Registration Dossier

Administrative data

Description of key information

No data on acute toxicity is available for"Fatty acids C18 unsat, reaction products with triethylenetetramine" (or TO + TETA). Read-across is performed with "Fatty acids C18 unsat, reaction products with diethylenetetramine" (or TO + DETA).

A combined repeated dose/reproduction screening toxicity study according to OECD 422 with Fatty acids, C18 unsat, reaction products with diethylenetriamine (AAI-DETA) resulted to a NOAEL of 10 mg/kg bw/day based on the increased incidence/severity of macrophage foci in the mesenteric lymph node observed at 30 or 100 mg/kg bw/day, the highest dose tested. All already available data from the group of AAI substances, including 90 -day studies in rat and dogs on a similar substance, also indicate low repeated dose toxicity.

The 90-day NOAEL with AAI-DETA is considered to be 10 mg/kg bw/d. At higher dose levels an increase of the presence of foamy macrophages in the lamina propria of the small intestines and mesenteric lymph nodes is observed, as well as lower mean body weight and body weight gain, especially in the males, with lower food intake, essentially during the second half of the treatment period.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 November 2013 to 18 February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2012 by MHLW (0331 No.7), METI (No. 5) and MOE (No. 110331009)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Wistar (Han)
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 6 weeks old).
- Weight at study initiation: mean weight range at start of treatment was 147-148 gr (males) or 122-125 gr (females).
- Housing: Group housing of 5 animals per cage in labeled Macrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days.

Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 18 November 2013 to 18 February 2014
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for specific gravity of the test substance and specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.

VEHICLE
- Justification for use and choice of vehicle: The same vehicle was used under project 491556 (combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of Tall oil diethylenetriamine imidazoline in rats by oral gavage, followed by a 14-Day recovery period).

DOSE VOLUME:
5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted during the treatment phase. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations, in Weeks 1, 6 and 13). Stability in vehicle over 8 days in the refrigerator under nitrogen was also determined (highest and lowest concentration, in Week 1). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation was = 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily, 7 d/w.
Remarks:
Doses / Concentrations:
0, 10, 30, 100 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of Tall oil diethylenetriamine imidazoline in rats by oral gavage, followed by a 14-Day recovery period.

Note: “Tall oil diethylenetriamine imidazoline” is the same test substance as “Fatty acids, C18 unsat, reaction products with diethylenetriamine”.

In this study rats were dosed by oral gavage at dose levels of 10, 30 and 100 mg/kg. At 100 mg/kg, histopathology revealed foamy macrophage foci in the ileum of all selected males and females, a slightly increased incidence and degree of macrophage foci in the mesenteric lymph node of both sexes, and increased incidence of lymphoid atrophy in the thymus of females. At the end of the 14-day recovery period for males, foamy macrophage foci in the ileum had completely resolved, whilst macrophage foci in the mesenteric lymph node persisted at higher severity than observed at the end of treatment. Given the persistence of this finding it was considered to be of an adverse nature. However, it was considered that a dose of 100 mg/kg would be tolerated in a 90-day study. No toxicologically relevant changes were noted at 10 and 30 mg/kg.
Positive control:
Not required.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily from start of treatment onwards in all animals between 1-2 hours after dosing.

BODY WEIGHT:
- Time schedule for examinations: Weekly.

FOOD CONSUMPTION
- Time schedule for examinations: Weekly.

WATER CONSUMPTION: No quantitative investigation (subjective appraisal).

OPHTHALMOSCOPIC EXAMINATION
- Time schedule for examinations: at pretest : All animals (including spare animals), at week 13 : control and high dose.

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes (maximally 24 hours)
- How many animals: all animals
- Parameters checked: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes (maximally 24 hours)
- How many animals: all animals
- Parameters checked: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: During Week 12 of treatment, between 1 and 3 hours after dosing.
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength, locomotor activity test.

ESTROUS CYCLE DETERMINATION
All females had a daily lavage from Day 72 up to and including Day 92 to determine the stage of estrous.
Sacrifice and pathology:
GROSS PATHOLOGY:
- All animals were fasted overnight with a maximum of 24 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: all groups
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS:
Organs checked according to test guidelines

HISTOPATHOLOGY:
According to test guidelines
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Based on subjective appraisal.
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, treatment-related
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
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY AND CLINICAL SIGNS
No mortality occurred during the study period. No clinical signs of toxicity or abnormalities during weekly arena observations were noted during the observation period.

BODY WEIGHT AND WEIGHT GAIN
Males at 30 and 100 mg/kg showed a lower mean body weight and body weight gain from Week 5 of the treatment period onwards, being statistically significant at 100 mg/kg. The lower mean body weight at 100 mg/kg was considered to be primarily due to a lower weight gain of two males. Mean body weight and body weight gain of females at 100 mg/kg also appeared slightly lower than controls during the last weeks of treatment.

FOOD CONSUMPTION
Males at 30 and 100 mg/kg showed a slightly lower food intake during the larger part of the treatment period. After correction for body weight, food consumption of these males was similar to control levels. Food consumption before or after correction for body weight for females remained similar to control means over the study period.

OPHTHALMOSCOPIC EXAMINATION
No ophthalmology findings were noted that were considered to be related to treatment.

HAEMATOLOGY
Haematological parameters of treated rats were considered not to have been affected by treatment.

CLINICAL CHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher alanine aminotransferase activity (ALAT) in males and females (most notably for one female) at 100 mg/kg,
- Higher aspartate aminotransferase activity (ASAT) in males (most notably for one male) and females at 100 mg/kg,
- Lower total protein levels in males (most notably for one male) and females at 100 mg/kg,
- Lower albumin levels in males (most notably for one male) and females at 100 mg/kg,
- Higher glucose levels in males at 100 mg/kg (upon excluding one male),
- Lower urea levels in males at 100 mg/kg (a trend towards an increase was also apparent among female dose groups),
- Higher bile acid levels in females at 100 mg/kg,
- Lower calcium levels in females at 100 mg/kg.
One male at 100 mg/kg also showed a lower glucose value and a higher total bilirubin, bile acid and inorganic phosphate level.

