Methyl isothiocyanate

Endpoint summary

Description of key information

In a combined oral repeated dose and reproduction / developmental screening test (OECD 422) in rats, the NOAEL for methyl isothiocynate was 8 mg/kg bw/d for sytemic toxicity.
In a 4-week inhalation rat study (OECD 412), at doses of 100 mg methyl isothiocyanate/m3, lung weight was increased, and was associated with bronchopneumonia and epithelial proliferation in bronchi and bronchiole. At this dose level, proliferation in tracheal epithelial cells and inflammatory changes in the nasal cavity and atrophy of the olfactory epithelium as well as focal metaplasia of squamous epithelial cells in the area of respiratory epithelium were reported. The NOAEC for respiratory tract irritation and for systemic effects was 19.9 mg/m3.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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)

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 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

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

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%).

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.

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

Dose descriptor:

NOEL

Remarks:

gastric irritation

Effect level:

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

Sex:

male

Critical effects observed:

not specified

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

8 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Takashima's study is reliable and performed in accordance with the OECD guidelines.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

(food consumption was not monitored)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. K. Thomae GmbH, D-7950 Biberach, FRG
- Age at study initiation: 8 weeks
- Weight at study initiation:
Test group 0: 280.2 ± 5.8 (males), 170.0 ±2.4 (females);
Test group 1: 278 ± 13.6 (males), 170.6 ± 6.8 (females);
Test group 2: 274.9 ± 6.0 (males), 171.3 ± 6.9 (females);
Test group 3: 280.2 ± 9.7 (males), 173.2 ± 4.5 (females).
- Fasting period before study: no
- Housing: during the non-exposure period: singly in type D III wire mesh cages supplied by Becker, Castrop-Rauxel, FRG; during the exposure period: singly in wire mesh cages in the inhalation chambers.
- Diet (e.g. ad libitum): during the exposure free period: Kliba Laboratory diet, rat/mouse maintenance Kliba 24-343-4, 10 mm pellets, Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland; The feed used was from batche: Nos. Ba 33-85 and Ba 34-85.
- Water (e.g. ad libitum): during the exposure free period: tape water ad libitum
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: nitrogen/air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation chamber: glass/steel construction) with a volume of about 500 liters.
- Method of holding animals in test chamber: the rats in the test groups were housed singly in wire mesh cages in the inhalation chamber.
- Source and rate of air: supply air about 8000 L/h, Nitrogen/air quantity ratios for the thermostatic water bath were set in average at: 0 L/h (Test group 0), 0.28 L/h (Test group 1), 1.91 L/h (Test group 2) and 7.98 L/h (Test group 3).
- Method of conditioning air: Methyl isothiocyanate was contained within a fritted glass flask placed in a thermostatic water bath. A stream of metered nitrogen took up the vapors and together with a stream of supply air, was introduced into a whole body exposure system (inhalation chamber: glass/steel construction) with a volume of about 500 liters after passing through a thermostatic mixing device arranged upstream. The inhalation system was also connected to an exhaust air system.
- System of generating particulates/aerosols: not applicable
- Temperature, humidity, pressure in air chamber: 22°C, 50% humidity, and a pressure below atmospheric of -2 Pa in chambers 1+2 (Test groups 1+2), and of -5 Pa in chamber 3 (Test group 3) and an excess pressure of 1 or 2 Pa in the chamber 0 (Test group 0).
The temperature and all air flows, as well as the supply air and exhaust air, were continuously measured for all test groups (digital thermometer, rotameter) and recorded 3 times during each exposure.
The pressure in the chambers was measured continuously (inclined-tube manometer) and recorded once daily. The humidity in the chamber was checked twice during each exposure by means of a humidity measuring probe (Vaisala) and was recorded.
- Air flow rate of the sample taken for gas chromatographic analysis: 1.5 L/min
- Air change rate: not reported
- Method of particle size determination: not applicable
- Treatment of exhaust air:
- Test group 0: The exhaust air system was set by about 200 L/h lower as compared to the supply air system (excess pressure) (7800 L/h). This ensured that no laboratory air could reach the control animals.
- Test groups 1-3: The exhaust air system was set by 100 and 300 L/h higher as compared to the supply air system (pressure below atmospheric) (8300 L/h (Test group 1+2) and 8110 L/h (Test group 3)) . This was to ensure that no contamination of the laboratory occurred as the result of possible leakages from the inhalation chambers.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations for the test groups were measured after absorption of methyl isothiocyanate in 2-propanol gas chromatographically.
- Samples taken from breathing zone: yes, 6 vapor samples per concentration group were taken from the breathing zones of the animals daily.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sampling from inhalation chambers and gas chromatographic analysis:
Equipment for vapor sampling:
- Elster experimental gas meter, converted for counting impulses
- Neuberger diaphragm pump
- BASF impulse counter with automatic pump switching
- Absorption set (glass)
- Sampling probe: 4 mm diameter
The flow rate of the sample was 1.5 L/min.

The samples were collected in 2 absorption vessels connected in series with a downstream fritted glass flask, each of which being filled with 15 - 20 ml 2-propanol.
The following sample volumes were drawn from the inhalation chambers: 50 L (Test group 1), 15 L (Test group 2), and 5 L (Test group 3).
0.5 ml internal standard was added to the samples and filled with 2-propanol in 50-ml graduated flasks up to the calibration mark. Then 2 ml of the samples were filled in tubes for the automatic sampler and analyzed. The volume injected was 2.5 µL.
Methyl isothiocyanate quantities were calculated from the corresponding area integral values, which, under conversion of the sample volumes, yielded the methyl isothiocyanate concentrations for test groups 1 - 3.
Concentration measured were: 5.1 ± 0.53, 19.9 ± 1.27, and 100.0 ± 5.33

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6hr/day, 5d/wk

Remarks:

Doses / Concentrations:
5, 20 and 100 mg/m3
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1.7, 6.8 and 34 ppm
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
5.1, 19.9 and 100 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

5 rats/sex/dose

Control animals:

yes

Details on study design:

- Dose selection rationale: 1)The highest concentration should be in a range that would make it possible to recognize any toxic effects (toxic profile of action). Within the frame of a 1-week preliminary study (Proj. No. 3010231/8113) only minimal toxic effects were observed at 30 mg/m³ [body weight gain unaffected; clinicochem. parameters and hematology unaffected; pathologic findings in the lungs: pinhead-sized focuses (activated alveolar epithelium; alveoli filled with macrophages)]. The highest selected concentration was therefore 100 mg/m³.
2) The intermediate concentration was selected in such a way that concentration-response relationships could be recognized. The concentration selected was 20 mg/m³.
3) The low concentration should present a NOEL. The concentration selected was 5 mg/m³.
- Rationale for animal assignment: the animals were randomized on a weight basis (randomization program WTALOC of Instern). Groups of the same weight were obtained.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: several times each working day and once workdaily on exposure free days.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: several times each working day and once workdaily on exposure free days.


