Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992-02-19 to 1993-01-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Principles of method if other than guideline:
No guideline stated, broadly similar to OECD 408.
GLP compliance:
yes
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 Labs, Portage, MI, USA
- Age at study initiation: approx 23 days
- Housing: 1/wire mesh cage
- Diet: standard diet (Purina Certified Rodent Chow #5002
- Water: drinking water ad libitum
- Acclimation period: 20 days, including pretest

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 67-75 deg F
- Humidity (%): 29-88 (said not to have affected results)
- Air changes (per hr): 12h/ 12h
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: From: 1992-02-19 To: 1992-05-22
Route of administration:
oral: feed
Vehicle:
other: standard diet
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): diet offered fresh weekly


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Three samples were taken from the 500 and 10,000 ppm diets to determine homogeneity, stability (for 7 and 14 days). Samples were taken from the middle of each control and test diet on the day of preparation during weeks 0, 1, 2, 3, 7 and 12 to determine concentration of test substance.

Individual food consumption was measured weekly and the mean daily dose calculated for each dose group using body weight.
Duration of treatment / exposure:
91-93 days
Frequency of treatment:
diet ad libitum
Dose / conc.:
35 mg/kg bw/day (actual dose received)
Remarks:
males: equivalent to 500ppm
Dose / conc.:
69 mg/kg bw/day (actual dose received)
Remarks:
males: equivalent to 1000ppm
Dose / conc.:
339 mg/kg bw/day (actual dose received)
Remarks:
males: equivalent to 5000ppm
Dose / conc.:
673 mg/kg bw/day (actual dose received)
Remarks:
males: equivalent to 10000ppm
Dose / conc.:
42 mg/kg bw/day (actual dose received)
Remarks:
females: equivalent to 500ppm
Dose / conc.:
84 mg/kg bw/day (actual dose received)
Remarks:
females: equivalent to 1000ppm
Dose / conc.:
401 mg/kg bw/day (actual dose received)
Remarks:
females: equivalent to 5000ppm
Dose / conc.:
781 mg/kg bw/day (actual dose received)
Remarks:
females: equivalent to 10000ppm
No. of animals per sex per dose:
20
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment: computer randomized
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (from wk -1)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and week 12
- Dose groups that were examined: all groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy (13 wk)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10/sex/dose
- Parameters checked in table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals: 10/sex/dose
- Parameters checked in table No.1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (see table 2)
Statistics:
All analyses were conducted using 2-tailed tests for minimum significance levels 1% and 5% comparing treated groups with controls by sex. Body weights, food consumption, clinical laboratory parameters, absolute and relative organ weights: 1-way analyis of variance and Dunnett's test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
no deaths, rectal mucous exudate.
Mortality:
mortality observed, treatment-related
Description (incidence):
no deaths, rectal mucous exudate.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
significant reduction in final body weight in males at 1000 ppm and above and in females at 5000 ppm and above
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
reduced food consumption at 5000 ppm in males and 10000 ppm in both sexes
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
dose-related effects at 5000 ppm and above in both sexes
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
dose-related effects at 5000 ppm and above in both sexes
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
lower testis, epididimal and prostate weight (5000 and/or 10,000 ppm)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
testis: small and/or soft at 5000 ppm and above
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
testis: dose-related degenerationof germinal epithelium of seminiferous tubules and interstitial oedema at 5000 ppm and above. Adhesions of the spleen at these doses and capsular fibrosis (focal) sporadically in all treated groups.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No deaths.
Rectal mucous exudate sporadically at 5000 ppm and above.

BODY WEIGHT AND WEIGHT GAIN
Significant reduction in final body weight in males at 1000 ppm and above and in females at 10,000 ppm (p<0.01 in both cases). Dose-related decreases (statistically significant in some cases) in mean body weights (compared to control) and body weight gains in all treated male groups (500 ppm and above) and in females in the top two dose groups.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Reduced food consumption at 5000 ppm in males and 10,000 ppm in both sexes. Dose-related decrease in mean food consumption in treated male groups.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related effects.

