Registration Dossier

Administrative data

Description of key information

Oral:

- OECD 422; GLP; rat (Wistar); 40, 125, 375 mg/kg bw/day; no mortality observed; NOAEL 375 mg/kg bw/day (2018)


Dermal:
-
Equivalent to OECD 411; GLP not specified; Sprague-Dawley rats; 20, 66.6, 200 mg/kg/day; no mortality observed; NOAEL (local) 20 mg/kg/day; NOAEL (systemic) 66.6 mg/kg/day based on reduced body weight (1982)


Inhalation:
No data available

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Mar - Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliance
Reason / purpose:
reference to same study
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)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed on August 2019
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: ReachCentrum SA
- Batch number of test material: 180003P040
- Expiration date of the lot/batch: 31. Dec 2018
- Purity/Composition: 100% (UVCB)
- Appearance: clear, colorless liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Stability under storage conditions: until 31. Dec 2018
- Stability of the test substance in the vehicle: Stability for at least 24 hours at room temperature protected from light, stability for at least 8 days in the refrigerator, and stability of 0.5 mL samples for at least 3 weeks in the freezer (≤ -15°C) is confirmed over the concentration range 1 to 200 mg/mL (solutions)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han), outbred, SPF-Quality
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source.
This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (males), 13 weeks (females)
- Weight at study initiation: males: 251- 322 g; females: 198- 263 g
- Housing: individually (females during the post-mating phase and lactation phase with the pups) and grouping (pretest, males during the post-amting phase
- Diet: ad libitum; pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum
- Acclimation period: for 5 days prior to start of the pretest period (females) or 5 days before the commencement of dosing (males).

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water is performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40-70 (daily mean relative humidity of 36 to 60%)
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 8, 25, 75 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure. Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Duration of treatment / exposure:
males: 29 days; females: 50-62 days
The duration of treatment covered a 2-week premating period and mating in both sexes as well as entire gestation and the duration of pregnancy and at least 14 days after delivery,
up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 40-53 days.
Frequency of treatment:
daily (7d/week)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
375 mg/kg bw/day (nominal)
No. of animals per sex per dose:
- in total 10 animals/ sex/ dose
- 5 animals /sex/ dose were selected for functional tests (males only), clinical pathology, collection of full list of organs/tissues at macroscopic examination, organ weights (full list) and histopathology (full list)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 16-day dose range finder with oral gavage administration of the test item.


Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
F0 animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily (F0 and pubs)

BODY WEIGHT: Yes
- Time schedule for examinations: F0: First day of treatment (prior to dosing) and weekly thereafter (after dosing); F1: Live pups were weighed individually on PND 1, 4, 7 and 13.
- Body Weight Gains: Calculated against the body weight on Day 1 (premating, mating and lactation periods) or Day 0 (postcoitum period).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Time schedule for examinations: quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
- Relative Food Consumption Calculated against the body weight for scheduled intervals.

WATER CONSUMPTION: not quantitative
- Subjective appraisal was maintained during the study

HAEMATOLOGY: Yes, F0 animals
- Time schedule for collection of blood: day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, males only (maximum of 24 hours)
- How many animals: 5/sex/group
- Parameters checked in table 1 were examined.

COAGULATION
Blood plasma of F0 animals was analyzed for Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, males only (maximum of 24 hours)
- How many animals: 5/sex/group
- Parameters checked in table 2 were examined.

OTHER:
Functional observational battery (FOB): Males only
- Functional tests were performed on the selected 5 males (F0) during Week 4 of treatment (after dosing, after completion of clinical observations)
- Examined parameters:
• Hearing ability
• Pubillary reflex
• Static righting reflex
• Fore- and hind-limb grip strength
• Locomotor activity

Estrous cycle:
- Daily vaginal lavage was performed for all females (F0) beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.

Female reproduction and delivery data
From the mating period onwards, the following parameters were recorded for each female (F0): male number paired with, mating date, confirmation of pregnancy and delivery day.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care

Thyroid hormones
- Time schedule for collection of blood: All F0 animals on scheduled necropsy, PND 14-16 and PND 4 for 2 pubs per litter
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: males only ( mximum of 24 h)
- How many animals: All F0 animals, 2 pubs per litter on PND 4 and PND 4-16
- For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no adverse changes in T4 were noted in F0- males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight were recorded
- Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16

OTHER:
Sex was externally determined for all pups on PND 1 and 4.
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
All male pups in each litter were examined for the number of areola/nipples on PND 13.


Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3 and 4, Organ weights)