BODY WEIGHT: Yes
- Time schedule for examinations: The animals' body weight was checked at the beginning of preflow and of the exposure periods, thereafter once weekly, and at the end of exposure. In general, the weighings were carried out at the same time of day.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning between 07.00 and 12.00 hours
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 5 animals per test group and sex
- Parameters checked in table [1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning between 07.00 and 12.00 hours
- Animals fasted: No
- How many animals: 5 animals per test group and sex
- Parameters checked in table [No.1] were examined.

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No

OTHER EXAMINATIONS:
- The activities of enzymes:
- glutamate pyruvate transaminase (ALAT);
- alkaline phosphatase (AP);
- glutamate oxalacetate transaminase (AST).
- Clotting analysis: - thromboplastin time (Hepato Quick's test)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)

Other examinations:

no data

Statistics:

The data were evaluated statistically using the computer systems of the Department of Toxicology (responsible: Dr. Hoffmann, ZNT) or in the Computer Center of BASF Aktiengesellschaft (responsible: Mr. Helmstadter, ZLI/NI).
Clinical examinations:
For the statistical evaluation of the study means and standard deviations were calculated for the variables (body weight and body weight change) of the animals in each test group, and printed in the form of tables together with the individual values (body weight). Statistical analysis was carried out using the ANOVA test and Dunnett's test.
Blood and plasma examinations: Means and standard errors were calculated and tabulated together with the individual values.
In order to test significances, the individual dose groups were compared with the control group using the t- test.
Pathology: In the statistical evaluation of the study, means and standard deviation (of the individual values) were calculated for the variable (absolute organ weights) of the animals of each test group and were tabulated together with the individual values.
The statistical evaluation was carried out using a t test generalized by WILLIAMS for the simultaneous comparison of several dose groups with a control group.

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):

not examined

Food efficiency:

not examined

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:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No deaths occurred during the study.
Clinical observations at the high dose revealed marked mucous membrane and respiratory tract irritation (reedish nasal discharge, salivation, eye discharge) resulting in a change in breathing pattern and whooping respiration. Intensified cleaning and stretched posture were also noted. As the study progressed, certain signs (ruffled fur and respiratory sounds) ceased being reversible. less severe signs (eyelid closure, somnolence, and ruffled fur) were noted at the mid dose from day 3 onward. However, unlike the high dose animals, mid dose animals began to recover following the completion of each day's dosing. incidence rates for these clinical signs were not reported.

BODY WEIGHT AND WEIGHT GAIN
High dose males suffered a substantial weight loss during the first week (-19.2g, compared to a gain of 23-5g in control males, p<0.01). High dose females gained less weight than control during the first week (1.1g vs. 13.1 in controls), though statistical significance was not etablished. These differences were maintained throughout the study in both sexes. High dose male body weights on days7, 14, 21 and 27 were lower than controls by statistically significant margins. No convincing weight differences were observed at either of the two lower doses in either sex.
There were no measurements of food consumption, making it difficult to determine the reasons for the body weight effects at the high dose.

HAEMATOLOGY
Mid and high dose males exhibited increased numbers of neutrophilic polymorphonuclear granulocytes. The mean concentration in giga/L at increasing doses was 0.43±0.05, 0.48±0.07, 0.69±0.07*, and 1.42±0.18** (*p<0.05, **p<0.01, Student’s t test on logtransformed data [Dunnett’s parametric t test was positive at the high dose only]). Females showed this increase only at the high dose, with mean concentrations in giga/L at increasing doses of 0.52±0.09, 0.52±0.10, 0.55±0.08, and 1.44±0.28**. Overall leukocyte counts were also increased in high dose females (mean concentration in giga/L: 5.19, 4.72, 4.54, 6.73). These changes were considered related to inflammatory processes occurring in the respiratory tract.

CLINICAL CHEMISTRY
Clinical chemistry revealed statistically significant decreases in serum urea (22%), glucose (17%), triglyceride (58%), and albumin (9%) in high dose males. The urea and glucose concentrations in high dose females were likewise statistically depressed (30% and 14% respectively). Total bilirubin concentrations and thromboplastin time (a measure of blood clotting ability) were markedly increased in high dose males (92% and 16% respectively), though not in females. These changes were difficul to ascribe to a particular clinical picture. Smaller changes in urea (-17%), glucose (-5%), bilirubin (+41%), and thromboplastin time (+14%) were also present mid dose males, though these did not achieve statistical significance.

ORGAN WEIGHTS
Organ weight determinations showed statistically significant decreases in liver (18%) and kidney (17%) weights and an increase in lung (70%) weight among high dose males. Females showed only the increase in lung weight (83%).

GROSS PATHOLOGY
Gross pathology showed the lungs of all high dose males and three females to be pale, rigid, and of a puffy consistency.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathology revealed an increase in incidence and severity of rhinitis in the nasal cavity at the high dose in both sexes (incidence in males: 2/5, 2/5, 2/5, 5/5; females: 0/5, 3/5, 1/5, 5/5). Other histopathologic findings at the high dose included: metaplasia of the nasal epithelium, tracheal epithelial proliferation and single cell necrosis (all high dose animals), bronchopneumonia and bronchial and bronchiolar epithelial proliferation (5 males, 2 females), and emphysema (3 males, 2 females). One finding, nasal epithelial atrophy, may have been induced at the low dose. This was based on increases in focal atrophy at that dose, and increases in total atrophy. The latter was evident both in the number of animals exhibiting focal + non-focal atrophy and in the fraction of section planes, particularly among females, showing atrophy. The absence of a further increase at the mid dose may be due in part to a process by which focal atrophy at lower doses was replaced by “non-focal” atrophy, considered to be a more general (i.e., diffuse) and potentially more serious lesion, at higher doses. Such a process was very clear at the high dose, where virtually all reported atrophy was non-focal.