NEUROBEHAVIOUR
Males at 100 mg/kg showed a statistically significantly lower motor activity (based on counts for total movements). Females also showed a trend towards lower motor activity at 30 and 100 mg/kg, but means did not achieve a level of statistical significance.

ESTROUS CYCLE DETERMINATION
No treatment related changes in estrous cycle length were noted across the dose groups during the period in which estrous cycle length was determined (Day 72 up to and including Day 92). One control female, and three females at 30 mg/kg showed an irregular estrous cycle length. All other females showed a normal (regular) estrous cycle of 4 days. The incidence of irregular estrous cycle length showed no relationship to the dose, and was therefore considered unrelated to treatment.

ORGAN WEIGHTS
The following (statistically significant) changes in absolute organ weights and relative organ weights (organ to body weight ratio) were considered to be related to treatment:
- Lower liver weights in males at 100 mg/kg (with a decreasing trend for absolute liver weights across other male groups),
- Lower thymus weights in males at 100 mg/kg (with a decreasing trend for absolute thymus weights across other male groups),
- Lower spleen weights in males at 100 mg/kg (with a decreasing trend for absolute spleen weights across other male groups).
- Lower prostate weights at 100 mg/kg.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

HISTOPATHOLOGY
The following microscopic findings were considered to be related to treatment:
- Lung (alveolar (mainly foamy) macrophage aggregations):
At 100 mg/kg: increased incidence and severity in females (6/10 females, 4 minimal, 2 slight).
- Small intestines (foamy macrophages in the lamina propria):
At 10 mg/kg: in jejunum in 1/10 males and 3/10 females and in ileum in 3/10 males and 3/9 females (all minimal).
At 30 mg/kg: in duodenum in 1/10 males (slight), in jejunum in 1/10 males (minimal) and 4/10 females (minimal) and in ileum in 8/10 males (6: minimal, 2: slight) and females 10/10 females (5: minimal, 5: slight).
At 100 mg/kg: in duodenum in 8/10 males (7: minimal, 1:slight) and in 5/10 females (4: minimal, 1: slight), in jejunum in 10/10 males (2: minimal, 8: slight) and 8/10 females (6: minimal, 2: slight) and in ileum in 10/10 males (1: minimal, 7: slight, 2: moderate) and in 10/10 females (8: slight, 2: moderate).
- Kidneys (foamy macrophages in the glomeruli):
At 100 mg/kg: in 7/10 males (6: minimal, 1: slight) and in 9/10 females (minimal).
- Mesenteric lymph node (foamy macrophages):
At 10 mg/kg: in 1/10 female (slight),
At 30 mg/kg: in 2/10 males (minimal) and in 3/10 females (minimal)
At 100 mg/kg: in 10/10 males (7: slight, 2: moderate, 1: marked) and in 8/10 females (1: slight, 7: moderate).
- Mesenteric lymph node (pigmented macrophage foci):
At 30 mg/kg: slightly increased incidence and/or severity in 9/10 males (4: minimal, 5: slight) and in 9/10 females (7: minimal, 2: slight).
At 100 mg/kg: slightly increased incidence and/or severity in 10/10 males (5: minimal, 5: slight) and in 7/10 females (3: minimal, 4 slight).
The spermatogenic staging profiles were normal for all males of the control group and the 100 mg/kg group, except for one male at 100 mg/kg (all stages missing). The macroscopic and microscopic findings recorded for this male at 100 mg/kg included effects in the reproductive organs (flaccid and reduced size of testes, microscopic correlate: undeveloped testes; reduced size of epididymides, microscopic correlate: reduced sperm; reduced size of prostate gland, microscopic correlate: immature) and liver (reduced size, no microscopic correlate and accentuated lobular pattern, microscopic correlate: difference in cell size periportal-centrilobular). These findings for this single male at 100 mg/kg were considered to be incidental findings and unrelated to treatment.
Dose descriptor:
NOAEL
Effect level:
ca. 10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Accuracy of preparation: The concentrations analysed in the formulations for the 10, 30 and 100 mg/kg dose groups were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). A small response at the retention time of the test substance was observed in the chromatograms of the control formulations. It was considered to derive from carry over since a similar response was obtained in the analytical blanks.

Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation = 10%).

Stability: Formulations at the entire range were stable when stored under nitrogen in a refrigerator for at least 8 days.

Conclusions:
The 90-day NOAEL is considered to be 10 mg/kg bw/d. At higher dose levels an increase of the presence of foamy macrophages in the lamina propria of the small intestines and mesenteric lymph nodes is observed, as well as lower mean body weight and body weight gain, especially in the males, with lower food intake, essentially during the second half of the treatment period.
Executive summary:

Tall oil diethylenetriamine imidazoline was administered by daily oral gavage to groups of 10 male and 10 female Wistar Han rats for 90 days at dose levels of 0, 10, 30 and 100 mg/kg/day. The study was performed under GLP and based on OECD TG 408.

 

Evaluated parameters:

Chemical analyses of formulations preparations; clinical signs daily; functional observation tests in Week 12; body weight and food consumption weekly; ophthalmoscopy at pretest and in Week 13; estrous cycle determination; clinical pathology and macroscopy at termination; organ weights and histopathology (including spermatogenesis staging) on a selection of tissues.

 

Results

Formulation analyses confirmed that formulations of test substance in propylene glycol were prepared accurately and homogenously, and were stable over at least 8 days.

All animals survived up until scheduled termination. No toxicologically significant clinical signs were noted during the observation period.