HAEMATOLOGY
Dose-related effects at 5000 ppm and above in both sexes: decreased haemoglobin, MCH, MCHC and platelet counts. Slightly reduced MCV values in females at 5000 ppm and above.

CLINICAL CHEMISTRY
Dose-related effects at 5000 ppm and above in both sexes: dose-related reduction in total protein and globulin, and increased A/G ratio at 5000 ppm and above in both sexes. Albumin was reduced in females at 10,000 ppm.

ORGAN WEIGHTS
Reduced testis and epididimal weight (mean absolute and relative) at 5000 ppm and above in males, lower prostate weight (mean absolute and relative) at 10,000 ppm (all p<0.01 or 0.05, except mean relative testis weight at 5000 ppm). Absolute seminal vesicle weights were lower at 5000 ppm and above, and absolute prostate weight was lower at 1000 and above. Differences in liver weights were not considered treatment-related due to inconsistency between the responses of the sexes. No other treatment-related differences reported.

GROSS PATHOLOGY
Testis: small and/or soft at 5000 ppm and above

HISTOPATHOLOGY: NON-NEOPLASTIC
Testis: dose-related degeneration of germinal epithelium of seminiferous tubules and interstitial (peritubular) oedema at 5000 ppm and above.
Epididymides: empty or fluid filled tubules with accumulation of cellular debris seen in all 10,000 ppm males and some 5000 ppm males.
Spleen: adhesion to mesentery, abdominal wall or adipose tissue sporadically at 5000 ppm and above (both sexes). Capsular fibrosis (focal) sporadically in all treated groups. The investigators noted that while this effect may have been treatment-related its toxicological significance was not known.
Key result
Dose descriptor:
NOAEL
Effect level:
500 ppm
Based on:
other: in the diet
Sex:
male
Basis for effect level:
other: mean measured dose 35 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Based on:
other: in the diet
Sex:
female
Basis for effect level:
other: mean measured dose 84 mg/kg bw/day
Critical effects observed:
not specified
Conclusions:
A well reported 90-day dietary study, conducted in the main according to the current guideline and in accordance with GLP, identified NOAELs of 500 ppm and 1000 ppm in the diet in male and female rats, respectively. This dietary intake was equivalent to mean measured intakes of 35 and 84 mg THFA/kg bw/day, in males and females. Reduced body weights were reported at the next highest dietary concentration in both cases. The testis was the major target organ.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
35 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
In addition to the key study two further oral studies in the rat (MHLW, 2004a, MHLW, 2004b) provided results that closely support the findings of the key oral study. Both were conducted according to a standard guideline and GLP, meriting a reliability score of 1 (reliable without restriction).

A 28-day oral gavage study (MHLW, 2004a) reported a NOAEL of 40 mg/kg bw/day in both sexes, with effects on the testis, thymus, spleen, kidney, or clinical chemistry and haematology parameters at 150 mg/kg bw/day, and above. A combined repeated-dose and reproductive/developmental toxicity screening test (MHLW, 2004b), in which rats were treated via gavage for around 50 days, identified a NOAEL for both sexes in the parental generation of 50 mg/kg bw/day in both sexes, with effects to the thymus, spleen, testis and body weights at 150 mg/kg bw/day, and above.

Two further studies involving 90-day adminsitration of THFA in the diet to rats and dogs (Industrial Biotest, 1970) were considered unreliable and not considered.
System:
male reproductive system
Organ:
testes

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994-10-03 to 1995-01-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes
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 Labs, Portage, MI, USA
- Age at study initiation: 6 wk
- Weight at study initiation: 155-203 (m); 129-159 (f) g
- Fasting period before study: no data
- Housing: 1/wire mesh cage
- Diet: standard diet at libitum
- Water: drinking water ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 +/- 4 deg F
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: To: 1994-10-03 to around 1995-01-03
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 0.5 m3 stainless steel and glass inhalation chambers, dynamic operation
- Source and rate of air: filtered air, 12-15 chamber volumes/hr
- Temperature, humidity, pressure in air chamber: 19.7-25.8 deg C; 19-85 % RH
- Air change rate: 12-15 chamber volumes/hr

