Methyl isothiocyanate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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 oral 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%.

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, manufactruer : nacalai Tesque Inc
- Purity: no data

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

Doses / concentrations

Remarks:

Doses / Concentrations:
0.5, 2 and 8 mg/kg bw/d
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

Observations and examinations performed and frequency:

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).

Sacrifice and pathology:

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.

Other examinations:

no

Statistics:

Fisher direct probability was performed on the results from the examination of functions (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%).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

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 the recovery period, changes to the overall condition, including salivation, were not noted in any of the groups.

DETAILLED CLINICAL OBSERVATION
During both the administration period and the recovery period, noteworthy changes were not noted in any of the groups, and findings that suggest nerve toxicity were not noted.

FUNCTION OBSERVATION BATTERY
At the end of both the administration period and the recovery period, there was nothing abnormal noted in any of the groups.

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 blood chemistry 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 a increase in the relative weight of the epididymis of males in the 8 mg/kg group when compared to the control group but no effect on the absolute weight. 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 mesogastrium 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 jejunum 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.
A thickening of the proventriculus was noted in 2 males in the 8 mg/kg group at the end of the recovery period.

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.
A thickening of the proventriculus was noted in 1 female in the 8 mg/kg group at the end of the recovery period.

HISTOPATHOLOGY: NON-NEOPLASTIC
(1) Males at the End of the Administration Period
Nothing abnormal was noted in the testes.
In the epididymis, visual observation noted nodules on the right end section on one animal in the 8 mg/kg group, and spermatic granuloma was observed on the right end section but there was no difference in the frequency of occurrence and the degree when compared to the control group.
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. These results indicated papillary hyperplasia of the squamous epithelium in the proventriculus (Photo 1, 2) on all animals in the 2 and 8 mg/kg group, and were significantly higher in frequency of occurrence and degree when compared to the control group. Also, the extent of changes in the proventriculus in the 8 mg/kg group exhibited a strong trend when compared to the 2 mg/kg group. There was nothing abnormal noted in the proventriculus in the 0.5 mg/kg group. The necropsy findings confirmed white nodules in the proventriculus in one animal in the 2 mg/kg group but the results of the histopathological examination did not exhibit visual findings that would suggest abnormalities.
All animals in the control group and the 8 mg/kg group exhibited fatty changes around the portal vein in the liver, but differences in the degree were not noted with either group. Also, small granulomas were noted in the control group and the 8 mg/kg group but differences in the degree were not noted with either group. Furthermore, visual inspection revealed renal edema in the 0.5 and 2 mg/kg groups as well as fatty changes around the portal vein and small granulomas.
Three animals in the control group and one animal in the 8 mg/kg group exhibited acidophilic bodies in the cortical proximal renal tubules, and four animals in the control group and one animal in the 8 mg/kg group exhibited the expression of cortical basic renal tubules but all of these were minor changes and differences in the frequency and degree with the control group were not noted. Furthermore, visual examination revealed one animal in the control group with cortical depressions, along with limited saturation of the lymph nodes.
Additionally, all of the animals in the control group and the 8 mg/kg group exhibited blood production outside the marrow and brown residue in the spleen, along with saturation of the neutrophil and lymph nodes in the epithelium of the prostate but differences in the degree between the groups was not noted. Also, in the control group and 8 mg/kg group, aggregate foamy cells and mineral deposits in the artery walls were observed in the alveolar cavity. Changes and fibrosis of the cardiac muscle in the heart were observed, along with saturation of the lymph nodes in the mucous membranes of the bladder. One animal in the 8 mg/kg group exhibited star shaped glioma in the brain but none of these changes exhibited differences in the frequency of occurrence and degree with the control group. One animal in the 0.5 mg/kg group exhibited diverticulum of the jejune, and the one animal in the 8 mg/kg group that had edema of the submandibular lymph nodes did not exhibit abnormal findings in the submandibular joint.
No abnormal findings were noted in the other organs subject to histopathological examination.

