0, and the distribution was not uniform, Kruskal-Wallis analysis of variance was performed (standard of significance: 5%), while if there was significance between the groups, multiple comparisons were conducted using the Dunnett method (standard of significance: 5%).

Reproductive indices:

The copulation rate, the implantation rate and the fertilization rate were calculated.

Offspring viability indices:

the delivery rate, the live pup delivery rate and the live birth rate were calculated.

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY
There were no deaths or moribund animals in any of the groups.
During the administration period, both sexes in the 8 mg/kg group experienced temporary salivation immediately after administration, which was observed from day 6 of administration throughout the administration period. This salivation was noted in 11 of the 12 males and 8 of the 17 females, and the frequency of occurrence was greater for males than for females. Additionally, one male in the 2 mg/kg group was noted to experience temporary salivation immediately after administration on one occasion.
During both the administration period and the recovery period, changes were not noted in any of the groups, and findings that suggest nerve toxicity were not noted. At the end of both the administration period and the recovery period, there was nothing abnormal noted in any of the groups concerning functions.

BODY WEIGHT AND WEIGHT GAIN
When compared to the control group, there were significantly low values in the amount of weight gain during week 1 or 2 of administration for the males in the 8 mg/kg group during the administration period, as well as the overall weight gain throughout the administration period. On the other hand, a significant difference with the control group was not noted for the body weights of females in any of the groups during the administration period.
During the recovery period, there were significantly low values in the amount of total weight gain for males in the 8 mg/kg group when compared to the control group during week 1 of recovery, which continued throughout the recovery period. On the other hand, the body weights for the females did not demonstrate any significant differences between the control group and any of the administration groups during the recovery period.

FOOD CONSUMPTION
During the administration period, males in the 8 mg/kg group exhibited significantly lower values for food consumption on days 7~8 and days 41~42 of administration when compared to the control group. On the other hand, the amount of food consumed by the females did not demonstrate any significant differences between the control group and any of the administration groups.
During the recovery period, neither of the sexes demonstrated any significant differences between the control group and any of the administration groups.

HAEMATOLOGY
The hematological examination conducted at the end of the administration period revealed increases in the red blood cells, hemoglobin and platelet count among males in the 8 mg/kg group. On the other hand, there were no significant differences between the females in any of the administration groups and the control group.
The examination conducted at the end of the recovery period did not reveal any significant differences between either of the sexes in any of the administration groups when compared to the control group.

CLINICAL CHEMISTRY
The hematological examination conducted at the end of the administration period revealed a significant increase in the glucose concentration among males in the 8 mg/kg group when compared to the control group. On the other hand, the females did not demonstrate any significant differences between the control group and any of the administration groups.
During the recovery period, neither of the sexes demonstrated any significant differences between the control group and any of the administration groups.

ORGAN WEIGHTS
The examination conducted at the end of the administration period revealed significant increases in the absolute weight of the epididymis of males in the 8 mg/kg group when compared to the control group. On the other hand, the females did not demonstrate any significant differences between the control group and any of the administration groups.
During the recovery period, neither of the sexes demonstrated any significant differences between the control group and any of the administration groups

GROSS PATHOLOGY (males)
A thickening of the proventriculus was noted in 7 animals in the 2 mg/kg group and all animals in the 8 mg/kg group. Of these, edema of the proventriculus was seen in 5 animals in the 8 mg/kg group, while whitish residue and yellow sections were observed in the other animal. Furthermore, white nodules were noted on the megogastrium mucosa of one animal in the 2 mg/kg group. Renal swelling was noted in one animal in the 0.5 mg/kg group and 5 animals in the 2 mg/kg group, but the same findings were not evident in the 8 mg/kg group. Additionally, depressions on the right kidney were noted in one animal in both the control group and the 8 mg/kg group, and diverticulum of the jejune was observed in one animal in the 0.5 mg/kg group, and nodules on the right epididymis as well as edema of the submandibular lymph nodes were noted in one animal in the 8 mg/kg group.

GROSS PATHOLOGY (females)
A thickening of the proventriculus was noted in 4 animals in the 2 mg/kg group and 6 animals in the 8 mg/kg group. Of these, edema of the proventriculus was seen in 4 animals in the 8 mg/kg group, while ragged edges were observed in the other animal. Additionally, black spots on the proventriculus were noted on one animal in each of the 0.5 and 2 mg/kg groups. Contraction of the thymus was noted in one animal in each of the control group and 8 mg/kg group, and the animal in the 8 mg/kg group also experienced contraction of the spleen as well as rough surfaces on the kidneys.