TEST ATMOSPHERE
- Brief description of analytical method used: infrared spectoscopic method , examination 3 times daily (no findings given in Volumer 1 of report)

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
6 hr/day, 5 days/wk, at least 65 exposures (equivalent to 13 weeks)
Frequency of treatment:
daily on weekdays
Dose / conc.:
50 ppm (nominal)
Dose / conc.:
150 ppm (nominal)
Dose / conc.:
500 ppm (nominal)
No. of animals per sex per dose:
10, with a further 4 males per concentration to examine spermatogenic effects
Control animals:
yes, concurrent vehicle
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once/week

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: weeks 0 and 12

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 3, 13
- Anaesthetic used for blood collection:No data
- Animals fasted: Yes
- How many animals: all survivors from main groups (10/sex/concentration)
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals: all survivors from main groups (10/sex/concentration)
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: examination of groups of 4 males at week 6 and 10 males at week 13, at each concentration, for effects on spermatogenic endpoints
Sacrifice and pathology:
GROSS PATHOLOGY: Yes see table 2
HISTOPATHOLOGY: Yes see table 2
Statistics:
2-tailed tests for minimum significance levels of 1% and 5%, comparing treatment groups to the vehicle control. Body weight, weight change, food consumption, clinical laboratory, spermatogenic and organ weight data were subjected to a 1-way analysis of variance and Dunnett’s test.
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
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
testis
Details on results:
CLINICAL SIGNS AND MORTALITY
1/10 females died in at 50 ppm during week 0, the cause of death was not found to be treatment-related. The predominant clinical finding was intermittent whole-body spasms in all treated groups that were frequent and exposure-related. Hypoactivity and excessive grooming were occasionally observed in males and females at 500 ppm. One hour after exposure, concentration-related hyperactivity was reported in all treated groups, and yellow urogenital matting and salivation at 500 ppm.

BODY WEIGHT AND WEIGHT GAIN
Mean body weight gains were lower than controls in males at 150 and 500 ppm (9.2% and 13.3%, respectively).

FOOD CONSUMPTION
Mean food consumption was generally lower in mid and high exposure males throughout the study

OPHTHALMOSCOPIC EXAMINATION
No treatment-related observations.

HAEMATOLOGY
Several statistically significant treatment-related changes were reported after exposure to 500 ppm. Platelet and haemoglobin means were significantly reduced in both sexes (study weeks 3 and/or 13). MCH (mean cell haemoglobin) was reduced in males (weeks 3 and 13) and females (week 13); MCHC (mean corpuscular haemoglobin concentration) was reduced in males (week 13) and females (week 3).

CLINICAL CHEMISTRY
No treatment-related effect.

ORGAN WEIGHTS
Prostate weight (absolute and relative) were lower than controls in 150 and 500 ppm groups. The weights of seminal vesicles (absolute) and epididymides (absolute and relative) were lower at 500 ppm.

GROSS PATHOLOGY
No macroscopic lesions in any exposure group.

HISTOPATHOLOGY: NON-NEOPLASTIC
Spermatogenic findings: reduced epididymal sperm numbers and motility, and a higher incidence of morphological abnormalities were seen in 500 ppm males at weeks 6 and 13. Multifocal atrophy of the testes was reported in a single male at 500 ppm.
Dose descriptor:
NOAEC
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: whole body spasm and hyperactivity, and other effects.
Remarks on result:
not determinable
Remarks:
no NOAEC identified
Dose descriptor:
LOAEC
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: around 0.2 mg/l
Critical effects observed:
not specified
Conclusions:
A well reported 90-day rat inhalation study, conducted according to the current guideline and in accordance with GLP, did not identify a NOAEC value. Clinical observations (including spasm) were evident at the lowest tested nominal concentration of 50 ppm (around 0.2 mg/l).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
209 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
No other inhalation studies were identified.
System:
male reproductive system
Organ:
testes

