Methyl isothiocyanate

Toxicity to reproduction

Administrative data

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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

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

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:

Source, batch number : not precised
Purity : 99.8%
Storage : in a refrigerator (2-8°C)

Test animals

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)

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

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.

Effect levels (P0)

1

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.

2

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.

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

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.