The treatment-related lower motor activity of males at 100 mg/kg, and a trend towards lower motor activity for females at 30 and 100 mg/kg were considered not to represent an adverse effect on neurobehaviour. These results were not supported by clinical observations or other functional observation tests, were slight in nature (within the normal range for rats of this age and strain), and had no supportive morphological correlates in examined neuronal tissues.

 

Males at 30 and 100 mg/kg showed a lower mean body weight and body weight gain with lower food intake, essentially during the second half of the treatment period, with a slightly lower body weight and body weight gain of females at 100 mg/kg during the last week of treatment.

No treatment-related ophthalmology findings, changes in haematological parameters or macroscopic findings were noted.

Test item-related microscopic findings consisted of:

-  foamy macrophages in the lamina propria of the small intestines (males and females starting at 10 mg/kg/day),

- foamy macrophages in the mesenteric lymph nodes (females starting at 10 mg/kg/day, males starting at 30 mg/kg/day),

- increased incidence and severity of pigmented macrophage foci in the mesenteric lymph nodes (males and females starting at 30 mg/kg/day),

- increased incidence and severity of alveolar (mainly foamy) macrophage aggregations in the lung (females at 100 mg/kg/day),

- foamy macrophages in the glomeruli of the kidneys (males and females at 100 mg/kg/day). 

The findings in the lamina propria of the small intestines were considered to have caused reduced protein uptake, and to correlate with lower total protein and albumin levels in males and females at 100 mg/kg.

 

Other treatment-related changes in clinical biochemistry parameters at 100 mg/kg consisted of higher alanine and aspartate aminotransferase activity in males and females, higher total bilirubin and glucose levels in males, lower urea levels in males (with a trend towards an increase among female dose groups), higher bile acid levels in females, and lower calcium levels in females at 100 mg/kg (possibly secondary to the lower albumin levels).

The lower liver, thymus and spleen weights in males at 100 mg/kg (with a decreasing trend across other male groups) had no histopathological correlates, and were therefore ascribed to the lower terminal body weights for these males. As such, these changes were considered to be of no toxicological relevance.

 

One male at 100 mg/kg showed various microscopic (and correlating macroscopic and organ weight) changes in reproductive organs including undeveloped testes (correlating to absence of all spermatogenesis stages) with reduced sperm content in the epididymides and immature prostate, and a difference in cell size in the periportal-centrilobular area of the liver. In addition, this animal showed a lower weight gain during treatment, and blood analyses showed a lower glucose value and a higher bile acid and inorganic phosphate level. Since these findings were confined to this single animal, these were considered unrelated to treatment with the test substance.

 

There were no indications of possible reproductive toxicity based on the parameters determined in this study. No treatment related changes in estrous cycle length were noted across the dose groups during the period in which estrous cycle length was determined (Day 72 up to and including Day 92), and histopathological examination of the male and female reproductive organs (including spermatogenesis staging) did not show treatment-related lesions.

 

Conclusion

The results from this study are comparable to those obtained from the earlier OECD 422 study.

The first effects to occur is the presence of foamy macrophages in the lamina propria of the small intestines and mesenteric lymph nodes. These effects are considered to represent a local, porte d’entrée related effect due to the route of application, rather than a systemic effect.