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994-10-03 to 1995-01-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes
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 Labs, Portage, MI, USA
- Age at study initiation: 6 wk
- Weight at study initiation: 155-203 (m); 129-159 (f) g
- Fasting period before study: no data
- Housing: 1/wire mesh cage
- Diet: standard diet at libitum
- Water: drinking water ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 +/- 4 deg F
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: To: 1994-10-03 to around 1995-01-03
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 0.5 m3 stainless steel and glass inhalation chambers, dynamic operation
- Source and rate of air: filtered air, 12-15 chamber volumes/hr
- Temperature, humidity, pressure in air chamber: 19.7-25.8 deg C; 19-85 % RH
- Air change rate: 12-15 chamber volumes/hr

TEST ATMOSPHERE
- Brief description of analytical method used: infrared spectoscopic method , examination 3 times daily (no findings given in Volumer 1 of report)

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
6 hr/day, 5 days/wk, at least 65 exposures (equivalent to 13 weeks)
Frequency of treatment:
daily on weekdays
Dose / conc.:
50 ppm (nominal)
Dose / conc.:
150 ppm (nominal)
Dose / conc.:
500 ppm (nominal)
No. of animals per sex per dose:
10, with a further 4 males per concentration to examine spermatogenic effects
Control animals:
yes, concurrent vehicle
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once/week

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: weeks 0 and 12

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 3, 13
- Anaesthetic used for blood collection:No data
- Animals fasted: Yes
- How many animals: all survivors from main groups (10/sex/concentration)
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals: all survivors from main groups (10/sex/concentration)
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: examination of groups of 4 males at week 6 and 10 males at week 13, at each concentration, for effects on spermatogenic endpoints
Sacrifice and pathology:
GROSS PATHOLOGY: Yes see table 2
HISTOPATHOLOGY: Yes see table 2
Statistics:
2-tailed tests for minimum significance levels of 1% and 5%, comparing treatment groups to the vehicle control. Body weight, weight change, food consumption, clinical laboratory, spermatogenic and organ weight data were subjected to a 1-way analysis of variance and Dunnett’s test.
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
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
testis
Details on results:
CLINICAL SIGNS AND MORTALITY
1/10 females died in at 50 ppm during week 0, the cause of death was not found to be treatment-related. The predominant clinical finding was intermittent whole-body spasms in all treated groups that were frequent and exposure-related. Hypoactivity and excessive grooming were occasionally observed in males and females at 500 ppm. One hour after exposure, concentration-related hyperactivity was reported in all treated groups, and yellow urogenital matting and salivation at 500 ppm.

BODY WEIGHT AND WEIGHT GAIN
Mean body weight gains were lower than controls in males at 150 and 500 ppm (9.2% and 13.3%, respectively).

FOOD CONSUMPTION
Mean food consumption was generally lower in mid and high exposure males throughout the study

OPHTHALMOSCOPIC EXAMINATION
No treatment-related observations.

HAEMATOLOGY
Several statistically significant treatment-related changes were reported after exposure to 500 ppm. Platelet and haemoglobin means were significantly reduced in both sexes (study weeks 3 and/or 13). MCH (mean cell haemoglobin) was reduced in males (weeks 3 and 13) and females (week 13); MCHC (mean corpuscular haemoglobin concentration) was reduced in males (week 13) and females (week 3).

CLINICAL CHEMISTRY
No treatment-related effect.

ORGAN WEIGHTS
Prostate weight (absolute and relative) were lower than controls in 150 and 500 ppm groups. The weights of seminal vesicles (absolute) and epididymides (absolute and relative) were lower at 500 ppm.

GROSS PATHOLOGY
No macroscopic lesions in any exposure group.