(2) Females at the End of the Administration Period
No abnormal findings were noted 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. These results indicated papillary hyperplasia of the squamous epithelium in the proventriculus on all animals in the 2 and 8 mg/kg group, and were significantly higher in frequency of occurrence and degree when compared to the control group. Also, the extent of changes in the proventriculus in the 8 mg/kg group exhibited a strong trend when compared to the 2 mg/kg group.
All animals in the control group and the 8 mg/kg group exhibited fatty changes around the portal vein in the liver, but differences in the degree were not noted with either group. Also, small granulomas and limited intracapsular necrosis were noted in one animal in the control group.
Two animals in the control group and one animal in the 8 mg/kg group exhibited the expression of cortical basic renal tubules. Mineral deposits were noted in two animals in the control group and one animal in the 8 mg/kg group. Two animals in the 8 mg/kg group exhibited changes in the cortical proximal renal tubules. However, the frequency of occurrence and degree of these renal changes did not vary from that of the control group. Also, in addition to the moderate basic renal tubule on the one animal in the 8 mg/kg group that exhibited rough kidney surfaces during the visual examination, deposit of fatty droplets and papillary hyperplasia of the squamous epithelium was observed in the proximal renal tubules to a very mild or mild degree, and hyaline droplets were noted in the papillary area.
All of the animals in the control group and the 8 mg/kg group exhibited blood production outside the marrow and brown residue in the spleen, but there was no difference in the degree between either group. Additionally, the one animal in the 8 mg/kg group that exhibited contraction of the spleen during visual examination also had a reduction in the red pulp.
Atrophy of the thymus was noted in one animal in the control group and two animals in the 8 mg/kg group but there were no differences in the frequency of occurrence or degree between the two groups. Also, in one of the animals in both the control group and the 8 mg/kg group where contraction of the thymus was noted during visual examination, the atrophy was severe in the animal in the control group.
Aggregate foamy cells and mineral deposits in the artery walls were observed in the alveolar cavity in the control group and 8 mg/kg group but there were no differences in the frequency of occurrence or degree between the two groups.
No abnormal findings were noted in the other organs subject to histopathological examination.

(3) Males and Females at the End of the Recovery Period
Papillary hyperplasia of the squamous epithelium in the proventriculus was noted on all animals in the 8 mg/kg group, and this change was significantly higher in frequency of occurrence and degree when compared to the control group. When compared to the animals sacrificed at the end of the administration period, the changes exhibited a reducing trend.
No abnormal findings were noted in the testes.
Relative to the epididymis, saturation in the lymph nodes was noted in one animal each in the control group and the 8 mg/kg group but there were no differences in the frequency of occurrence or degree between the two groups.
No abnormal findings were noted in the ovaries.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

8 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: decrease of the body weight gain and the food consumption, and reversible effects on some haematological parameters in males, which could be considered as secondary to a severe (partially reversible) gastric irritation

2

Dose descriptor:

NOEL

Remarks:

gastric irritation

Effect level:

0.5 mg/kg bw/day (actual dose received)

Sex:

male

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Relative to repeated dose toxicity, suppression of body weight gain and reduction in food consumption was noted in males in the 8 mg/kg group, and in the groups administered 2 mg/kg and higher, since salivation and organic changes of the proventriculus attributed to the irritation of the MITC.
The NOAEL value was 8 mg/kg bw for systemic toxicity and the NOEL was 0.5 mg/kg bw for local (gastric) irritation.

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. Two satellite groups of five un-mated females were administered dose levels of 0 and 8 mg/kg/day and were not subject to any administration for 14 days after 42 days of administration. After 42 days of treatment, five males from the 0 and 8 mg/kg/day groups were keep without treatment for a 14 -day treatment-free period. There were no mortalities or moribund animals in any of the groups. Changes attributed to methylisothiocynate administration included observation of transient salivation after administration in one male in the 2 mg/kg administration group and both sexes in the 8 mg/kg group, and suppression of body weight gain and reduction in food consumption was noted in males in the 8 mg/kg group. The hematological examination conducted at the end of the administration period revealed slight increases in the red blood cells and hemoglobin among males in the 8 mg/kg group. At necropsy, macroscopic examination revealed a thickening or edema of the proventriculus in both sexes in the 2 and 8 mg/kg group, and histologically, papillary hyperplasia of the squamous epithelium in the proventriculus was noted. During the 14 day period without administration, a significant increase in the body weights of the 8 mg/kg group compared to the control group was noted. No differences with the control group were noted for red blood cell count or hemoglobin. On the other hand, the histologicalc changes of the proventriculus showed a decreasing trend when compared to the end of the administration period, and complete recovery was not achieved. Additionally, during the blood chemical examination and based on the results of the organ weights, changes deemed to be the result of methylisothiocyanate administration were not noted. Furthermore, there was nothing abnormal noted in the detailed observation of symptoms or examination of functions, and there were no findings that suggest nerve toxicity. From the aforementioned results, the NOAEL for methylisothiocyanate was 8 mg/kg/day for systemic toxicity, based on a decrease of the body weight gain and the food consumption, and reversible effects on some haematological parameters in males, which could be considered as secondary to a severe (partially reversible) gastric irritation and the NOEL for gastric irritation was 0.5 mg/kg/day.