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

19.9 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: increased neutrophilic polymorphonuclear granulocytes in the blood of males secondary to the respiratory tract irritation

Dose descriptor:

LOAEC

Remarks:

systemic toxicity

Effect level:

100 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: lower body weight gain

Dose descriptor:

NOAEC

Remarks:

extrathoracic (ET) region

Effect level:

19.9 mg/m³ air (analytical)

Sex:

male/female

Dose descriptor:

LOAEC

Remarks:

extrathoracic (ET) region

Effect level:

100 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: pathological changes of the nasal cavity (metaplasia of respiratory epithelium and atrophy of the olfactory epithelium)

Dose descriptor:

NOAEC

Remarks:

tracheabronchial (TB) region

Effect level:

19.9 mg/m³ air (analytical)

Sex:

male/female

Dose descriptor:

LOAEC

Remarks:

tracheabronchial (TB) region

Effect level:

100 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: pathological changes (tracheal epithelial proliferation and single cell necrosis, bronchopneumonia and bronchial and bronchiolar epithelial proliferation)

Critical effects observed:

not specified

Table 3:Mean Body Weights and Body Weight Gains

	Concentration (mg/m^3)
Treatment day	0	5	20	100
	Mean body weights (g)
Males
-7	280.2	278.0	274.9	280.2
0	307.9	311.0	302.9	310.7
7	331.4	335.8	322.3	291.5**
14	350.5	356.6	340.7	288.7**
21	370.8	378.3	358.8	282.6**
27	387.6	394.6	375.7	295.8**
Weight gain (g)
Day 7	23.5	24.7	19.3	-19.2**
Day 14	42.6	45.6	37.8	-22.0**
Day 21	62.9	67.2	55.8	-28.2**
Day 27	79.7	83.6	72.7	-14.9**
Females
-7	170.0	170.6	171.3	173.2
0	184.9	183.4	185.3	184.8
7	198.0	198.9	198.8	185.9
14	206.4	207.0	207.0	195.4
21	209.0	216.0	218.2	201.4
27	220.6	223.7	226.0	207.1
Weight gain (g)
Day 7	13.1	15.5	13.5	1.1
Day 14	21.5	23.6	21.7	10.7
Day 21	24.1	32.6	32.9	16.6
Day 27	35.7	40.3	40.7	22.4

Table 4 :Hematology and Clinical Chemistry

	Concentration (mg/m^3)
Parameter	0	5	20	100
Males
Thromboplastin time (sec)	35.900	36.580	40.780	41.840*
Thrombocytes (10^9/L)	983.6	934.0	1006.4	1034.4
Leucocytes (10^9/L)	7.568	8.106	6.360	7.188
Neutrophilic polymorphonuclear granulocytes (10^9/L)	0.43	0.48	0.69	1.42
Lymphocytes (10^9/L)	6.64	6.93	5.08	5.16
Creatinine (µmol/L)	64.592	61.288	52.583*	55.421
Potassium (mmol/L)	6.303	5.464**	5.823	5.834
Inorganic phosphate (mmol/L)	2.791	2.415**	2.581	2.479
Total bilirubin (mmol/L)	1.302	1.358	1.833	2.501**
Urea (mmol/L)	8.448	7.906	7.019	6.641*
Glucose (mmol/L)	7.802	7.681	7.437	6.493**
Triglycerides (mmol/L)	3.248	2.718	3.139	1.351**
Albumin (g/L)	39.880	40.200	37.900	36.520*
ALT (µkat/L)	1.216	1.100	1.070	1.130
AP (µkat/L)	8.086	8.054	8.074	8.002
Females
Thromboplastin time (sec)	39.980	38.140	35.760	41.140
Thrombocytes (10^9/L)	1012.4	1012.75	1060.8	1065.0
Leucocytes (10^9/L)	5.192	4.722	4.544	6.732**
Neutrophilic polymorphonuclear granulocytes (10^9/L)	0.52	0.52	0.55	1.44
Lymphocytes (10^9/L)	4.23	3.89	3.62	4.60
Creatinine (µmol/L)	62.528	59.507	65.013	53.171
Potassium (mmol/L)	5.778	6.085	5.741	5.427
Inorganic phosphate (mmol/L)	1.997	2.267	2.229	2.178
Total bilirubin (mmol/L)	2.142	1.709	1.724	1.789
Urea (mmol/L)	8.744	8.580	8.107	6.143*
Glucose (mmol/L)	7.094	7.387	7.115	6.079*
Triglycerides (mmol/L)	2.042	2.083	2.079	1.129
Albumin (g/L)	42.040	40.440	41.300	36.950
ALT (µkat/L)	0.872	1.000	0.920	1.104*
AP (µkat/L)	5.318	7.282*	5.940	6.958**

Table 5:Absolute Organ Weights

Body / Organ weights (g)	Concentration (mg/m^3)
	0	5	20	100
Males
Body weight day 27	387.6	394.6	375.7	295.8**
Kidneys	2.328	2.274	2.172	1.944**
Liver	12.028	11.734	10.694	8.882**
Lungs	0.962	1.028	0.930	1.650**
Females
Body weight day 27	220.6	223.7	226.0	207.1
Kidneys	1.414	1.448	1.348	1.366
Liver	6.458	6.506	6.564	6.524
Lungs	0.624	0.706	0.686	1.062*

Table 6:Histopathology

	Concentration (mg/m^3)
Findings	0		5		20		100
	M	F	M	F	M	F	M	F
Lungs
emphysema	0/5^1)	0/5	0/5	0/5	0/5	0/5	3/5	2/5
bronchopneumonia	0/5	0/5	0/5	0/5	0/5	0/5	5/5	2/5
bone metaplasia	1/5	0/5	0/5	1/5	0/2	1/5	3/5	0/5
epithelial proliferation	0/5	0/5	0/5	0/5	0/5	0/5	5/5	2/5
Trachea
inflammation	0/5	0/5	0/5	0/5	0/5	0/5	1/5	1/5
epithelial proliferation	0/5	0/5	0/5	0/5	0/5	0/5	5/5	5/5
single cell necrosis	0/5	0/5	0/5	0/5	0/5	0/5	5/5	5/5
Nasal cavity
rhinitis	2/5	0/5	1/5	2/5	1/5	1/5	5/5	5/5
focal atrophy	1/5	1/5	2/5	3/5	2/5	1/5	1/5	1/5
atrophy	2/5	0/5	1/5	2/5	2/5	2/5	5/5	5/5
metaplasia	0/5	0/5	0/5	0/5	0/5	0/5	3/5	5/5