HISTOPATHOLOGY: NON-NEOPLASTIC
Spermatogenic findings: reduced epididymal sperm numbers and motility, and a higher incidence of morphological abnormalities were seen in 500 ppm males at weeks 6 and 13. Multifocal atrophy of the testes was reported in a single male at 500 ppm.
Dose descriptor:
NOAEC
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: whole body spasm and hyperactivity, and other effects.
Remarks on result:
not determinable
Remarks:
no NOAEC identified
Dose descriptor:
LOAEC
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: around 0.2 mg/l
Critical effects observed:
not specified
Conclusions:
A well reported 90-day rat inhalation study, conducted according to the current guideline and in accordance with GLP, did not identify a NOAEC value. Clinical observations (including spasm) were evident at the lowest tested nominal concentration of 50 ppm (around 0.2 mg/l).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994-09-08 to 1994-12-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes
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 Labs, Portage, MI, USA
- Weight at study initiation g: 168-209 (m); 137-164 (f)
-age: 6 weeks
- Housing: 1/ wire mesh cage
- Diet: standard diet ad libitum
- Water: drinking water ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 70-76 deg F
- Humidity (%): 37-66
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: To: 1994-09-15 to 1994-12-15
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure:
- % coverage: 20-25% shaved; coverage (maximum possible) 1.7-4.1% and 1.6-4.2% coverage of body for males, and females, respectively
- Type of wrap if used: gauze binder, impervious plastic wrap, dermiform tape
- Time intervals for shavings or clipplings: day prior to dosing and weekly

REMOVAL OF TEST SUBSTANCE
- Washing (if done): wiped with wet paper towel moistened with tepid tap water
- Time after start of exposure: 6h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.095, 0.29, 0.96 ml/kg bw/day; 100, 300, 1000 mg/kg bw/day
- Concentration: neat
- Constant volume or concentration used: no

VEHICLE
Control: 0.9% saline, 0.95 ml/kg bw/day

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (Elizabethan restraint collars)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
application neat; purity reported
Duration of treatment / exposure:
6h/day, 5 days/week, 13 weeks
Frequency of treatment:
daily (weekdays)
Dose / conc.:
100 mg/kg bw/day
Remarks:
analytical per unit body weight
Dose / conc.:
300 mg/kg bw/day
Remarks:
analytical per unit body weight
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
analytical per unit body weight
No. of animals per sex per dose:
17 males, 12 females (5 males terminated at 7 weeks for examination of spermatogenic endpoints)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random): computer randomization
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly

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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: week 12
- slit lamp and indirect ophthalmoscope

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 4, 13
- Anaesthetic used for blood collection: /No data
- Animals fasted: Yes
- How many animals: 12/sex/dose
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 4, 13
- Animals fasted: Yes
- How many animals: 12/sex/dose
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:

Spermatogenic endpoints: 5 males/dose; terminated at week 7: sperm motility, morphology
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table2)
Statistics:
All analyses were conducted using two-tailed tests for significance levels of 5 % and 1%.
Body weight, body weight change, food consumption, clinical laboratory, spermatogenic and absolute and relative organ weight data were subjected to a one-way analysis of variance followed by Dunnett's Test. Clinical laboratory values for white blood cell types that occur at a low incidence (i.e. monocytes, eosinophils, basophils and unsegmented neutrophils) were not subjected to statistical analysis.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
very minimal dermal irritation
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced mean body weight in both sexes at 1000 mg/kg bw/day
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:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortatily or treatment-related clinical signs.

BODY WEIGHT AND WEIGHT GAIN
8-10% lower weight gain in 1000 mg/kg bw/day males comapred to controls (p<0.05 or 0.1; weeks 9-13). 7% lower weight gain in 1000 mg/kg bw/day females compared to controls (p<0.05; weeks 12, 13).

FOOD CONSUMPTION
No effect.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related effects at week 12 examination.