HISTOPATHOLOGY: Yes (see table 5 for collected tissue)
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
- Selected animals: Tissues identified in Text Table 5 (except animal identification, aorta, nasopharynx, esophagus, harderian
gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue).
- Males that failed to sire (except for males which were selected), females that failed to deliver pups and females with total litter loss: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.
- Females with total litter loss: Mammary gland.
- Remaining animals: Gross lesions/masses.
Statistics:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation seen after dosing among animals of all dose groups was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Further observed clinical signs affecting the skin/fur (alopecia, scabs), the eye (Chromodacryorrhoea) and breathing (rales) were observed.
Mortality:
no mortality observed
Description (incidence):
One female of the control group (no. 49) was euthanized on Lactation Day 1, as she had a total litter loss (at first litter check she had only dead pups).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weight gain was increased in males in the second week of treatment after dosing of 125 and 375 mg/kg, and in the fifth week of treatment after dosing with 375 mg/kg. These changes in body weight gain were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment and values were within the historical control range
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The statistically significant change in relative food consumption in 40 mg/kg females during post-coitum Days 17-20 was considered to be unrelated to treatment, since no trend was apparent regarding dose and duration of treatment.
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A decreased number of reticulocytes was observed in females at 125 mg/kg, which was considered unrelated to administration of the test item due to absence of a dose-related trend response.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males: The following statistically significant increases were noted for treated males (relative changes in mean values as compared to the concurrent control group are indicated between parentheses):
-increased total protein at 375 mg/kg (6%)
-increased albumin at 375 mg/kg (6%)
-increased calcium at 375 mg/kg (4%)
-increased urea at 40 and 375 mg/kg (27% and 30%, respectively)
The changes in total protein, albumin and calcium were minimal and remained within the historical control range. No dose related trend was observed for the increase in urea and values remained within the historical control range. In addition, a slight increase in sodium was observed at 125 mg/kg (1%), which occurred in the absence of a dose related trend.

No treatment-related changes were noted in clinical chemistry parameters in females
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher liver weights (absolute and relative to body weights) were noted in the 375 mg/kg/day group males.
There were no other test item-related organ weight changes.
The significant relative prostate gland weight decrease and liver weight increase in the 40 mg/kg/day treated males was considered incidental and not related to treatment in absence of a dose-related trend.

for details see table 6
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related irregular surface was observed in the (fore)stomach in 2/10 males treated at 125 mg/kg/day and in 10/10 males and 2/10 females treated at 375 mg/kg/day.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Watery fluid in the uterus, found in one control female and 3 females treated with 125 mg/kg, is related to a stage in the estrous cycle and is a normal finding.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the (fore)stomach of males and females and the liver and kidneys of males.

Stomach, squamous cell hyperplasia was present in 2/5 males starting at 125 mg/kg/day up to marked degree and in females at 375 mg/kg/day up to moderate degree. This correlated with the macroscopic irregular surface.
Stomach, ulcer forestomach was present in males starting at 125 mg/kg/day at minimal degree.
Stomach, inflammation forestomach was present in males starting at 125 mg/kg/day up to moderate degree.

Liver, hepatocellular hypertrophy was present in males treated at 375 mg/kg/day at minimal degree. This correlated with the increased liver weight. Due to the absence of any indicators of cellular degeneration. The changes in the liver were not considered adverse at current severities.

Kidneys, an increased incidence and severity of hyaline droplet accumulation was present in males treated at 375 mg/kg/day up to slight degree. The increased hyaline droplet accumulation in the male kidneys was not accompanied by indicators of tubular damage and therefore this was considered to be nonadverse.

Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
- Functional observation parameters were not considered to be affected by treatment in males up to 375 mg/kg.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Forelimb and hind limb grip strength was similar between control and treated animals. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
- Coagulation parameters (prothrombin time and activated partial thromboplastin time) of treated rats were considered not to have been affected by treatment.
-Serum levels of T4 in F0 males were increased at 125 and 375 mg/kg (35% and 37% increase in mean values compared to concurrent control, respectively). These values remained within the historical control range and the control value was on the lower limit of this range. In addition, no effect was observed in respect to thyroid weight, therefore, this effect was considered not toxicologically relevant.
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
375 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects up to and including the highest tested dose
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no

The various analyses confirmed

- Accuracy: The concentrations analyzed 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%). No test item was detected in the Group 1 formulation.

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

Table 6: Mean Percent Liver Weight Differences from Control Groups

Dose level in mg/kg bw/d

Males

Females

40

125

375

40

125

375

LIVER

 

absolute

13

4

31**

-7

-4

1

Relative to bodyweight

9*

5

25**

-6

-1

0

*: P < 0.05, **: P < 0.01

Table 7: Summary Test Item-Realted Microcopic Findings-(fore)stomach

Dose level in mg/kg bw/d

Males

Females

0

40

125

375

0

40

125

375

STOMACHa

5

5

5

10

5

5

5

5

Hyperplasia squamous cell

 

Slight

-

-

-

-

-

-

-

1

Moderate

-

-

2

6

-

-

-

1

Marked

-

-

-

4

-

-

-

-

Ulcer forestomach

 

minimal

-

-

1

2

-

-

-

-

Inflammation forestomach

 

Minimal

-

-

1

6

-

-

-

-

Moderate

-

-

1

-

-

-

-

-

a = Number of tissues examined from each group

Table 8 Summary Test Item-Related Microscopic Findings – Liver and Kidneys – Males

 

Males

Dose level in mg/kg bw/d

0

40

125

375

LIVERa

5

5

5

5

Hepatocellular hypertrophy

 

minimal

-

-

-

4

KIDNEYa

5

5

5

5

Hyaline droplet accumulation

 

minimal

1

1

2

3

slight

-

-

-

2

a= Number of tissues examined from each group

Conclusions:
Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) were established:
Parental local NOAEL: 40 mg/kg (based on findings in the (fore)stomach)
Parental systemic NOAEL: at least 375 mg/kg due to the absence of adverse toxicity in the study for both sexes.
Executive summary:

Wistar Han rats were treated with the test item by daily oral gavage at dose levels of 40, 125 and 375 mg/kg according to OECD 422 and in compliance with GLP. The rats of the control group received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-62 days). Females that failed to deliver pups were treated for 40-53 days.

Parental toxicity was observed in the (fore)stomach of males from 125 mg/kg and in females at 375 mg/kg.