The magnitude of these effects as observed at the lowest dose level of 10 mg/kg, is often also observed in control groups in general, and was also seen in the control group of the OECD 422 study performed before on the same substance. These effects are therefore not considered adverse. As no other effects were observed, this dose level is considered to represent the NOAEL. At higher dose levels an increase in foamy macrophages is observed beyond levels that can occur in control groups, as well as lower mean body weight and body weight gain, especially in the males, with lower food intake, essentially during the second half of the treatment period.
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: limited reporting, EPA evaluated: Reliable without restriction; guideline study.
Qualifier:
equivalent or similar to
Guideline:
EPA OPP 82-1 (90-Day Oral Toxicity)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
Diet and water were available ad libitum
Weight of animals: Male animals weighed between 9.1 to 11.6 kg and females weighed 6.5 to 8.8 kg.
Route of administration:
oral: feed
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Exposure period: 91 to 93 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
4000, 12000 and 40000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
Four male and four female dogs
Control animals:
yes, plain diet
Details on study design:
No post-exposure recovery period
Observations and examinations performed and frequency:
Observations were conducted at least twice daily for mortality and overt toxicity.
Detailed observations, body weights and food consumption were recorded weekly.
Ophthalmological examinations were performed on all animals prior to study initiation and at study termination.
Physical examinations, as well as hematological clinical chemistry and urological evaluations were conducted on all animals prior to study initiation and at monthly intervals during the study.
Sacrifice and pathology:
At study termination, a thorough post-mortem examination was conducted on all dogs. A complete set of all major tissues and organs was harvested and selected organs were weight. The saved tissues were processed histologically and microscopic examination was conducted.
Statistics:
Analysis of variance, Bartlett’s Test for Homogeneity of Variance, T-statistic as described by Steel and Torrie, Ostle and Dunnett’s Tables with a Bonferroni correctio
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional 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
Histopathological findings: neoplastic:
no effects observed
Details on results:
Actual dose received: 142, 366 and 1322 mg/kg/day for males; 144, 632 and 1948 mg/kg/day for females
During the 13-week treatment period, one male and one female dog administered diets containing 40,000 ppm test substance lost 1.4 and 1.1 kg of body weight, respectively. The body weight gains of all other male and female dogs administered test substance in their diet were considered to be comparable to the body weight gains of the respective control animals in this study. During the first week of the study, there was a clear reduction in food consumption, indicating an aversion to the treated diets, in both the male and female dogs administered diets containing 40,000 ppm test substance. Thereafter, there continued to be evidence of aversion to the treated diets. In males, this was evidenced by a slight reduction in food consumption in all treatment groups. In females, the aversion was evidenced by an apparent increase in food spillage, which was considered to at least partially account for the difference in actual received dosages between males and females. All dogs survived to study termination. No changes noted in physical condition or appearance were considered to be related to treatment with the test substance. At termination, body weights appeared to be reduced for males and females receiving 40,000 ppm of the test substance in the diet. However, the reduction was due to the body weight loss of the one male and one female dog noted above. Small reductions in mean values for erythrocyte, hemoglobin and hematocrit were observed in both male and female dogs in the 40,000 ppm treatment group relative to the corresponding mean control values at one or more intervals of analysis. The differences were slight, a slight difference was also observed in the pre-study measurements, and the values were within historical control range. Therefore, the toxicological significance of the changes in hematology measurements was unclear. At all analysis intervals during the treatment period, mean cholesterol values for male and female dogs in the 40,000 ppm treatment group were reduced relative to the corresponding control group. The mean cholesterol values for the male dogs in the 12,000 ppm treatment group were also reduced relative to the corresponding controls. No treatment-related changes in urinalysis measurements were observed. No treatment related ophthalmologic changes or clinical observations, organ weight changes, or gross necropsy observations were seen at termination. A small number of macroscopic lesions were seen in both male and female animals across dietary concentrations. These lesions were considered to be spontaneous and not related to the administration of the test article. The ratio of the weight of the pituitary gland to the body weight of males at the 12,000 ppm dietary concentration was significantly decreased relative to the control group. The ratio of the weight of the pituitary gland to the body weight of females in the 4,000 ppm dietary concentration also was significantly decreased compared to the control group. The ratio of the weight of the right adrenal gland to the brain weight of the females was significantly increased at the 4,000 ppm dietary concentration compared to the control group. These findings were not consistent, could not be correlated with microscopic findings and were considered to be either spurious or due to biological variation, and not related to the administration of the test article. A small number of non-neoplastic findings were evident in this study. Many of them occurred in single animals. Some of the more common lesions included interstitial pneumonia, parathyroid cysts, pituitary cysts, thymic atrophy, c-cell hyperplasia of the thyroid gland and mineralization of the kidneys. Multifocal mineralization of the renal medulla of the kidneys was present in both male (16/16) and female (15/16) dogs of all dietary concentrations. The above lesions are considered to be common spontaneous findings in a 13-week beagle dog study, and none of the microscopic findings were considered to be related to the administration of the test article. Reproductive organs were examined meeting the requirements for SIDS/HPV reproductive screening.
Dose descriptor:
NOAEL
Effect level:
4 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 4000 ppm: 144 mg a.i./kg/day for males and 144 mg a.i./kg/day for females
Critical effects observed:
not specified
Conclusions:
Dietary admixture to groups of 4 male and 4 female Beagle dogs up to 4% (40,000 ppm) into the diet for 91 days, resulted to questionable toxicity. The EPA concluded to NOAEL ~1635 mg/kg-bw/day (males and females).
Executive summary:

The study evaluated the subchronic oral toxicity of Varisoft 475 (75%) in a 13-week study in dogs. Four male and four female beagle dogs were offered the test substance in the diet at concentrations of 0, 4000, 12000 and 4000 ppm active ingredient for 13 weeks. Diet and water were available ad libitum, except prior to clinical pathology testing and necropsy, when diet and/or water were withheld overnight. Male animals weighed between 9.1 to 11.6 kg and females weighed 6.5 to 8.8 kg. Observations were conducted at least twice daily for mortality and overt toxicity. Detailed observations, body weights and food consumption were recorded weekly. Ophthalmological examinations were performed on all animals prior to study initiation and at study termination. Physical examinations, as well as hematological clinical chemistry and urological evaluations were conducted on all animals prior to study initiation and at monthly intervals during the study. At study termination, a thorough post-mortem examination was conducted on all dogs. A complete set of all major tissues and organs was harvested and selected organs were weight. The saved tissues were processed histologically and microscopic examination was conducted.

 

Actual dose received: 142, 366 and 1322 mg/kg/day for males; 144, 632 and 1948 mg/kg/day for females

During the 13-week treatment period, one male and one female dog administered diets containing 40,000 ppm test substance lost 1.4 and 1.1 kg of body weight, respectively. The body weight gains of all other male and female dogs administered test substance in their diet were considered to be comparable to the body weight gains of the respective control animals in this study. During the first week of the study, there was a clear reduction in food consumption, indicating an aversion to the treated diets, in both the male and female dogs administered diets containing 40,000 ppm test substance. Thereafter, there continued to be evidence of aversion to the treated diets. In males, this was evidenced by a slight reduction in food consumption in all treatment groups. In females, the aversion was evidenced by an apparent increase in food spillage, which was considered to at least partially account for the difference in actual received dosages between males and females. All dogs survived to study termination. No changes noted in physical condition or appearance were considered to be related to treatment with the test substance. At termination, body weights appeared to be reduced for males and females receiving 40,000 ppm of the test substance in the diet. However, the reduction was due to the body weight loss of the one male and one female dog noted above. Small reductions in mean values for erythrocyte, hemoglobin and hematocrit were observed in both male and female dogs in the 40,000 ppm treatment group relative to the corresponding mean control values at one or more intervals of analysis. The differences were slight, a slight difference was also observed in the pre-study measurements, and the values were within historical control range. Therefore, the toxicological significance of the changes in hematology measurements was unclear. At all analysis intervals during the treatment period, mean cholesterol values for male and female dogs in the 40,000 ppm treatment group were reduced relative to the corresponding control group. The mean cholesterol values for the male dogs in the 12,000 ppm treatment group were also reduced relative to the corresponding controls. No treatment-related changes in urinalysis measurements were observed. No treatment related ophthalmologic changes or clinical observations, organ weight changes, or gross necropsy observations were seen at termination. A small number of macroscopic lesions were seen in both male and female animals across dietary concentrations. These lesions were considered to be spontaneous and not related to the administration of the test article. The ratio of the weight of the pituitary gland to the body weight of males at the 12,000 ppm dietary concentration was significantly decreased relative to the control group. The ratio of the weight of the pituitary gland to the body weight of females in the 4,000 ppm dietary concentration also was significantly decreased compared to the control group. The ratio of the weight of the right adrenal gland to the brain weight of the females was significantly increased at the 4,000 ppm dietary concentration compared to the control group. These findings were not consistent, could not be correlated with microscopic findings and were considered to be either spurious or due to biological variation, and not related to the administration of the test article. A small number of non-neoplastic findings were evident in this study. Many of them occurred in single animals. Some of the more common lesions included interstitial pneumonia, parathyroid cysts, pituitary cysts, thymic atrophy, c-cell hyperplasia of the thyroid gland and mineralization of the kidneys. Multifocal mineralization of the renal medulla of the kidneys was present in both male (16/16) and female (15/16) dogs of all dietary concentrations. The above lesions are considered to be common spontaneous findings in a 13-week beagle dog study, and none of the microscopic findings were considered to be related to the administration of the test article. Reproductive organs were examined meeting the requirements for SIDS/HPV reproductive screening.