HAEMATOLOGY
No treatment-related effect on haematological parameters. Mean white blood cell counts and absolute numbers of lymphocytes were lower (p<0.01 or 0.05) in 1000 mg/kg bw/day males at weeks 4 and 13 examinations, No similar effects were seen in females and the white cell counts were within WIL Research Laboratories historic control ranges for this species, strain and age. Statistically significant differences in red blood cell parameters did not show trends to indicate they were treatment-related.

CLINICAL CHEMISTRY
No treatment-related effect on serum chemistry parameters. Statistically significant differences (p<0.01 or <0.05) in serum protein parameters were seen in both sexes at 1000 mg/kg bw/day at weeks 4 or 13 but these were small, their means within the normal range of biologic variation and without trend to indicate an association with treatment. Other statistically significant differences were limited to one sex at a single evaluation, showed no dose-response or were not considered toxicologically significant.

ORGAN WEIGHTS
No treatment-related effect.

GROSS PATHOLOGY
No treatment-related observations.

HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment-related effects at any dose including effects on skin and male reproductive organs

OTHER FINDINGS
dermal: very limited dermal irritation - single occasions of very slight erythema for one, two and one males in the 100, 300 and 1000 mg/kg bw/day groups, respectively, and or one female in the 100 mg/kg bw/day group. Desquamation was observed on one day for single females in the control and 100 mg/kg bw/day group and thus was apparently not test article-related. No other dermal irritation was observed.

Spermatogenic endpoints:
Administration of up to 1000 mg THFA/kg bw/day for 7 weeks (interim necropsy) had no effect on spermatogenic endpoints (testicular epididymal sperm numbers, sperm production rate, sperm motility, sperm morphology).

Mean numbers of sperm present in testicular homogenates and mean sperm production rate were lower than controls in 300 and 1000 mg/kg bw/day males after 13 weeks treatment (p<0.01). One male in the 1000 mg/kg bw/day group had markedly lower values for these parameters but the the group mean was still significantly reduced (p<0.01) when the values for this male were omitted. Sperm motility was reduced at 1000 mg/kg bw/day (p<0.05).

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: spermatogenesis
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: lower body weight gain
Critical effects observed:
not specified
Conclusions:
A 90-day dermal study, conducted according to the current guideline (OECD 411) and GLP, identified NOAEL values of 100 mg/kg bw/day in male rats and 300 mg/kg bw/day female rats. Effects on body weight were evident at 1000 mg/kg bw/day in both sexes and spermatogenic effects were reported in males at 300 mg/kg bw/day.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
No other relevant studies were identified
System:
male reproductive system
Organ:
testes

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994-09-08 to 1994-12-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes
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 Labs, Portage, MI, USA
- Weight at study initiation g: 168-209 (m); 137-164 (f)
-age: 6 weeks
- Housing: 1/ wire mesh cage
- Diet: standard diet ad libitum
- Water: drinking water ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 70-76 deg F
- Humidity (%): 37-66
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: To: 1994-09-15 to 1994-12-15
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure:
- % coverage: 20-25% shaved; coverage (maximum possible) 1.7-4.1% and 1.6-4.2% coverage of body for males, and females, respectively
- Type of wrap if used: gauze binder, impervious plastic wrap, dermiform tape
- Time intervals for shavings or clipplings: day prior to dosing and weekly

REMOVAL OF TEST SUBSTANCE
- Washing (if done): wiped with wet paper towel moistened with tepid tap water
- Time after start of exposure: 6h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.095, 0.29, 0.96 ml/kg bw/day; 100, 300, 1000 mg/kg bw/day
- Concentration: neat
- Constant volume or concentration used: no