These changes consisted of ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females.

Other treatment-related but non-adverse changes were observed in the liver at microscopic examination. An absolute increase of 31% and a relative increase of 25% in liver weight was observed at dose 375 mg/kg. At microscopic examination, hepatocellular hypertrophy in the liver was observed at minimal severity and was in the absence of any indicators of cellular degeneration. The changes in the liver were not considered adverse at current severities. In the kidneys an increase in hyaline droplet accumulation was recorded in males which was considered to likely represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules (Alden et al., 1991). This male rat specific protein is not present in female rats nor in higher mammals, including man (Sahota et al., 2013). The increased hyaline droplet accumulation in the male kidneys at 375 mg/kg/day was not accompanied by indicators of tubular damage and therefore this was considered to be nonadverse.

Functional observations were not performed for females and therefore, possible treatment related effects on the functional parameters could not be evaluated.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations (males), body weight, food consumption, clinical laboratory investigations (including male T4 thyroid hormone levels), macroscopic examination and organ weights).

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, no systemic no-observed-adverse-effect level (NOAEL) were established under the conditions of this study. The Parental local NOAEL of 40 mg/kg is based on the findings in the (fore)stomach. Due to the absence of systemic toxicity up to and including the highest tested dose of 375 mg/kg bw/d no systemic NOAEL could be derived.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
375 mg/kg bw/day
Study duration:
subacute
Species:
rat

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

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jun - Sep 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 424 (Neurotoxicity Study in Rodents)
Principles of method if other than guideline:
The subchronic dermal application of the test substance to the backs of Sprague-Dawley rats was studied to assess potential neurotoxic and other local and systemic effects.
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): C-178
- Physical state: liquid
- Analytical purity: 100% active ingredient
- Storage condition of test material: room temperature
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 41 days (age: 28 days at receipt)
- Weight at study initiation: week 0 males: 157-163 g; week 0 females: 133-139 g
- Housing: individually in elevated stainless steel cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
other: not occluded
Vehicle:
corn oil
Details on exposure:
TEST SITE
- Area of exposure: the back of the rats
- % coverage: no data
- Type of wrap if used: not occluded no further data
- Time intervals for shavings or clipplings: all animals were clipped ca. 23 h prior to initial dose. The animals were reclipped when necessary.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.077 ml/kg
- Concentration (if solution): 1.0, 3.33 and 10.0 %
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): immiscible with water
- Amount(s) applied (volume or weight with unit): 2.077 ml 7kg of the test substance in corn oil
- Concentration (if solution): 1.0, 3.33 and 10.0 % of the test substance in corn oil

USE OF RESTRAINERS FOR PREVENTING INGESTION: no data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
the remaining samples of weekly dosing solutions for each dose were returned to the sponsor for analysis, no further data
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily, 5 days/week
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
66.666 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: for mortality and gross signs of toxicologic or pharmacologic effects

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly including signs of local or systemic toxicity, pharmacologic effects and palpation for tissue masses

BODY WEIGHT: Yes
- Time schedule for examinations: twice pretest, weekly during treatment and terminally (after fasting)