*:            significantly different from control (p < 0.05);**:  significantly different from control (p < 0.01)

Conclusions:

Methylisothiocyanate caused nasal epithelial atrophy at the low dose. At the middle dose, this atrophy was seen in addition to clinical signs in both sexes andan increase in neutrophilic polymorphonuclear granulocytes in males. The latter sign, which also occurred in females at the high dose, was considered to reflect an inflammatory process in the lungs. Such a process was clearly evident at the high dose, where increased lung weight and severe histopathologic signs were observed.

Executive summary:

In a 28 day inhalation toxicity study (OECD 421), methyl isothiocyanate [96.9 % a. i. ] was administered to 5/sex/dose of SPF Wistar/Chubb: THOM rats by whole body exposure at analytical concentrations of 0, 5.0, 20, or 100 mg/m3 (measured concentrations 0, 5.1, 19.9 or 100 mg/m3) and (equivalent to concentrations of 0, 1.7, 6.8, and 34 ppm) for 6 hours per day, 5 days/week for a total of 28 days (Klimisch et al., 1987; Rubin, 2002; Lowit and Facey, 2005; Lowit, 2006). All animals survived to study termination. Mid and high dose rats demonstrated clinical signs during exposure from the third exposure period day. No clinical signs were observed in the low dose animals. According to the study report, “During exposure, the animals of test group 2 showed eyelid closure, somnolence, and ruffled fur from the third day of exposure onwards. On the next morning before exposure nothing abnormal was found in the animals. At 20 mg/m3 the animals showed first indications of an irritating effect of the test substance and a slightly deteriorated general state of health. ” Additional clinical signs observed at the high exposure concentration included reddish nasal discharge, salivation, eye discharge, and difficulty in breathing or whooping respiration, and stretched posture. In the high dose rats, although signs recovered between exposures at the beginning of the study, towards the end of the study ruffled fur and respiratory sounds were no longer reversible. Body weight and body weight gain were significantly decreased (p<0.05) at the high dose. Food consumption and feed efficiency were not measured. There were decreases in plasma urea, glucose, triglyceride, and albumin the high dose males. In high dose females, urea and glucose were also decreased. In the males of mid exposure group, there was a decrease in urea concentration in the plasma. At the mid and high exposure concentrations, increase in neutrophilic polymorphonuclear granulocytes in the peripheral blood was observed in males; this was also observed in the high exposure concentration for females. There was increased lung weight at the high exposure concentration. Histopathology revealed an increase in incidence and severity of rhinitis in the nasal cavity at the high exposure concentration in both sexes (incidence in males: 2/5, 2/5, 2/5, 5/5; females: 0/5, 3/5, 1/5, 5/5). Other histopathologic findings at the high exposure concentration included: atrophy of the olfactory epithelium, metaplasia of the nasal respiratory epithelium (3 males in section plane 1 only, 5 females in section planes 1 and, to a lesser extent, section plane 2), tracheal epithelial proliferation and single cell necrosis (all high exposure concentration), bronchopneumonia and bronchial and bronchiolar epithelial proliferation (5 males, 2 females), and emphysema (3 males, 2 females). The systemic LOAEL is 100 mg/m3 (34 ppm), based on effects on the body weight gain. The systemic NOAEL is 19.9 mg/m3 (6.8 ppm ) based on an increase of neutrophilic polymorphonuclear granulocytes in the blood of males, which was not observed the oral OECD 422 and 90-day inhalation studies, and which could be considered as a secondary effect of the respiratory tract inflammation. The LOAEL for effects in the extrathoracic (ET) region is 100 mg/m3 (34 ppm), based on observation of pathological changes of the nasal cavity (metaplasia of respiratory epithelium and atrophy of the olfactory epithelium). The ET NOAEL is 19.9 mg/m3 (6.8 ppm). The LOAEL for effects in the tracheabronchial (TB) region is 100 mg/m3 (34 ppm), based on observation of pathological changes (tracheal epithelial proliferation and single cell necrosis, bronchopneumonia and bronchial and bronchiolar epithelial proliferation). The TB NOAEL is 19.9 mg/m3 (6.8 ppm).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

19.9 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch's study is reliable and performed in accordance with the OECD guidelines.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