 

NOEL = 4,000 ppm (143 mg/kg/day)

LOAEL (LOEL) 12,000 ppm (366 and 632 mg/kg/day for males and females, respectively)

 

Evaluation by EPA concluded that:

Decreased food consumption observed in all treatment groups, most likely reflects decreased palatability in the treated diet. A significant decrease in body weight was observed in one male and one female at the highest dose. Study authors attributed observed decreases in mean cholesterol levels at or above 366 mg/kg-bw/day to decreased food intake. Macroscopic lesions (type not specified) were observed in all treatment groups; however, these lesions were not dose-related. Other findings reported in this study include significantly increased relative adrenal gland weight in females treated at 144 mg/kgbw/ day and significantly decreased relative pituitary gland weight in males and females at 1322 and 144 mg/kg-bw/day, respectively (level of significance not specified). These effects most likely reflect biological variation. No treatment- related histopathology was observed.

NOAEL ~ 1635 mg/kg-bw/day (males and females)

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:
01 September 2009 - 15 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD 422 guidelines and GLP principles.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 12 weeks.
At start treatment, animals were approximately 12 weeks old instead of approximately 10 weeks. A slight deviation in age does not affect the study integrity. Mating started shortly after the animals had attained full sexual maturity according to the OECD 422 guideline.
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm). This also accounts for the Recovery males for the complete treatment period.
Mating: Females were caged together with Main males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Main males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/sex/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During activity monitoring, animals were housed individually in Macrolon cages (MIII type; height 15 cm) with sterilised sawdust as bedding material. No cage-enrichment was provided during overnight activity monitoring.

- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 – 22.0°C
- Humidity (%): 28 - 79%
Temporary deviations from the minimum level of relative humidity occurred in the animal room. Laboratory historical data do not indicate an effect of the deviations.
- Air changes (per hr): approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour and 6 minutes) occurred due to performance of functional observations in the room.

On Day 2 or 4 of lactation, the maximum allowed deviation from the dark period of 1 hour was exceeded with a maximum of approximately 6 minutes for the 5 selected females with live pups per group. These temporary deviations from the light/dark cycle were considered not to have affected the study outcome.

IN-LIFE DATES: From: 01 September 2009 to 15 October 2009
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for density of the test substance and specific gravity of the vehicle (1.036 ).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.
- Concentration in vehicle: 2, 6 and 20 mg/mL

- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during treatment phase (02 October 2009) according to a validated method (NOTOX project 491557). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation was = 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

RESULTS:
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation = 10%). Formulations at the entire range were stable when stored at room temperature for at least 6 hours.
Duration of treatment / exposure:
Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination or up to the day prior to start of the recovery period for Recovery males. Females were exposed for at least 42 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Remarks:
Doses / Concentrations:
0, 10, 30 and 100 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 and an extra 5 males for Group 1 and 4. The study included a recovery phase for males only. These animals were not mated and, consequently, were not used for the assessment of reproduction/developmental toxicity.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of the dose range finding/MTD study (NOTOX Project 491555; see endpoint study record 7.5.1: repeated dose toxicity: oral.rat_NOTOX 491555)
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (early morning/late afternoon)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, between approximately 1 and 2 hours after dosing and once daily during the recovery period, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually.

On Day 27 of the repro period, clinical signs of females were inadvertently recorded under the online data collection project number for males (NOTOX project 491961). For males a second observation was conducted on this day, together with clinical observations of the females. Females: clinical observations were conducted; no clinical signs were noted for the females. Males: additional information.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
Weekly, for males and females. Food consumption was not recorded during the mating period, except for Recovery males. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY: Yes
(average food consumption [per animal per day]/average body weight per cage)x1000.

WATER CONSUMPTION: No
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes, iso-flurane
- Animals fasted: Yes, but water was available
- How many animals: 5 animals/sex/group (females with live offspring only).
- Parameters examined were: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes,eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes, iso-flurane
- Animals fasted: Yes, but water was available
- How many animals: 5 animals/sex/group (females with live offspring only).
- Parameters examined were: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity.
Sacrifice and pathology:
SACRIFICE
All animals were fasted overnight (with a maximum of approximately 21 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy were anaesthetised using iso-flurane (Abbott Laboratories Ltd., Hoofddorp, The Netherlands) and subsequently exsanguinated.