HISTOPATHOLOGY: NON-NEOPLASTIC
Nothing abnormal was noted in the testes and in the ovaries.
During histopathological examination that included animals that had abnormalities confirmed in the stomach during necropsy, there were 5 animals in the control and 0.5 mg/kg groups, 10 animals in the 2 mg/kg group and 7 animals in the 8 mg/kg group (in males and females).
Effects on epididymis, liver, renal tubules, heart, bladder and on spleen were observed in males and females.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All the animals in each of the groups successfully copulated and fertilization occurred. furthermore, copulation occurred during the first heat in all animals.
During the administration period but prior to mating, with the exception of one animal in the control group that did not go into heat, the administration did not impact the sexual cycles in any of the administration groups.

Dose descriptor:

NOAEL

Remarks:

(maternal toxicity)

Effect level:

8 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed at this dose. Only an irritation of stomach (local effect) was observed. This local effect explained therefore haematologic changes, decrease of body weight and food consumption which were observed in males only.

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

> 8 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: No effects on reproductive performance or on estrous cycle were observed at any dose.

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Findings delivery and lactation :
All of the pregnant animals delivered pups, and no significant differences in gestation time were noted between the administration groups and the control group. On the other hand, there were two animals in the 8 mg/kg group where poor delivery/lactation state not directly observed was noted.

VIABILITY (OFFSPRING)
There were no significant differences in the number of corpus luteum and number of implantations noted between the control group and the administration groups.
In the 0.5 and 2 mg/kg groups, the number of female pups delivered was significantly higher when compared to the control group at days 0 and 4 of lactation but since significant changes in the sex ratio were not confirmed, this was not deemed to be impacted by administration of the test substance.
One female (8 mg/kg group) did not gather the pups after delivery, and all of the pups perished by day 2 of lactation. Additionally, in the 8 mg/kg group, the number of surviving pups at day 4 of lactation and the survival rate for newborn pups showed a lower trend when compared to the control group.
Relative to reproduction/developmental toxicity, a poor delivery/lactation state was noted in the 8 mg/kg bw group, it's not possible to conclude if this decrease of live pup is related to MITC. It can noted that the repartition of foetal mortality is not homogeneous in all groups, high SD were observed in the control group and in the higher dose group (8 mg/kg bw/d). Individual data is not available, therefore no NOAEL for developmental toxicity can be choosen.


CLINICAL SIGNS (OFFSPRING)
Nothing adnormal was noted in the delivery status, or the overall condition of the newborn pups.

BODY WEIGHT (OFFSPRING)
Differences in the body weights of the lactating pups were not noted between the control group and the administration groups.

GROSS PATHOLOGY (OFFSPRING)
During the physical examinations on surviving newborn pups and during necropsy on day 4 of lactation, one female pup from animal (animal number FB04002) in the 8 mg/kg group was tailless but since other physical deformities were not noted on any of the other surviving pups from the same litter, it was considered a naturally occurring deformity. None of the pups that perished exhibited any physical deformity.

Reproductive effects observed:

not specified

Conclusions:

Based, pups mortality secondary to a poor delivery/lactation condition of some dams noted at 8 mg/kg/day group, the NOAEL for female fertility was 2 mg/kg/day. Since males did not have any effects caused by administration of methylisothiocyanate, the NOAEL for male fertility was 8 mg/kg/day or higher. The NOAEL for developmental toxicity was 8 mg/kg/day.

Executive summary:

In a combined oral repeated dose and reproduction/developmental screening test (OECD 422), dose levels of 0 (corn oil), 0.5, 2 and 8 mg/kg/day of methylisothiocyanate were repeatedly orally administered to groups of 12 males and 12 females Sprague-Dawley rats,for a continuous period of 42 days for the males and from two weeks prior to mating, the mating period until copulation, the gestation period and until day 4 of lactation for the females.