(food consumption was not monitored)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. K. Thomae GmbH, D-7950 Biberach, FRG
- Age at study initiation: 8 weeks
- Weight at study initiation:
Test group 0: 280.2 ± 5.8 (males), 170.0 ±2.4 (females);
Test group 1: 278 ± 13.6 (males), 170.6 ± 6.8 (females);
Test group 2: 274.9 ± 6.0 (males), 171.3 ± 6.9 (females);
Test group 3: 280.2 ± 9.7 (males), 173.2 ± 4.5 (females).
- Fasting period before study: no
- Housing: during the non-exposure period: singly in type D III wire mesh cages supplied by Becker, Castrop-Rauxel, FRG; during the exposure period: singly in wire mesh cages in the inhalation chambers.
- Diet (e.g. ad libitum): during the exposure free period: Kliba Laboratory diet, rat/mouse maintenance Kliba 24-343-4, 10 mm pellets, Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland; The feed used was from batche: Nos. Ba 33-85 and Ba 34-85.
- Water (e.g. ad libitum): during the exposure free period: tape water ad libitum
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: nitrogen/air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation chamber: glass/steel construction) with a volume of about 500 liters.
- Method of holding animals in test chamber: the rats in the test groups were housed singly in wire mesh cages in the inhalation chamber.
- Source and rate of air: supply air about 8000 L/h, Nitrogen/air quantity ratios for the thermostatic water bath were set in average at: 0 L/h (Test group 0), 0.28 L/h (Test group 1), 1.91 L/h (Test group 2) and 7.98 L/h (Test group 3).
- Method of conditioning air: Methyl isothiocyanate was contained within a fritted glass flask placed in a thermostatic water bath. A stream of metered nitrogen took up the vapors and together with a stream of supply air, was introduced into a whole body exposure system (inhalation chamber: glass/steel construction) with a volume of about 500 liters after passing through a thermostatic mixing device arranged upstream. The inhalation system was also connected to an exhaust air system.
- System of generating particulates/aerosols: not applicable
- Temperature, humidity, pressure in air chamber: 22°C, 50% humidity, and a pressure below atmospheric of -2 Pa in chambers 1+2 (Test groups 1+2), and of -5 Pa in chamber 3 (Test group 3) and an excess pressure of 1 or 2 Pa in the chamber 0 (Test group 0).
The temperature and all air flows, as well as the supply air and exhaust air, were continuously measured for all test groups (digital thermometer, rotameter) and recorded 3 times during each exposure.
The pressure in the chambers was measured continuously (inclined-tube manometer) and recorded once daily. The humidity in the chamber was checked twice during each exposure by means of a humidity measuring probe (Vaisala) and was recorded.
- Air flow rate of the sample taken for gas chromatographic analysis: 1.5 L/min
- Air change rate: not reported
- Method of particle size determination: not applicable
- Treatment of exhaust air:
- Test group 0: The exhaust air system was set by about 200 L/h lower as compared to the supply air system (excess pressure) (7800 L/h). This ensured that no laboratory air could reach the control animals.
- Test groups 1-3: The exhaust air system was set by 100 and 300 L/h higher as compared to the supply air system (pressure below atmospheric) (8300 L/h (Test group 1+2) and 8110 L/h (Test group 3)) . This was to ensure that no contamination of the laboratory occurred as the result of possible leakages from the inhalation chambers.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations for the test groups were measured after absorption of methyl isothiocyanate in 2-propanol gas chromatographically.
- Samples taken from breathing zone: yes, 6 vapor samples per concentration group were taken from the breathing zones of the animals daily.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sampling from inhalation chambers and gas chromatographic analysis:
Equipment for vapor sampling:
- Elster experimental gas meter, converted for counting impulses
- Neuberger diaphragm pump
- BASF impulse counter with automatic pump switching
- Absorption set (glass)
- Sampling probe: 4 mm diameter
The flow rate of the sample was 1.5 L/min.

The samples were collected in 2 absorption vessels connected in series with a downstream fritted glass flask, each of which being filled with 15 - 20 ml 2-propanol.
The following sample volumes were drawn from the inhalation chambers: 50 L (Test group 1), 15 L (Test group 2), and 5 L (Test group 3).
0.5 ml internal standard was added to the samples and filled with 2-propanol in 50-ml graduated flasks up to the calibration mark. Then 2 ml of the samples were filled in tubes for the automatic sampler and analyzed. The volume injected was 2.5 µL.
Methyl isothiocyanate quantities were calculated from the corresponding area integral values, which, under conversion of the sample volumes, yielded the methyl isothiocyanate concentrations for test groups 1 - 3.
Concentration measured were: 5.1 ± 0.53, 19.9 ± 1.27, and 100.0 ± 5.33

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6hr/day, 5d/wk

Remarks:

Doses / Concentrations:
5, 20 and 100 mg/m3
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1.7, 6.8 and 34 ppm
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
5.1, 19.9 and 100 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

5 rats/sex/dose

Control animals:

yes

Details on study design:

- Dose selection rationale: 1)The highest concentration should be in a range that would make it possible to recognize any toxic effects (toxic profile of action). Within the frame of a 1-week preliminary study (Proj. No. 3010231/8113) only minimal toxic effects were observed at 30 mg/m³ [body weight gain unaffected; clinicochem. parameters and hematology unaffected; pathologic findings in the lungs: pinhead-sized focuses (activated alveolar epithelium; alveoli filled with macrophages)]. The highest selected concentration was therefore 100 mg/m³.
2) The intermediate concentration was selected in such a way that concentration-response relationships could be recognized. The concentration selected was 20 mg/m³.
3) The low concentration should present a NOEL. The concentration selected was 5 mg/m³.
- Rationale for animal assignment: the animals were randomized on a weight basis (randomization program WTALOC of Instern). Groups of the same weight were obtained.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: several times each working day and once workdaily on exposure free days.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: several times each working day and once workdaily on exposure free days.


BODY WEIGHT: Yes
- Time schedule for examinations: The animals' body weight was checked at the beginning of preflow and of the exposure periods, thereafter once weekly, and at the end of exposure. In general, the weighings were carried out at the same time of day.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning between 07.00 and 12.00 hours
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 5 animals per test group and sex
- Parameters checked in table [1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning between 07.00 and 12.00 hours
- Animals fasted: No
- How many animals: 5 animals per test group and sex
- Parameters checked in table [No.1] were examined.

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No

OTHER EXAMINATIONS:
- The activities of enzymes:
- glutamate pyruvate transaminase (ALAT);
- alkaline phosphatase (AP);
- glutamate oxalacetate transaminase (AST).
- Clotting analysis: - thromboplastin time (Hepato Quick's test)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)

Other examinations:

no data

Statistics:

The data were evaluated statistically using the computer systems of the Department of Toxicology (responsible: Dr. Hoffmann, ZNT) or in the Computer Center of BASF Aktiengesellschaft (responsible: Mr. Helmstadter, ZLI/NI).
Clinical examinations:
For the statistical evaluation of the study means and standard deviations were calculated for the variables (body weight and body weight change) of the animals in each test group, and printed in the form of tables together with the individual values (body weight). Statistical analysis was carried out using the ANOVA test and Dunnett's test.
Blood and plasma examinations: Means and standard errors were calculated and tabulated together with the individual values.
In order to test significances, the individual dose groups were compared with the control group using the t- test.
Pathology: In the statistical evaluation of the study, means and standard deviation (of the individual values) were calculated for the variable (absolute organ weights) of the animals of each test group and were tabulated together with the individual values.
The statistical evaluation was carried out using a t test generalized by WILLIAMS for the simultaneous comparison of several dose groups with a control group.