VEHICLE
Control: 0.9% saline, 0.95 ml/kg bw/day

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (Elizabethan restraint collars)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
application neat; purity reported
Duration of treatment / exposure:
6h/day, 5 days/week, 13 weeks
Frequency of treatment:
daily (weekdays)
Dose / conc.:
100 mg/kg bw/day
Remarks:
analytical per unit body weight
Dose / conc.:
300 mg/kg bw/day
Remarks:
analytical per unit body weight
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
analytical per unit body weight
No. of animals per sex per dose:
17 males, 12 females (5 males terminated at 7 weeks for examination of spermatogenic endpoints)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random): computer randomization
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly

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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: week 12
- slit lamp and indirect ophthalmoscope

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 4, 13
- Anaesthetic used for blood collection: /No data
- Animals fasted: Yes
- How many animals: 12/sex/dose
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 4, 13
- Animals fasted: Yes
- How many animals: 12/sex/dose
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:

Spermatogenic endpoints: 5 males/dose; terminated at week 7: sperm motility, morphology
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table2)
Statistics:
All analyses were conducted using two-tailed tests for significance levels of 5 % and 1%.
Body weight, body weight change, food consumption, clinical laboratory, spermatogenic and absolute and relative organ weight data were subjected to a one-way analysis of variance followed by Dunnett's Test. Clinical laboratory values for white blood cell types that occur at a low incidence (i.e. monocytes, eosinophils, basophils and unsegmented neutrophils) were not subjected to statistical analysis.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
very minimal dermal irritation
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced mean body weight in both sexes at 1000 mg/kg bw/day
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:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortatily or treatment-related clinical signs.

BODY WEIGHT AND WEIGHT GAIN
8-10% lower weight gain in 1000 mg/kg bw/day males comapred to controls (p<0.05 or 0.1; weeks 9-13). 7% lower weight gain in 1000 mg/kg bw/day females compared to controls (p<0.05; weeks 12, 13).

FOOD CONSUMPTION
No effect.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related effects at week 12 examination.

HAEMATOLOGY
No treatment-related effect on haematological parameters. Mean white blood cell counts and absolute numbers of lymphocytes were lower (p<0.01 or 0.05) in 1000 mg/kg bw/day males at weeks 4 and 13 examinations, No similar effects were seen in females and the white cell counts were within WIL Research Laboratories historic control ranges for this species, strain and age. Statistically significant differences in red blood cell parameters did not show trends to indicate they were treatment-related.

CLINICAL CHEMISTRY
No treatment-related effect on serum chemistry parameters. Statistically significant differences (p<0.01 or <0.05) in serum protein parameters were seen in both sexes at 1000 mg/kg bw/day at weeks 4 or 13 but these were small, their means within the normal range of biologic variation and without trend to indicate an association with treatment. Other statistically significant differences were limited to one sex at a single evaluation, showed no dose-response or were not considered toxicologically significant.

ORGAN WEIGHTS
No treatment-related effect.

GROSS PATHOLOGY
No treatment-related observations.

HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment-related effects at any dose including effects on skin and male reproductive organs

OTHER FINDINGS
dermal: very limited dermal irritation - single occasions of very slight erythema for one, two and one males in the 100, 300 and 1000 mg/kg bw/day groups, respectively, and or one female in the 100 mg/kg bw/day group. Desquamation was observed on one day for single females in the control and 100 mg/kg bw/day group and thus was apparently not test article-related. No other dermal irritation was observed.

Spermatogenic endpoints:
Administration of up to 1000 mg THFA/kg bw/day for 7 weeks (interim necropsy) had no effect on spermatogenic endpoints (testicular epididymal sperm numbers, sperm production rate, sperm motility, sperm morphology).

Mean numbers of sperm present in testicular homogenates and mean sperm production rate were lower than controls in 300 and 1000 mg/kg bw/day males after 13 weeks treatment (p<0.01). One male in the 1000 mg/kg bw/day group had markedly lower values for these parameters but the the group mean was still significantly reduced (p<0.01) when the values for this male were omitted. Sperm motility was reduced at 1000 mg/kg bw/day (p<0.05).