OTHER:
- blood was obtained from over night fasted rats via venipuncture of the orbital sinus under light ether anestehsia, the same animals were used that were intended for formalin-fixation (5/sex/dose).
- hematology upon termination: hemoglobin, hematocrit, erythrocytes, clotting time, total and differential, leukocytes, erythrocytes morphology
- clinical chemistry upon termination: serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, blood urea nitrogen, fasting glucose, total protein, total bilirubin, sodium, potassium, calcium, inorganic phosphorus
- urinanalysis 6 days before termination: gross appearance, specific gravity, pH, protein, glucose, occult blood
Sacrifice and pathology:
One-half of the animals were sacrificed by exsanguination under light ether anesthesia, and selected organs and tissues were fixed
in formalin. Organ and organ-body weight ratios (adrenals, brain, liver, kidney, heart, spleen, testes with epididymides, ovaries) were determined on all of these animals only. Histopathological evaluations of 10 organs or tissues (liver, kidney, lung, heart, stomach, adrenal, pituitary, testes, ovaries,
spleen and skeletal muscle) were conducted on all formalin-fixed control animals and the high dose animals. The remaining animals were perfused
intravenously with glutaraldehyde under sodium pentobarbital anesthesia . Quantitative assessments of teased tibial nerve preparations were
performed on all glutaraldehyde-perfused animals in control animals and the high dose animals. In addition, brain, spinal cord and sciatic nerve were evaluated microscopically (hematoxylin and eosin and Luxol-fast blue staining) from these same animals.
Other examinations:
- neurologic functions were evaluated monthly
- Parameters examined according to a scoring system: posture, gait, muscular tone, reflexes (corneal), righting and toe-pinch
- no further data
Statistics:
Body weight, hematology and clinical chemistry parameters, organ weights and organ/body weight ratios were analyzed. Mean values of
all dose groups were compared to control at each time interval.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Weekly physical examinations of all animals failed to indicate any toxic effects of the test material other than irritation at the application site. Alopecia observed on the forepaws and legs was the most common finding in both sexes; however, the incidence was spread over all test groups, including control.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
The test substance produced moderate levels of irritation, in a dose-related manner, beginning the first week of the study. Males were generally more susceptible than females to the dermal effects of the test substance throughout the study. Dermal effects (erythema and eschar formation) were only scored as present or absent therefore, the number of times per week the effects were noted was used as a general indication of the severity of the dermal observations.
- 20 mg/kg/day: erythema noted occassionally during the final 2 months; more frequently in the initial 3 weeks; exfoliation observed in approximately one-half during weeks 2 and 3 with diminishing frequency
- 66.6 mg/kg/day: erythema noted with a somewhat higher frequency than 20 mg/kg/day, frequency in females comparable to control. Exfoliation and eschar recorded for most animals by week 3.
- 200 mg/kg/day: erythema, exfoliation, and eschar seen in most animals of both sexes beginning in week 1. Atonia was observed in one males and one female, fissures present in one female and four males. A persistant fissuring was observed in one male rat from week 2 through week 7.
Males appeared somewhat more sensitive than females to erythema and eschar formation.
Mortality:
no mortality observed
Description (incidence):
All animals survived to the scheduled termination of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significantly reduced body weights in high dose male rats (versus control male rats) were noted from weeks 3 through 12, except during weeks 10 and 11. Male rat weights of the low and mid dose groups were also reduced in dose-related fashion, but the differences were not statistically significant in comparison to control values. No statistically significant effects on weight were seen in treated female rats, however, high dose female animals never exceeded 95% of the mean weights of control females after 2 weeks on test.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
None of the hematologic parameters evaluated differed significantly from control values.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Of the clinical chemical parameters which were determined at the study termination, none were affected in a manner suggestive of a treatment-effect.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Protein (2+, 100 mg/dl) was confirmed to be present in the urine of one mid-dose male and female, in two high-dose males and one high-dose female. A large amount of occult blood was also present in the urine of this one high-dose female. The specific gravity of this high-dose female and one mid-dose female was also high (>1.090). These findings suggested a possible effect of the test substance on the kidneys, but no alterations were observed in the histopathological examination to support this conclusion. Additionally, without relation to urinary volumes and creatinine, the relevance of in urinary protein concentration is questionable. All in all, the singular changes are not considered adverse by the registrant.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There was a significant downward trend in male liver weights; however, this was not evident in the liver/body weight ratio and is therefore of doubtful significance. Other organ weights and organ/body weight ratios were comparable across all groups.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Formalin-fixed rats:
No changes, gross or microscopic, were evident which could be attributed to a systemic toxic effect of the test substance. The most common spontaneous gross necropsy findings, occurring across all groups, were inflammations around the ear tags, and slight hair loss on the extremities. No unusual microscopic pathological findings were evident which could be attributed to the topical administration of the test substance.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Month 1 neurological function tests showed two high-dose males with slightly reduced corneal response. All other evaluations were normal.
Month 2 neurological function tests were unremarkable in control, low and mid dose animals (both sexes). Four high dose males and three high dose females showed slightly abnormal gait described as "stilted". A slight decreased corneal reflex was observed in four males and one female. A moderately decreased toe pinch response (hindtoes only) was also present in one male rat.
Month 3 neurological function tests showed a slightly stilted gait and altered righting reflex in one male control rat and a slightly relaxed body tone in one mid dose male rat. One male high dose animal continued to exhibit a moderately decreased toe-pinch response (hindtoes only) . All neurological observations were normal in both the control and treated female rats at Month 3.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Glutaraldehyde-perfused rats:
Histopathological examinations of hematoxylin and eosin and Luxol-fast Blue stained slides of brain, spinal cord and sciatic nerve from ten rats (5/sex) treated with the high dose of the test substance failed to reveal any treatment-related lesions when these tissues were compared to similar ones from ten control rats (5/sex). In addition, microscopic examination of 50 teased nerve fibers from the tibial nerve of ten high-dose animals (5/sex) were comparable to those of the controls. When quantitative measurements were taken of myelinated nerve fibers in cross-section of the distal sciatic nerve, a slight shift to larger diameters could be detected in high-dose males when compared to the controls. However, there was also a slight decrease in fiber diameters in treated females. The relatively large standard deviation in data from both males and females suggest that these slight changes in fiber diameters are not significant. Moreover, the absence of lesions by more conventional histopathological examinations substantiate this conclusion.
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
LOAEL
Remarks:
skin irritation
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation
Dose descriptor:
NOAEL
Remarks:
for systemic toxicity
Effect level:
66.66 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Remarks:
for systemic toxicity
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed
Critical effects observed:
yes
Lowest effective dose / conc.:
20 mg/kg bw/day (nominal)
System:
integumentary
Organ:
skin
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
The test substance did not induce neurotoxicity and no sub-chronic toxicity other than dermal irritation and decreased body weight was observed in animals, under the conditions tested. Thus, the NOAEL for local effects was considered to be 20 mg/kg/day and the NOAEL for systemic effects (based on body weight) was concluded to be 66.6 mg/kg/day.
Executive summary:

The sub-chronic dermal application of the test substance to the backs of Sprague-Dawley rats was studied to assess potential neurotoxic and other local and systemic toxic effects. Three groups of 20 rats each (10/sex/level) were treated topically with 20, 66 2/3, and 200 mg/kg/day 5 days per week for 3 months. Twenty control animals (10/sex) were treated with corn oil. Solutions at appropriate levels were prepared in corn oil and a constant dose volume (2.077 ml/kg) was applied to all animals. Treatment sites were not occluded. Dermal observations were performed pretest and 5 times/week throughout the study. Clinical laboratory studies were performed at termination. Neurological function evaluations were performed at months 1, 2, and 3. At three months, all animals were terminated. One-half of the animals were sacrificed by exsanguination under light ether anesthesia, and selected organs and tissues were fixed in formalin. Organ and organ body weight ratios were determined on all of these animals only. Histopathological evaluations of 10 organs or tissues were conducted on all formalin-fixed Group 1 and 4 animals. The remaining animals were perfused intravenously with glutaraldehyde under sodium pentobarbital aesthesia. Quantitative assessments of teased tibial nerve preparations were performed on all glutaraldehyde-perfused animals in Group 1 and 4. In addition, brain, spinal cord and sciatic nerve were evaluated microscopically (hematoxylin and eosin and Luxol-fast Blue staining) from these same animals. Erythema, eschar and exfoliation were recorded during the initial week of the study and maximum frequency of these effects were seen during Weeks 2 and 3. A dose-response was noted, with males being slightly more susceptible. In the last 2 months of the study most treated animals developed an apparent tolerance to the irritant effects of the test substance. Significantly lower body weights were recorded throughout the study for Group IV males (200 mg/kg/day). Body weights for female rats and Group II and III male rats were not significantly affected. Routine toxicologic and pharmacologic signs were unremarkable throughout the study. Month 2 neurologic evaluations showed an effect on gait in 4 of 10 male and 3 of 10 female Group IV rats. Reduced corneal reflex was also seen in some rats in this group; however, Month 3 evaluations failed to show these effects. Hematological and clinical chemistry parameters appeared unaffected by treatment with the test substance. However, there was a dose-related increase in urinary protein values in both sexes. Organ weights, gross necropsy observations and microscopic studies did not reveal any systemic toxic effects. Using tibial nerve teasing techniques, no morphometric differences were found between Group land Group IV glutaraldehyde perfused rats. Quantitative assessment of tibial nerve fiber cross-sections showed slightly increased diameters for males and slightly decreased diameters for females. These changes were not considered to be treatment-related.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
66.7 mg/kg bw/day
Study duration:
subchronic
Species:
rat

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:
Jun - Sep 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 424 (Neurotoxicity Study in Rodents)
Principles of method if other than guideline:
The subchronic dermal application of the test substance to the backs of Sprague-Dawley rats was studied to assess potential neurotoxic and other local and systemic effects.
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): C-178
- Physical state: liquid
- Analytical purity: 100% active ingredient
- Storage condition of test material: room temperature
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 41 days (age: 28 days at receipt)
- Weight at study initiation: week 0 males: 157-163 g; week 0 females: 133-139 g
- Housing: individually in elevated stainless steel cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
other: not occluded
Vehicle:
corn oil
Details on exposure:
TEST SITE
- Area of exposure: the back of the rats
- % coverage: no data
- Type of wrap if used: not occluded no further data
- Time intervals for shavings or clipplings: all animals were clipped ca. 23 h prior to initial dose. The animals were reclipped when necessary.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.077 ml/kg
- Concentration (if solution): 1.0, 3.33 and 10.0 %
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): immiscible with water
- Amount(s) applied (volume or weight with unit): 2.077 ml 7kg of the test substance in corn oil
- Concentration (if solution): 1.0, 3.33 and 10.0 % of the test substance in corn oil

USE OF RESTRAINERS FOR PREVENTING INGESTION: no data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
the remaining samples of weekly dosing solutions for each dose were returned to the sponsor for analysis, no further data
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily, 5 days/week
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
66.666 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: for mortality and gross signs of toxicologic or pharmacologic effects

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly including signs of local or systemic toxicity, pharmacologic effects and palpation for tissue masses

BODY WEIGHT: Yes
- Time schedule for examinations: twice pretest, weekly during treatment and terminally (after fasting)