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):

not examined

Food efficiency:

not examined

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:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No deaths occurred during the study.
Clinical observations at the high dose revealed marked mucous membrane and respiratory tract irritation (reedish nasal discharge, salivation, eye discharge) resulting in a change in breathing pattern and whooping respiration. Intensified cleaning and stretched posture were also noted. As the study progressed, certain signs (ruffled fur and respiratory sounds) ceased being reversible. less severe signs (eyelid closure, somnolence, and ruffled fur) were noted at the mid dose from day 3 onward. However, unlike the high dose animals, mid dose animals began to recover following the completion of each day's dosing. incidence rates for these clinical signs were not reported.

BODY WEIGHT AND WEIGHT GAIN
High dose males suffered a substantial weight loss during the first week (-19.2g, compared to a gain of 23-5g in control males, p<0.01). High dose females gained less weight than control during the first week (1.1g vs. 13.1 in controls), though statistical significance was not etablished. These differences were maintained throughout the study in both sexes. High dose male body weights on days7, 14, 21 and 27 were lower than controls by statistically significant margins. No convincing weight differences were observed at either of the two lower doses in either sex.
There were no measurements of food consumption, making it difficult to determine the reasons for the body weight effects at the high dose.

HAEMATOLOGY
Mid and high dose males exhibited increased numbers of neutrophilic polymorphonuclear granulocytes. The mean concentration in giga/L at increasing doses was 0.43±0.05, 0.48±0.07, 0.69±0.07*, and 1.42±0.18** (*p<0.05, **p<0.01, Student’s t test on logtransformed data [Dunnett’s parametric t test was positive at the high dose only]). Females showed this increase only at the high dose, with mean concentrations in giga/L at increasing doses of 0.52±0.09, 0.52±0.10, 0.55±0.08, and 1.44±0.28**. Overall leukocyte counts were also increased in high dose females (mean concentration in giga/L: 5.19, 4.72, 4.54, 6.73). These changes were considered related to inflammatory processes occurring in the respiratory tract.

CLINICAL CHEMISTRY
Clinical chemistry revealed statistically significant decreases in serum urea (22%), glucose (17%), triglyceride (58%), and albumin (9%) in high dose males. The urea and glucose concentrations in high dose females were likewise statistically depressed (30% and 14% respectively). Total bilirubin concentrations and thromboplastin time (a measure of blood clotting ability) were markedly increased in high dose males (92% and 16% respectively), though not in females. These changes were difficul to ascribe to a particular clinical picture. Smaller changes in urea (-17%), glucose (-5%), bilirubin (+41%), and thromboplastin time (+14%) were also present mid dose males, though these did not achieve statistical significance.

ORGAN WEIGHTS
Organ weight determinations showed statistically significant decreases in liver (18%) and kidney (17%) weights and an increase in lung (70%) weight among high dose males. Females showed only the increase in lung weight (83%).

GROSS PATHOLOGY
Gross pathology showed the lungs of all high dose males and three females to be pale, rigid, and of a puffy consistency.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathology revealed an increase in incidence and severity of rhinitis in the nasal cavity at the high dose in both sexes (incidence in males: 2/5, 2/5, 2/5, 5/5; females: 0/5, 3/5, 1/5, 5/5). Other histopathologic findings at the high dose included: metaplasia of the nasal epithelium, tracheal epithelial proliferation and single cell necrosis (all high dose animals), bronchopneumonia and bronchial and bronchiolar epithelial proliferation (5 males, 2 females), and emphysema (3 males, 2 females). One finding, nasal epithelial atrophy, may have been induced at the low dose. This was based on increases in focal atrophy at that dose, and increases in total atrophy. The latter was evident both in the number of animals exhibiting focal + non-focal atrophy and in the fraction of section planes, particularly among females, showing atrophy. The absence of a further increase at the mid dose may be due in part to a process by which focal atrophy at lower doses was replaced by “non-focal” atrophy, considered to be a more general (i.e., diffuse) and potentially more serious lesion, at higher doses. Such a process was very clear at the high dose, where virtually all reported atrophy was non-focal.

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

19.9 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: increased neutrophilic polymorphonuclear granulocytes in the blood of males secondary to the respiratory tract irritation

Dose descriptor:

LOAEC

Remarks:

systemic toxicity

Effect level:

100 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: lower body weight gain

Dose descriptor:

NOAEC

Remarks:

extrathoracic (ET) region

Effect level:

19.9 mg/m³ air (analytical)

Sex:

male/female

Dose descriptor:

LOAEC

Remarks:

extrathoracic (ET) region

Effect level:

100 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: pathological changes of the nasal cavity (metaplasia of respiratory epithelium and atrophy of the olfactory epithelium)

Dose descriptor:

NOAEC

Remarks:

tracheabronchial (TB) region

Effect level:

19.9 mg/m³ air (analytical)

Sex:

male/female

Dose descriptor:

LOAEC

Remarks:

tracheabronchial (TB) region

Effect level:

100 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: pathological changes (tracheal epithelial proliferation and single cell necrosis, bronchopneumonia and bronchial and bronchiolar epithelial proliferation)

Critical effects observed:

not specified

Table 3:Mean Body Weights and Body Weight Gains

	Concentration (mg/m^3)
Treatment day	0	5	20	100
	Mean body weights (g)
Males
-7	280.2	278.0	274.9	280.2
0	307.9	311.0	302.9	310.7
7	331.4	335.8	322.3	291.5**
14	350.5	356.6	340.7	288.7**
21	370.8	378.3	358.8	282.6**
27	387.6	394.6	375.7	295.8**
Weight gain (g)
Day 7	23.5	24.7	19.3	-19.2**
Day 14	42.6	45.6	37.8	-22.0**
Day 21	62.9	67.2	55.8	-28.2**
Day 27	79.7	83.6	72.7	-14.9**
Females
-7	170.0	170.6	171.3	173.2
0	184.9	183.4	185.3	184.8
7	198.0	198.9	198.8	185.9
14	206.4	207.0	207.0	195.4
21	209.0	216.0	218.2	201.4
27	220.6	223.7	226.0	207.1
Weight gain (g)
Day 7	13.1	15.5	13.5	1.1
Day 14	21.5	23.6	21.7	10.7
Day 21	24.1	32.6	32.9	16.6
Day 27	35.7	40.3	40.7	22.4