Necropsy was conducted on the following days:
Females which delivered: Lactation Day 5.
Females which failed to deliver: Post-coitum Day 26-27 (females with evidence of mating)
Males (Main): Following completion of the mating period (a minimum of 28 days of dose administration).
Males (Recovery): After a recovery phase of 14 days.

Several animals were necropsied later than after a maximum of 20 hours fasting, i.e. with a maximum of 1 hour and 59 minutes. The fasting period was only slightly longer and was considered not to have adversely affected the clinical laboratory, macroscopic or microscopic findings.

GROSS PATHOLOGY: Yes
All parental animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The number of former implantation sites and corpora lutea were recorded.

Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):

Selected 5 animals/sex/group and all Recovery males: Identification marks: not processed, Ovaries. Adrenal glands, Pancreas, Aorta, Peyer's patches (jejunum, ileum) if detectable, Brain (cerebellum, mid-brain, cortex), Pituitary gland, Caecum, Preputial gland, Cervix, Prostate gland, Clitoral gland, Rectum, Colon, (Salivary glands - mandibular, sublingual), Duodenum, Sciatic nerve, Epididymides*, Seminal vesicles including coagulating gland, Eyes with optic nerve (if detectable) and Harderian gland*, Skeletal muscle, (Skin), (Female mammary gland area), Spinal cord (cervical, midthoracic, lumbar), Femur including joint, Spleen, Heart, Sternum with bone marrow, Ileum, Stomach, Jejunum, Testes*, Kidneys, Thymus, (Lacrimal gland, exorbital), Thyroid including parathyroid (if detectable), (Larynx), (Tongue), Liver, Trachea, Lung, infused with formalin, Urinary bladder, Lymph nodes (mandibular, mesenteric), Uterus, (Nasopharynx), Vagina, Oesophagus, All gross lesions.

All remaining animals and females which failed to deliver$:

Identification marks: not processed, Prostate gland, Cervix, Seminal vesicles including coagulating glands, Clitoral gland, Testes*, Epididymides*, Uterus, Ovaries, Vagina, Preputial gland, All gross lesions.

* Fixed in modified Davidson's solution (prepared at NOTOX using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial)(all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.
$ In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique (Salewski, 1964) in order to detect any former implantation sites (Salewski staining prepared at NOTOX using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

ORGAN WEIGHTS: Yes
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:

Selected 5 animals/sex/group and all Recovery males: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix),
Kidneys, Prostate*, Liver, Seminal vesicles including coagulating glands*, Ovaries, Thyroid including parathyroid*.
* weighed when fixed for at least 24 hours.

All remaining males: Epididymides, Testes.

HISTOTECHNOLOGY:
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Of the selected 5 males/group of the control and high dose Main group additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
-The preserved organs and tissues of the selected 5 Main animals/sex of Groups 1 and 4.
-Mesenteric lymph nodes and ileum of selected Group 2 and 3 males and females and Recovery group 1 and 4 males, liver of selected Group 2 and 3 males and Recovery group 1 and 4 males and thymus of selected Group 2 and 3 females (based on (potentially) treatment-related histopathological changes in Group 4).
-The additional slides of the testes of the selected 5 Main males of Groups 1 and 4 to examine staging of spermatogenesis.
-The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testis, uterus, and vagina) of all Main animals that failed to conceive:
Group 1: One male and one female
Group 2: One male and one female
-All gross lesions of all animals (all dose groups).

Inadvertently, a few tissues were not available for histopathology. Reasons included that these tissues were not discernable at necropsy or trimming, or were erroneously not collected at necropsy. Tissues were listed in raw data and pathology report. Sufficient data was available for evaluation.

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Statistics:
The following statistical methods were used to analyse the data:
-If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
-The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
-The Fisher Exact-test (Fisher, 1950) was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional 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
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period.

There were no clinical signs of toxicity noted during the observation period.

One male and two females at 100 mg/kg/day showed lethargy, flat posture and/or piloerection on one or several days during treatment. Given the low incidence of these findings among these animals and absence of these findings among other animals of the same dose group, this was considered no be of no toxicological relevance.

Other incidental findings included salivation, rales, alopecia, a wound and scabs. These were considered to be within the normal range of biological variation for rats of this age and strain, and considered signs of no toxicological relevance.

No clinical signs were noted among control males and males at 10 and 30 mg/kg/day.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically significant changes in body weight (gain) occurred.

Males at 100 mg/kg/day showed a slight, but statistically significant, lower mean body weight and body weight gain during the treatment period, which recovered to control levels during the recovery phase. Some of these males showed (notable) weight loss during the premating period, which (largely) recovered during the mating/repro period. Overall, these changes were slight and reversible in nature. The statistically significant lower body weight gain on Day 4 of Lactation occurred in the absence of a dose- and time related trend, and was very slight in nature. Hence, these variations in body weight (gain) were not considered to be of toxicological relevance.

Body weight and body weight gain of males at 10 and 30 mg/kg/day was similar to control levels throughout the observation period.

FOOD CONSUMPTION
No toxicologically significant changes in food consumption before or after allowance for body weight occurred.

Food consumption before or after allowance for body weight of males at 100 mg/kg/day appeared slightly lower than controls during the premating and mating/repro phase. However during the recovery phase food intake levels were similar to controls. Therefore, these changes were not considered to be of toxicological relevance.

Males at 10 and 30 mg/kg/day, and females at 10, 30 and 100 mg/kg/day showed normal food consumption levels, before or after allowance for body weight. The statistically significant lower food consumption before or after allowance for body weight of females at 100 mg/kg/day over Days 4-7 of the post coitum phase was of a very slight nature and within the range considered normal for rats of this age and strain. Food intake of these females remained similar to control levels during the remainder of the post coitum and lactation phase.

HAEMATOLOGY
No toxicologically significant changes in haematological parameters were observed.