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: spermatogenesis
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: lower body weight gain
Critical effects observed:
not specified
Conclusions:
A 90-day dermal study, conducted according to the current guideline (OECD 411) and GLP, identified NOAEL values of 100 mg/kg bw/day in male rats and 300 mg/kg bw/day female rats. Effects on body weight were evident at 1000 mg/kg bw/day in both sexes and spermatogenic effects were reported in males at 300 mg/kg bw/day.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

In a 90-day oral study, THFA was administered to rats via the dietary route at 0, 500, 1000, 5000 or 10000 ppm. All animals survived the study period and there were no remarkable clinical signs. Lower body weight gain and food consumption were noted in all treated groups and were generally dose-related, the effects being considered adverse at concentrations of 1000 ppm and above in males and at 10 000 ppm at females. The other notable effects of treatment were recorded at 5000 and or 10000 ppm and included lower red cell parameters and total protein and albumin levels. At necropsy lower testes and epididimal, prostate and seminal vesicle weights were recorded for males and the testes were noted to be small and/or soft at these doses. Dose-related degeneration of germinal epithelium of seminiferous tubules and interstitial (peritubular) oedema in the testes and empty or fluid filled tubules with accumulation of cellular debris in the epididymides were noted. In the spleen, adhesions and focal capsular fibrosis were noted. The No-Observed-Adverse-Effect-Level was considered to be 500 ppm (corresponding to 35 mg/kg/day) in males and 1000 ppm (corresponding to 84 mg/kg/day) in females.

In a 28-day oral study, THFA was administered to rats by oral gavage at doses of 0, 10, 40, 150 or 600 mg/kg/day. Similar effects were noted generally at the high dose to those seen in the 90-day dietary study with histopathological changes, including necrosis of the seminiferous epithelium, again noted in the testes and capsular inflammatory cell infiltrate or fibrosis in the spleen. Based on these effects the No-Observed-Adverse-Effect-Level was considered to be 40 mg/kg/day.

A 90-day inhalation study was conducted in which THFA was administered to rats at 0, 50, 150 or 500 ppm. The predominant clinical finding was intermittent whole-body spasms in all treated groups that were frequent and exposure-related with hyperactivity noted 1 hour after dosing. Hypoactivity and excessive grooming were occasionally observed in males and females at 500 ppm. Body weight gain and food consumption were generally lower at the mid and high doses throughout the study. Lower red cell parameters were recorded at the high dose. At necropsy prostate and epididymides weight were lower at 150 and/or 500 ppm. Microscopically, reduced epididymal sperm numbers and motility and multifocal atrophy of the testes were recorded at 500 ppm. Based on the effects on the male reproductive organs and the clinical findings which were noted at all doses, a No-Observed-Adverse-Effect-Level was not established and the Lowest-Observed-Adverse-Effect-Level was considered to be 50 ppm.

A 90-day dermal study was conducted in which THFA was administered to rats at 0, 100, 300 or 1000 mg/kg/day. All animals survived and there were no remarkable clinical signs or effects on food consumption, haematology or clinical chemistry parameters or organ weight. Body weight gain was lower at the high dose. Mean numbers of sperm present in testicular homogenates and mean sperm production rate were lower than controls in 300 and 1000 mg/kg/day males and sperm motility was reduced at 1000 mg/kg/day. Based on the spermatogenic effects the No-Observed-Adverse-Effect-Level was considered to be 100 mg/kg/day.

.

Justification for classification or non-classification

All five studies (three key, two supporting) suggest that the testes are the main target organ. In accordance with Section 3.7.2.5.3 of Regulation (EC) 1272/2008, adverse effects seen in repeated dose toxicity studies which are judged likely to impair reproductive function and which occur in the absence of significant generalised toxicity may be used as a basis for classification for reproductive toxicity.

The observed effects on testes are considered as likely to impair reproductive function and, as such, the substance is classified for reproductive toxicity (Section 5.9). There is no need to include a separate classification for specific target organ toxicity following repeated exposures, in the absence of other significant adverse effects.