Table 4 :Hematology and Clinical Chemistry

	Concentration (mg/m^3)
Parameter	0	5	20	100
Males
Thromboplastin time (sec)	35.900	36.580	40.780	41.840*
Thrombocytes (10^9/L)	983.6	934.0	1006.4	1034.4
Leucocytes (10^9/L)	7.568	8.106	6.360	7.188
Neutrophilic polymorphonuclear granulocytes (10^9/L)	0.43	0.48	0.69	1.42
Lymphocytes (10^9/L)	6.64	6.93	5.08	5.16
Creatinine (µmol/L)	64.592	61.288	52.583*	55.421
Potassium (mmol/L)	6.303	5.464**	5.823	5.834
Inorganic phosphate (mmol/L)	2.791	2.415**	2.581	2.479
Total bilirubin (mmol/L)	1.302	1.358	1.833	2.501**
Urea (mmol/L)	8.448	7.906	7.019	6.641*
Glucose (mmol/L)	7.802	7.681	7.437	6.493**
Triglycerides (mmol/L)	3.248	2.718	3.139	1.351**
Albumin (g/L)	39.880	40.200	37.900	36.520*
ALT (µkat/L)	1.216	1.100	1.070	1.130
AP (µkat/L)	8.086	8.054	8.074	8.002
Females
Thromboplastin time (sec)	39.980	38.140	35.760	41.140
Thrombocytes (10^9/L)	1012.4	1012.75	1060.8	1065.0
Leucocytes (10^9/L)	5.192	4.722	4.544	6.732**
Neutrophilic polymorphonuclear granulocytes (10^9/L)	0.52	0.52	0.55	1.44
Lymphocytes (10^9/L)	4.23	3.89	3.62	4.60
Creatinine (µmol/L)	62.528	59.507	65.013	53.171
Potassium (mmol/L)	5.778	6.085	5.741	5.427
Inorganic phosphate (mmol/L)	1.997	2.267	2.229	2.178
Total bilirubin (mmol/L)	2.142	1.709	1.724	1.789
Urea (mmol/L)	8.744	8.580	8.107	6.143*
Glucose (mmol/L)	7.094	7.387	7.115	6.079*
Triglycerides (mmol/L)	2.042	2.083	2.079	1.129
Albumin (g/L)	42.040	40.440	41.300	36.950
ALT (µkat/L)	0.872	1.000	0.920	1.104*
AP (µkat/L)	5.318	7.282*	5.940	6.958**

Table 5:Absolute Organ Weights

Body / Organ weights (g)	Concentration (mg/m^3)
	0	5	20	100
Males
Body weight day 27	387.6	394.6	375.7	295.8**
Kidneys	2.328	2.274	2.172	1.944**
Liver	12.028	11.734	10.694	8.882**
Lungs	0.962	1.028	0.930	1.650**
Females
Body weight day 27	220.6	223.7	226.0	207.1
Kidneys	1.414	1.448	1.348	1.366
Liver	6.458	6.506	6.564	6.524
Lungs	0.624	0.706	0.686	1.062*

Table 6:Histopathology

	Concentration (mg/m^3)
Findings	0		5		20		100
	M	F	M	F	M	F	M	F
Lungs
emphysema	0/5^1)	0/5	0/5	0/5	0/5	0/5	3/5	2/5
bronchopneumonia	0/5	0/5	0/5	0/5	0/5	0/5	5/5	2/5
bone metaplasia	1/5	0/5	0/5	1/5	0/2	1/5	3/5	0/5
epithelial proliferation	0/5	0/5	0/5	0/5	0/5	0/5	5/5	2/5
Trachea
inflammation	0/5	0/5	0/5	0/5	0/5	0/5	1/5	1/5
epithelial proliferation	0/5	0/5	0/5	0/5	0/5	0/5	5/5	5/5
single cell necrosis	0/5	0/5	0/5	0/5	0/5	0/5	5/5	5/5
Nasal cavity
rhinitis	2/5	0/5	1/5	2/5	1/5	1/5	5/5	5/5
focal atrophy	1/5	1/5	2/5	3/5	2/5	1/5	1/5	1/5
atrophy	2/5	0/5	1/5	2/5	2/5	2/5	5/5	5/5
metaplasia	0/5	0/5	0/5	0/5	0/5	0/5	3/5	5/5

*:            significantly different from control (p < 0.05);**:  significantly different from control (p < 0.01)

Conclusions:

Methylisothiocyanate caused nasal epithelial atrophy at the low dose. At the middle dose, this atrophy was seen in addition to clinical signs in both sexes andan increase in neutrophilic polymorphonuclear granulocytes in males. The latter sign, which also occurred in females at the high dose, was considered to reflect an inflammatory process in the lungs. Such a process was clearly evident at the high dose, where increased lung weight and severe histopathologic signs were observed.

Executive summary:

In a 28 day inhalation toxicity study (OECD 421), methyl isothiocyanate [96.9 % a. i. ] was administered to 5/sex/dose of SPF Wistar/Chubb: THOM rats by whole body exposure at analytical concentrations of 0, 5.0, 20, or 100 mg/m3 (measured concentrations 0, 5.1, 19.9 or 100 mg/m3) and (equivalent to concentrations of 0, 1.7, 6.8, and 34 ppm) for 6 hours per day, 5 days/week for a total of 28 days (Klimisch et al., 1987; Rubin, 2002; Lowit and Facey, 2005; Lowit, 2006). All animals survived to study termination. Mid and high dose rats demonstrated clinical signs during exposure from the third exposure period day. No clinical signs were observed in the low dose animals. According to the study report, “During exposure, the animals of test group 2 showed eyelid closure, somnolence, and ruffled fur from the third day of exposure onwards. On the next morning before exposure nothing abnormal was found in the animals. At 20 mg/m3 the animals showed first indications of an irritating effect of the test substance and a slightly deteriorated general state of health. ” Additional clinical signs observed at the high exposure concentration included reddish nasal discharge, salivation, eye discharge, and difficulty in breathing or whooping respiration, and stretched posture. In the high dose rats, although signs recovered between exposures at the beginning of the study, towards the end of the study ruffled fur and respiratory sounds were no longer reversible. Body weight and body weight gain were significantly decreased (p<0.05) at the high dose. Food consumption and feed efficiency were not measured. There were decreases in plasma urea, glucose, triglyceride, and albumin the high dose males. In high dose females, urea and glucose were also decreased. In the males of mid exposure group, there was a decrease in urea concentration in the plasma. At the mid and high exposure concentrations, increase in neutrophilic polymorphonuclear granulocytes in the peripheral blood was observed in males; this was also observed in the high exposure concentration for females. There was increased lung weight at the high exposure concentration. Histopathology revealed an increase in incidence and severity of rhinitis in the nasal cavity at the high exposure concentration in both sexes (incidence in males: 2/5, 2/5, 2/5, 5/5; females: 0/5, 3/5, 1/5, 5/5). Other histopathologic findings at the high exposure concentration included: atrophy of the olfactory epithelium, metaplasia of the nasal respiratory epithelium (3 males in section plane 1 only, 5 females in section planes 1 and, to a lesser extent, section plane 2), tracheal epithelial proliferation and single cell necrosis (all high exposure concentration), bronchopneumonia and bronchial and bronchiolar epithelial proliferation (5 males, 2 females), and emphysema (3 males, 2 females). The systemic LOAEL is 100 mg/m3 (34 ppm), based on effects on the body weight gain. The systemic NOAEL is 19.9 mg/m3 (6.8 ppm ) based on an increase of neutrophilic polymorphonuclear granulocytes in the blood of males, which was not observed the oral OECD 422 and 90-day inhalation studies, and which could be considered as a secondary effect of the respiratory tract inflammation. The LOAEL for effects in the extrathoracic (ET) region is 100 mg/m3 (34 ppm), based on observation of pathological changes of the nasal cavity (metaplasia of respiratory epithelium and atrophy of the olfactory epithelium). The ET NOAEL is 19.9 mg/m3 (6.8 ppm). The LOAEL for effects in the tracheabronchial (TB) region is 100 mg/m3 (34 ppm), based on observation of pathological changes (tracheal epithelial proliferation and single cell necrosis, bronchopneumonia and bronchial and bronchiolar epithelial proliferation). The TB NOAEL is 19.9 mg/m3 (6.8 ppm).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

19.9 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch's study is reliable and performed in accordance with the OECD guidelines.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral route

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 methyl isothiocyanate 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 a14 -day treatment-free period. There were no mortalities or moribund animals in any of the groups. Changes attributed to methyl isothiocynate 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 histological 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 methyl isothiocyanate 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 afore mentioned results, the NOAEL for methyl isothiocyanate 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.

Dermal route

No reliable study is available.

Inhalation route

In a 28 day inhalation toxicity study (OECD 421), methyl isothiocyanate [96.9 % a. i. ] was administered to 5/sex/dose of SPF Wistar/Chubb: THOM rats by whole body exposure at analytical concentrations of 0, 5.0, 20, or 100 mg/m3 (measured concentrations 0, 5.1, 19.9 or 100 mg/m3) and (equivalent to concentrations of 0, 1.7, 6.8, and 34 ppm) for 6 hours per day, 5 days/week for a total of 28 days (Klimisch et al., 1987; Rubin, 2002; Lowit and Facey, 2005; Lowit, 2006). All animals survived to study termination. Mid and high dose rats demonstrated clinical signs during exposure from the third exposure period day. No clinical signs were observed in the low dose animals. According to the study report, “During exposure, the animals of test group 2 showed eyelid closure, somnolence, and ruffled fur from the third day of exposure onwards. On the next morning before exposure nothing abnormal was found in the animals. At 20 mg/m3 the animals showed first indications of an irritating effect of the test substance and a slightly deteriorated general state of health. ” Additional clinical signs observed at the high exposure concentration included reddish nasal discharge, salivation, eye discharge, and difficulty in breathing or whooping respiration, and stretched posture. In the high dose rats, although signs recovered between exposures at the beginning of the study, towards the end of the study ruffled fur and respiratory sounds were no longer reversible. Body weight and body weight gain were significantly decreased (p<0.05) at the high dose. Food consumption and feed efficiency were not measured. There were decreases in plasma urea, glucose, triglyceride, and albumin the high dose males. In high dose females, urea and glucose were also decreased. In the males of mid exposure group, there was a decrease in urea concentration in the plasma. At the mid and high exposure concentrations, increase in neutrophilic polymorphonuclear granulocytes in the peripheral blood was observed in males; this was also observed in the high exposure concentration for females. There was increased lung weight at the high exposure concentration. Histopathology revealed an increase in incidence and severity of rhinitis in the nasal cavity at the high exposure concentration in both sexes (incidence in males: 2/5, 2/5, 2/5, 5/5; females: 0/5, 3/5, 1/5, 5/5). Other histopathologic findings at the high exposure concentration included: atrophy of the olfactory epithelium, metaplasia of the nasal respiratory epithelium (3 males in section plane 1 only, 5 females in section planes 1 and, to a lesser extent, section plane 2), tracheal epithelial proliferation and single cell necrosis (all high exposure concentration), bronchopneumonia and bronchial and bronchiolar epithelial proliferation (5 males, 2 females), and emphysema (3 males, 2 females). The systemic LOAEL is 100 mg/m3 (34 ppm), based on effects on the body weight gain. The systemic NOAEL is 19.9 mg/m3 (6.8 ppm ) based on an increase of neutrophilic polymorphonuclear granulocytes in the blood of males, which was not observed the oral OECD 422 and 90-day inhalation studies, and which could be considered as a secondary effect of the respiratory tract inflammation. The LOAEL for effects in the extrathoracic (ET) region is 100 mg/m3 (34 ppm), based on observation of pathological changes of the nasal cavity (metaplasia of respiratory epithelium and atrophy of the olfactory epithelium). The ET NOAEL is 19.9 mg/m3 (6.8 ppm). The LOAEL for effects in the tracheabronchial (TB) region is 100 mg/m3 (34 ppm), based on observation of pathological changes (tracheal epithelial proliferation and single cell necrosis, bronchopneumonia and bronchial and bronchiolar epithelial proliferation). The TB NOAEL is 19.9 mg/m3 (6.8 ppm).

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only one repeated toxicity study is reliable on MITC by oral route.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Only one repeated toxicity study is reliable on MITC by inhalation.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Only one repeated toxicity study is reliable on MITC by inhalation.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
No reliable repeated toxicity study was available.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
No reliable repeated toxicity study was available.

Justification for classification or non-classification

Mandatory classification and self classification:

- Regulation (EC) No 1272/2008 and Directive 67/548/EEC: not classified