The statistically significant lower reticulocyte counts and higher mean corpuscular haemoglobin concentration (MCHC) in males at 100 mg/kg/day occurred in the absence of concurrent changes in red blood cell parameters. These changes, along with the statistically significant higher prothrombin time (PT) of these males were of a slight nature (i.e. within the range considered normal), and were absent at the end of the recovery phase. The statistically significant higher partial thromboplastin time (APTT) of females at 30 mg/kg/day occurred in the absence of a dose-related trend. No toxicological relevance was therefore ascribed to these changes.

CLINICAL CHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished animals at 100 mg/kg/day from control animals at the end of the treatment period:
- Higher alanine aminotransferase activity (ALAT) in males,
- Higher aspartate aminotransferase activity (ASAT) in males* and females ,
- Lower total protein level in males *,
- Higher creatinine level in males (not statistically significant),
- Lower urea level in females*.
The changes in males at 100 mg/kg/day had resolved at the end of the recovery period.
* mean within range considered normal.

The statistically significant lower alkaline phosphatase activity (ALP) in females at 100 mg/kg/day was within the range considered normal and control levels were considered to be slightly high. Moreover, a lower activity of this parameter is generally considered not to represent a change of toxicological relevance. The statistically significant lower calcium level of males at 100 mg/kg/day, lower albumin level of males at 10 mg/kg/day, and lower potassium level of females at 30 mg/kg/day occurred in the absence of a dose-related trend and the means remained within the range considered normal. The statistically significant higher inorganic phosphate level in males at 100 mg/kg/day at the end of the recovery period was absent at the end of the treatment period. No toxicological relevance was ascribed to these changes.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.

The variation in motor activity did not indicate a relation with treatment.

ORGAN WEIGHTS
Males at 100 mg/kg/day showed a statistically significant lower prostate and seminal vesicle weight, and lower prostate to body weight ratio at the end of the treatment period. These changes were absent at the end of the recovery period for males.

Organ weights and organ to body weight ratios of males at 10 and 30 mg/kg/day and of females at 10, 30 and 100 mg/kg/day were similar to those of control animals.

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations.

Incidental findings included yellowish-soft nodules on the epididymides, a hard nodule on the liver, reduced size of the preputial glands, prostate or thymus, red discolouration of the mesenteric lymph nodes, diaphragmatic hernia of the left median lobe of the liver, red foci on the lungs or kidneys, a cyst on the kidneys, watery-clear cysts on the ovaries, a red-brown focus on the clitoral glands, yellowish discolouration of the papillary process of the liver, a black focus on the thymus and alopecia. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological significance.

No macroscopic abnormalities were observed among males at 30 mg/kg/day.

HISTOPATHOLOGY:
The following microscopic findings were considered to be related to treatment:
- Ileum: foamy macrophage foci in 5/5 males (minimal to slight) and in 5/5 females (minimal to slight) at 100 mg/kg/day.
- Mesenteric lymph node: slightly increased incidence and degree of macrophage foci in males and females at 100 mg/kg/day.
- Thymus: increased incidence of lymphoid atrophy in females at 100 mg/kg/day.
At the end of the 14-day recovery period for males, foamy macrophage foci in the ileum had completely resolved, whilst macrophage foci in the mesenteric lymph node persisted at slightly increased incidence and severity, with the mean severity grade being higher than encountered at the end of treatment.

All other microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar rats of this age and strain.

No abnormalities were seen in the reproductive organs of non-fertile animals which could account for infertility.

The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.
Dose descriptor:
NOAEL
Remarks:
(F0)
Effect level:
>= 30 - < 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the increased incidence/severity of macrophage foci in the mesenteric lymph node at both the end of treatment and recovery period in males.
Critical effects observed:
not specified
Conclusions:
Based on the increased incidence/severity of macrophage foci in the mesenteric lymph node at both the end of treatment and recovery period in males, a parental No Observed Adverse Effect Level (NOAEL) of 30 mg/kg/day was derived.
Executive summary:

Tall oil diethylenetriamine imidazoline was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 10, 30 and 100 mg/kg/day. The males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). There was a 2 week recovery period for 5 animals of Group 1 and 4. The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation.

Formulation analysis showed that the formulations were prepared accurately and homogeneously and were stable for at least 6 hours at room temperature.

Parental results:

The changes in clinical biochemistry parameters in animals at 100 mg/kg/day at the end of the treatment period were generally slight in nature (i.e. mostly within the normal range) and were fully reversible after a 14-day recovery period in males. Moreover, no morphological changes were observed that would support these changes which included higher alanine and aspartate aminotransferase activity and creatinine level in males, and lower total protein and urea level in males and females respectively. Therefore, these changes were not considered to be of toxicological relevance.

The lower prostate and seminal vesicle weight, and lower prostate to body weight ratio at the end of the treatment period in males at 100 mg/kg/day were absent at the end of the recovery period in males, and were not supported by any histopathological lesions or reproductive toxicity. No toxicological relevance was ascribed to these findings.

Histopathology revealed foamy macrophage foci in the ileum of all selected males and females at 100 mg/kg/day, a slightly increased incidence and degree of macrophage foci in the mesenteric lymph node of both sexes at 100 mg/kg/day, and increased incidence of lymphoid atrophy in the thymus of females at 100 mg/kg/day. At the end of the 14-day recovery period for males, foamy macrophage foci in the ileum had completely resolved, whilst macrophage foci in the mesenteric lymph node persisted at higher severity than observed at the end of treatment. Given the persistence of this finding it was considered to be of an adverse nature.

No toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, haematology and macroscopic examination).

Based on the increased incidence/severity of macrophage foci in the mesenteric lymph node at both the end of treatment and recovery period in males, a parental No Observed Adverse Effect Level (NOAEL) of 30 mg/kg/day was derived.