OTHER:
- blood was obtained from over night fasted rats via venipuncture of the orbital sinus under light ether anestehsia, the same animals were used that were intended for formalin-fixation (5/sex/dose).
- hematology upon termination: hemoglobin, hematocrit, erythrocytes, clotting time, total and differential, leukocytes, erythrocytes morphology
- clinical chemistry upon termination: serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, blood urea nitrogen, fasting glucose, total protein, total bilirubin, sodium, potassium, calcium, inorganic phosphorus
- urinanalysis 6 days before termination: gross appearance, specific gravity, pH, protein, glucose, occult blood
Sacrifice and pathology:
One-half of the animals were sacrificed by exsanguination under light ether anesthesia, and selected organs and tissues were fixed
in formalin. Organ and organ-body weight ratios (adrenals, brain, liver, kidney, heart, spleen, testes with epididymides, ovaries) were determined on all of these animals only. Histopathological evaluations of 10 organs or tissues (liver, kidney, lung, heart, stomach, adrenal, pituitary, testes, ovaries,
spleen and skeletal muscle) were conducted on all formalin-fixed control animals and the high dose animals. The remaining animals were perfused
intravenously with glutaraldehyde under sodium pentobarbital anesthesia . Quantitative assessments of teased tibial nerve preparations were
performed on all glutaraldehyde-perfused animals in control animals and the high dose animals. In addition, brain, spinal cord and sciatic nerve were evaluated microscopically (hematoxylin and eosin and Luxol-fast blue staining) from these same animals.
Other examinations:
- neurologic functions were evaluated monthly
- Parameters examined according to a scoring system: posture, gait, muscular tone, reflexes (corneal), righting and toe-pinch
- no further data
Statistics:
Body weight, hematology and clinical chemistry parameters, organ weights and organ/body weight ratios were analyzed. Mean values of
all dose groups were compared to control at each time interval.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Weekly physical examinations of all animals failed to indicate any toxic effects of the test material other than irritation at the application site. Alopecia observed on the forepaws and legs was the most common finding in both sexes; however, the incidence was spread over all test groups, including control.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
The test substance produced moderate levels of irritation, in a dose-related manner, beginning the first week of the study. Males were generally more susceptible than females to the dermal effects of the test substance throughout the study. Dermal effects (erythema and eschar formation) were only scored as present or absent therefore, the number of times per week the effects were noted was used as a general indication of the severity of the dermal observations.
- 20 mg/kg/day: erythema noted occassionally during the final 2 months; more frequently in the initial 3 weeks; exfoliation observed in approximately one-half during weeks 2 and 3 with diminishing frequency
- 66.6 mg/kg/day: erythema noted with a somewhat higher frequency than 20 mg/kg/day, frequency in females comparable to control. Exfoliation and eschar recorded for most animals by week 3.
- 200 mg/kg/day: erythema, exfoliation, and eschar seen in most animals of both sexes beginning in week 1. Atonia was observed in one males and one female, fissures present in one female and four males. A persistant fissuring was observed in one male rat from week 2 through week 7.
Males appeared somewhat more sensitive than females to erythema and eschar formation.
Mortality:
no mortality observed
Description (incidence):
All animals survived to the scheduled termination of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significantly reduced body weights in high dose male rats (versus control male rats) were noted from weeks 3 through 12, except during weeks 10 and 11. Male rat weights of the low and mid dose groups were also reduced in dose-related fashion, but the differences were not statistically significant in comparison to control values. No statistically significant effects on weight were seen in treated female rats, however, high dose female animals never exceeded 95% of the mean weights of control females after 2 weeks on test.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
None of the hematologic parameters evaluated differed significantly from control values.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Of the clinical chemical parameters which were determined at the study termination, none were affected in a manner suggestive of a treatment-effect.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Protein (2+, 100 mg/dl) was confirmed to be present in the urine of one mid-dose male and female, in two high-dose males and one high-dose female. A large amount of occult blood was also present in the urine of this one high-dose female. The specific gravity of this high-dose female and one mid-dose female was also high (>1.090). These findings suggested a possible effect of the test substance on the kidneys, but no alterations were observed in the histopathological examination to support this conclusion. Additionally, without relation to urinary volumes and creatinine, the relevance of in urinary protein concentration is questionable. All in all, the singular changes are not considered adverse by the registrant.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There was a significant downward trend in male liver weights; however, this was not evident in the liver/body weight ratio and is therefore of doubtful significance. Other organ weights and organ/body weight ratios were comparable across all groups.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Formalin-fixed rats:
No changes, gross or microscopic, were evident which could be attributed to a systemic toxic effect of the test substance. The most common spontaneous gross necropsy findings, occurring across all groups, were inflammations around the ear tags, and slight hair loss on the extremities. No unusual microscopic pathological findings were evident which could be attributed to the topical administration of the test substance.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Month 1 neurological function tests showed two high-dose males with slightly reduced corneal response. All other evaluations were normal.
Month 2 neurological function tests were unremarkable in control, low and mid dose animals (both sexes). Four high dose males and three high dose females showed slightly abnormal gait described as "stilted". A slight decreased corneal reflex was observed in four males and one female. A moderately decreased toe pinch response (hindtoes only) was also present in one male rat.
Month 3 neurological function tests showed a slightly stilted gait and altered righting reflex in one male control rat and a slightly relaxed body tone in one mid dose male rat. One male high dose animal continued to exhibit a moderately decreased toe-pinch response (hindtoes only) . All neurological observations were normal in both the control and treated female rats at Month 3.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Glutaraldehyde-perfused rats:
Histopathological examinations of hematoxylin and eosin and Luxol-fast Blue stained slides of brain, spinal cord and sciatic nerve from ten rats (5/sex) treated with the high dose of the test substance failed to reveal any treatment-related lesions when these tissues were compared to similar ones from ten control rats (5/sex). In addition, microscopic examination of 50 teased nerve fibers from the tibial nerve of ten high-dose animals (5/sex) were comparable to those of the controls. When quantitative measurements were taken of myelinated nerve fibers in cross-section of the distal sciatic nerve, a slight shift to larger diameters could be detected in high-dose males when compared to the controls. However, there was also a slight decrease in fiber diameters in treated females. The relatively large standard deviation in data from both males and females suggest that these slight changes in fiber diameters are not significant. Moreover, the absence of lesions by more conventional histopathological examinations substantiate this conclusion.
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
LOAEL
Remarks:
skin irritation
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation
Dose descriptor:
NOAEL
Remarks:
for systemic toxicity
Effect level:
66.66 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Remarks:
for systemic toxicity
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed
Critical effects observed:
yes
Lowest effective dose / conc.:
20 mg/kg bw/day (nominal)
System:
integumentary
Organ:
skin
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
The test substance did not induce neurotoxicity and no sub-chronic toxicity other than dermal irritation and decreased body weight was observed in animals, under the conditions tested. Thus, the NOAEL for local effects was considered to be 20 mg/kg/day and the NOAEL for systemic effects (based on body weight) was concluded to be 66.6 mg/kg/day.
Executive summary:

The sub-chronic dermal application of the test substance to the backs of Sprague-Dawley rats was studied to assess potential neurotoxic and other local and systemic toxic effects. Three groups of 20 rats each (10/sex/level) were treated topically with 20, 66 2/3, and 200 mg/kg/day 5 days per week for 3 months. Twenty control animals (10/sex) were treated with corn oil. Solutions at appropriate levels were prepared in corn oil and a constant dose volume (2.077 ml/kg) was applied to all animals. Treatment sites were not occluded. Dermal observations were performed pretest and 5 times/week throughout the study. Clinical laboratory studies were performed at termination. Neurological function evaluations were performed at months 1, 2, and 3. At three months, all animals were terminated. One-half of the animals were sacrificed by exsanguination under light ether anesthesia, and selected organs and tissues were fixed in formalin. Organ and organ body weight ratios were determined on all of these animals only. Histopathological evaluations of 10 organs or tissues were conducted on all formalin-fixed Group 1 and 4 animals. The remaining animals were perfused intravenously with glutaraldehyde under sodium pentobarbital aesthesia. Quantitative assessments of teased tibial nerve preparations were performed on all glutaraldehyde-perfused animals in Group 1 and 4. In addition, brain, spinal cord and sciatic nerve were evaluated microscopically (hematoxylin and eosin and Luxol-fast Blue staining) from these same animals. Erythema, eschar and exfoliation were recorded during the initial week of the study and maximum frequency of these effects were seen during Weeks 2 and 3. A dose-response was noted, with males being slightly more susceptible. In the last 2 months of the study most treated animals developed an apparent tolerance to the irritant effects of the test substance. Significantly lower body weights were recorded throughout the study for Group IV males (200 mg/kg/day). Body weights for female rats and Group II and III male rats were not significantly affected. Routine toxicologic and pharmacologic signs were unremarkable throughout the study. Month 2 neurologic evaluations showed an effect on gait in 4 of 10 male and 3 of 10 female Group IV rats. Reduced corneal reflex was also seen in some rats in this group; however, Month 3 evaluations failed to show these effects. Hematological and clinical chemistry parameters appeared unaffected by treatment with the test substance. However, there was a dose-related increase in urinary protein values in both sexes. Organ weights, gross necropsy observations and microscopic studies did not reveal any systemic toxic effects. Using tibial nerve teasing techniques, no morphometric differences were found between Group land Group IV glutaraldehyde perfused rats. Quantitative assessment of tibial nerve fiber cross-sections showed slightly increased diameters for males and slightly decreased diameters for females. These changes were not considered to be treatment-related.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 µg/cm²
Study duration:
subchronic
Species:
rat
Quality of whole database:
Assuming an average b.w. of 200g and an exposed area of 20cm²

Additional information

Oral

Wistar Han rats were treated with the test item by daily oral gavage at dose levels of 40, 125 and 375 mg/kg according to OECD 422 and in compliance with GLP (2019). The rats of the control group received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-62 days). Females that failed to deliver pups were treated for 40-53 days. Parental toxicity was observed in the (fore)stomach of males from 125 mg/kg and in females at 375 mg/kg. These changes consisted of ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females. Other treatment-related but non-adverse changes were observed in the liver at microscopic examination. An absolute increase of 31% and a relative increase of 25% in liver weight was observed at dose 375 mg/kg. At microscopic examination, hepatocellular hypertrophy in the liver was observed at minimal severity and was in the absence of any indicators of cellular degeneration. The changes in the liver were not considered adverse at current severities. In the kidneys an increase in hyaline droplet accumulation was recorded in males which was considered to likely represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules (Alden et al., 1991). This male rat specific protein is not present in female rats nor in higher mammals, including man (Sahota et al., 2013). The increased hyaline droplet accumulation in the male kidneys at 375 mg/kg/day was not accompanied by indicators of tubular damage and therefore this was considered to be non-adverse. Functional observations were not performed for females and therefore, possible treatment related effects on the functional parameters could not be evaluated. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations (males), body weight, food consumption, clinical laboratory investigations (including male T4 thyroid hormone levels), macroscopic examination and organ weights). Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, no systemic no-observed-adverse-effect level (NOAEL) were established under the conditions of this study. The Parental local NOAEL of 40 mg/kg is based on the findings in the (fore)stomach. Due to the absence of systemic toxicity up to and including the highest tested dose of 375 mg/kg bw/d no systemic NOAEL could be derived.

 

Dermal

 