The males were subject to necropsy at week two after completion of the copulation period and the dams were subject to necropsy on day 5 of lactation after natural delivery, and the pups were subject to necropsy at day 4 of lactation.In the 8 mg/kg group, a poor delivery/lactation maternal condition was noted. A low trend in the live birth rate was noted at day 4 of lactation. It is believed that the result of the deterioration of the mother’s condition during delivery due to the administration of methylisothiocyanate was the death of the live pup that did not exhibit changes particular to their general condition. There was no impact of the administration of the test substance on the sexual cycle, ovulation, copulation, fertilization, implantation, gestation period, delivery rate, live birth rate and birth rate. Also, no changes were noted in the body weight of the live pups or the sex ratio, and no physical abnormalities thought to be attributed to administration were noted.

Based, pups mortality secondary to a poor delivery/lactation condition of some dams noted at 8 mg/kg/day group, the NOAEL for female fertility was 2 mg/kg/day. Since males did not have any effects caused by administration of methylisothiocyanate, the NOAEL for male fertility was 8 mg/kg/day or higher. The NOAEL for developmental toxicity was 8 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

8 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Takashima's study is considered to be reliable; it was performed in accordanc to the OECD guidelines.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a combined oral repeated dose and reproduction/developmental screening test (OECD 422) (Takashima et al., 2004), dose levels of 0 (corn oil), 0.5, 2 and 8 mg/kg/day of methylisothiocyanate were repeatedly orally administered to groups of 12 males and 12 females Sprague-Dawley rats, for a continuous period of 42 days for the males and from two weeks prior to mating, the mating period until copulation, the gestation period and until day 4 of lactation for the females.

The males were subject to necropsy at week two after completion of the copulation period and the dams were subject to necropsy on day 5 of lactation after natural delivery, and the pups were subject to necropsy at day 4 of lactation. In the 8 mg/kg group, a poor delivery/lactation maternal condition was noted. A low trend in the live birth rate was noted at day 4 of lactation. It is believed that the result of the deterioration of the mother’s condition during delivery due to the administration of methylisothiocyanate was the death of the live pup that did not exhibit changes particular to their general condition. There was no impact of the administration of the test substance on the sexual cycle, ovulation, copulation, fertilization, implantation, gestation period, delivery rate, live birth rate and birth rate. Also, no changes were noted in the body weight of the live pups or the sex ratio, and no physical abnormalities thought to be attributed to administration were noted.

Based, pups mortality secondary to a poor delivery/lactation condition of some dams noted at 8 mg/kg/day group, the NOAEL for female fertility was 2 mg/kg/day. Since males did not have any effects caused by administration of methylisothiocyanate, the NOAEL for male fertility was 8 mg/kg/day or higher. The NOAEL for developmental toxicity was 8 mg/kg/day.

Short description of key information:
In a combined oral repeated dose and reproduction/developmental screening test (OECD 422), based, pups mortality secondary to a poor delivery/lactation condition of some dams noted at 8 mg/kg/day group, the NOAEL for female fertility was 2 mg/kg/day. Since males did not have any effects caused by administration of methylisothiocyanate, the NOAEL for male fertility was 8 mg/kg/day or higher. The NOAEL for developmental toxicity was 8 mg/kg/day.

Justification for selection of Effect on fertility via oral route:
Only one reliable study is available to evaluate the effects of MITC on fertility.

Effects on developmental toxicity

Description of key information

In a developmental toxicity study performed following OECD 414 guideline, the maternal LOAEL/NOAEL was 10/3 mg/kg/day, based on salivation and decreased body weight gain and the developmental LOAEL/NOAEL was 30/10 mg/kg/day, based on reduced fetal weight and an increased incidence of the skeletal variation of unossified sternebra(e)

Link to relevant study records

Reference

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)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

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

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.

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

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

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.

Dose descriptor:

NOAEL

Effect level:

3 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

10 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Dose descriptor:

LOAEL

Effect level:

30 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

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

Dose descriptor:

NOAEL

Effect level:

> 30 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Stump's study is considered to be a reliable study, performed in accordance to the OECD guidelines.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a developmental toxicity study performed following OECD TG # 414 and according to GLP (Stump, 1998), methyl isothiocyanate (99.6% a.i.) was administered in corn oil by gavage twice daily from gestation day (GDs) 6 through 19 to 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.

Justification for selection of Effect on developmental toxicity: via oral route:
Stump's study (OECD 414) was the only one complete study performed on MITC to evaluation the developmental toxicity.

Justification for classification or non-classification

According to Directive 67/548/EEC and Regulation EC no.1272/2008 (CLP), no classification is warranted for reproductive toxicity.