As explained in the category justification, For cross-reading in general use is made with data of same or lower EA-length where available, and that of Tall oil + DETA representing the worst case.

This dossier is for the substance "Fatty acids C18 unsat, reaction products with triethylenetetramine" (or TO + TETA). As for the substance itself no toxicological information is available, cross-reading has been applied to TO + DETA.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Consistent results from all studies within the whole group of Amidoamine/imidazolines (AAI). Available studies are OECD Guidline compliant and performed under GLP.
System:
gastrointestinal tract

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

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

The available data available within the group of Amidoamines/imidazolines (AAI) substances indicate that for AAI substances based on shorter polyethyleneamines (EA), higher toxicity is observed compared to AAI based on longer EA. The forming of imidazoline itself does not seem to play a significant role. For cross-reading purposes between substances of AAI, Fatty acid reaction product with diethylene-triamine (AAI-DETA) therefore represents the worst case. In series of 28-day and combined repeated dose/reproduction screening toxicity studies (OECD 422) AAI-DETA has shown the highest level of toxicity. (See also document in support of category justification).

 

A combined repeated dose/reproduction screening toxicity study according to OECD 422 has been performed with AAI-DETA administered by daily oral gavage to rats at dose levels of 0, 10, 30 and 100 mg/kg/day. The males were exposed for 28 days. The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42-55 days). For the male also 14-day recovery groups were added to the control and HD group.

Results: The changes in clinical biochemistry parameters in animals at 100 mg/kg/day at the end of the treatment period were slight in and were fully reversible after a 14-day recovery period in males. Moreover, no morphological changes were observed that would support these changes which included higher alanine and aspartate aminotransferase activity and creatinine level in males, and lower total protein and urea level in males and females respectively.

Histopathology revealedan increased incidence and severity of foamy macrophage foci in the mesenteric lymph node at the end of treatment, and in males also after the after the recovery period.Also an increased incidence of lymphoid atrophy in the thymus of females was seen at 100 mg/kg/day.

No toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, haematology and macroscopic examination).

 

The study report concludes to a parental NOAEL of 30 mg/kg/day based on the increased incidence and severity of foamy macrophage foci in the mesenteric lymph node at the end of treatment, and in males also after the after the recovery period. However, considering that the incidence and severity of macrophage as observed at 30 mg is somewhat higher than at 10 mg and the control group, the NOAEL could also have been set at 10 mg/kg bw.

 

A subsequent 90-day (OECD 408, GLP) study was performed applying the same dose levels of 0, 10, 30 and 100 mg/kg/day AAI-DETA to groups of 10 animals/dose/sex.

The results from this study are comparable to those obtained from the earlier OECD 422 study: The first effect to occur is the presence of foamy macrophages in the lamina propria of the small intestines and mesenteric lymph nodes. These effects are considered to represent a local, porte d’entrée related effect due to the route of application, rather than a systemic effect.

The magnitude of these effects as observed at the lowest dose level of 10 mg/kg, is often also observed in control groups in general, and was also seen in the control group of the OECD 422 study performed before on the same substance. These effects are therefore not considered adverse. As no other effects were observed, this dose level is considered to represent the NOAEL.

At higher dose levels an increase in foamy macrophages is observed beyond levels that can occur in control groups, as well as lower mean body weight and body weight gain, especially in the males, with lower food intake, essentially during the second half of the treatment period.

 

Comparing the effects of foamy macrophages between the OECD 422 and the 90-day study, show that the NOAEL level around 10 is rather comparable between the two studies, but that with increase of the duration in the 90-day study, an increase in this response is seen at 30 and 100 mg/kg bw/d.

 

Other available data from the group of AAI substances, including 90-day studies in rat and dogs on a similar substance, indicate low toxicity.

 

Dermal route

For dermal exposures, effects are rather characterized by local corrosive effects that are related to duration, quantity and concentration, than by systemic toxicity due to dermal uptake. The mode of action of for AAI follows from its structure, consisting of an apolar fatty acid chain and a polar end of a primary amine from the polyethyleneamine. The structure can disrupt the cytoplasmatic membrane, leading to lyses of the cell content and consequently the death of the cell.

The AAI are protonated under environmental conditions which causes them to strongly adsorb to organic matter. This all leads to a low dermal absorption.

 

Inhalation:

Physical-chemical properties of polyamines indicate a low likelihood for exposure via inhalation, with a boiling point > 300 °C and low vapour pressure (0.00017 mPa at 25°C).Any inhalation exposures would therefore only be possible in the form of aerosol, consisting of larger droplets depositing in upper airways which could result to local irritation or corrosion

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Recent study of high quality and of longest duration.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

Likelihood of exposures via inhalation is low considering the high boiling point (> 300 °C) and very low vapour pressure (0.00017 mPa at 25°C),as well as low possibility of exposure to aerosols or droplets of an inhalable size. Furthermore, as the substance is classified as corrosive, local effects will be dose-limiting and preclude sufficient uptake for systemic effects to develop. The potential for inhalation is not significant to justify this study.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:

Lack of exposures

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

Substance is corrosive. Effects will be characterized by local corrosive effects that are related to duration, quantity and concentration, rather than by systemic toxicity due to dermal uptake. Because of the corrosive and sensitising properties, exposures are likely to be limited.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:

Lack of exposures: use is limited to industrial and professional users where because of its corrosive and sensitising properties sufficient measures will be taken to prevent dermal exposure.

Justification for classification or non-classification

Classification for STOT-RE Cat. 2 is required in case of significant toxic effects at levels = 100 mg/kgbw/d in case of standard 90-day study. In case of 28-day studies this can be multiplied by 3.

The available 90 -day study on AAI-DETA did not indicate severe toxicity at 100mg/kg/day. Also other available data do not suggest severe toxicity at levelsrequiring consideration for classification for STOTS-RE.