The sub-chronic dermal application of the test substance to the backs of Sprague-Dawley rats was studied to assess potential neurotoxic and other local and systemic toxic effects. The study was conducted equivalent to OECD 411. Compliance with GLP regulations was not specified (1982). Three groups of 20 rats each (10/sex/level) were treated topically with 20, 66 2/3, and 200 mg/kg/day 5 days per week for 3 months. Twenty control animals (10/sex) were treated with corn oil. Solutions at appropriate levels were prepared in corn oil and a constant dose volume (2.077 ml/kg) was applied to all animals. Treatment sites were not occluded. Dermal observations were performed pretest and 5 times/week throughout the study. Clinical laboratory studies were performed at termination. Neurological function evaluations were performed at months 1, 2, and 3. At three months, all animals were terminated. One-half of the animals were sacrificed by exsanguination under light ether anesthesia, and selected organs and tissues were fixed in formalin. Organ and organ body weight ratios were determined on all of these animals only. Histopathological evaluations of 10 organs or tissues were conducted on all formalin-fixed Group 1 and 4 animals. The remaining animals were perfused intravenously with glutaraldehyde under sodium pentobarbital aesthesia. Quantitative assessments of teased tibial nerve preparations were performed on all glutaraldehyde-perfused animals in Group 1 and 4. In addition, brain, spinal cord and sciatic nerve were evaluated microscopically (hematoxylin and eosin and Luxol-fast Blue staining) from these same animals. Erythema, eschar and exfoliation were recorded during the initial week of the study and maximum frequency of these effects were seen during Weeks 2 and 3. A dose-response was noted, with males being slightly more susceptible. In the last 2 months of the study most treated animals developed an apparent tolerance to the irritant effects of the test substance. Significantly lower body weights were recorded throughout the study for Group IV males (200 mg/kg/day). Body weights for female rats and Group II and III male rats were not significantly affected. Routine toxicologic and pharmacologic signs were unremarkable throughout the study. Month 2 neurologic evaluations showed an effect on gait in 4 of 10 male and 3 of 10 female Group IV rats. Reduced corneal reflex was also seen in some rats in this group; however, Month 3 evaluations failed to show these effects. Hematological and clinical chemistry parameters appeared unaffected by treatment with the test substance. However, there was a dose-related increase in urinary protein values in both sexes. Organ weights, gross necropsy observations and microscopic studies did not reveal any systemic toxic effects. Using tibial nerve teasing techniques, no morphometric differences were found between Group land Group IV glutaraldehyde perfused rats. Quantitative assessment of tibial nerve fiber cross-sections showed slightly increased diameters for males and slightly decreased diameters for females. These changes were not considered to be treatment-related. The test substance did not induce neurotoxicity and no sub-chronic toxicity other than dermal irritation and decreased body weight was observed in animals, under the conditions tested. Thus, the NOAEL for local effects was considered to be 20 mg/kg/day and the NOAEL for systemic effects (based on body weight) was concluded to be 66.6 mg/kg/day.

 

In a supporting study, the dermal toxicity of the test substance was evaluated after repeated topical applications for ten consecutive days on the abraded and intact dorsal skin of four groups of New Zealand White rabbits (five/sex/group). The study was performed in compliance with GLP regulations (1981a). The compound was applied at a dosage level of 500 mg/kg in each of the treated groups. The compound was applied undiluted to Group 2, in corn oil vehicle to Group 3, and in acetone to Group 4. The exposure area of three males and two females in each group was abraded prior to the first, third, sixth, and eighth doses. An additional group (Group 1) served as a control and was abraded in the same manner. Criteria used to evaluate for compound effects were mortality and moribundity, clinical observations, dermal responses, body weights, gross pathology, and histopathology. One Group 4 female was found dead on Day 9, and one Group 2 male and one Group 4 female were both sacrificed in extremis on Day 8. These deaths were not attributed to treatment. A higher incidence of depression, thinness, and anorexia was noted in the Group 3 animals. Erythema noted frequently during the treatment phase of the study ranged from severe to slight in Groups 2 and 4; while erythema ranged from moderate to slight in Group 3. Edema, noted most frequently during the treatment phase, was generally observed as slight in all treated groups. Other dermal effects were noted frequently in all treated groups and included: blanching, eschar formation, epi­ dermal scaling, fissuring, fissuring with bleeding, necrosis, raw areas, sloughing, and thickening. Mean body weights were significantly lower in Groups 3 and 4 males, and Groups 2, 3, and 4 females by the end of the treatment phase. At termination, the Group 3 male weight was still significantly lower while all treated female groups were lower than control but not significant. No treatment-related gross visceral lesions were noted. Microscopic evaluation did not reveal evidence of compound-related histomorphologic alteration. A variety of spontaneous disease lesions were observed in rabbits of all groups and lesions consistent with trauma were observed in the spinal cord of a Group 4 rabbit. In conclusion, treatment-related effects on body weight, clinical observations, and dermal observations were noted, with the response being somewhat more severe in the Group 3 (corn oil vehicle) animals. The LOAEL was concluded to be 500 mg/kg bw/day.

 

In another supporting study, repeated dermal toxicity was investigated in a 28 -Day dermal toxicity study in rabbits with the test substance. GLP compliance was not specified (1981b). The purpose of this study was to assess the toxicity of the test substance when administered repeatedly to the skin of rabbits for a total of ten applications (five per week) over a two-week period. Animals showed severe dermal damage, mortality, and body weight loss. No NOAEL could be determined. Thus, the LOAEL was concluded to be 500 mg/kg bw/day.

 

In a third supporting study, conducted in the absence of information concerning GLP regulations, New Zealand White rabbits were used (1981c). The subacute dermal toxicity study in rabbits with the test substance was conducted. The purpose of this study was to assess the toxicity of the test substance when applied to the skin of rabbits, wither neat, in corn or mineral oil, for ten consecutive days. Doses of 250 mg/kg/day (neat and in corn oil) and 500 mg/kg/day (neat, in corn oil, and in mineral oil) were administered. A vehicle control group received corn oil only. A four-week recovery period was allowed after termination of the ten-day dosing period to evaluate reversibility of effects. Severe dermal damage was observed, such as necrosis. Mortality was observed in animals treated with the neat substance, mixed in corn oil, and mineral oil. Thus, a NOAEL could not be determined. The LOAEL was concluded to be 250 mg/kg bw/day.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result, the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) 2018/1480 of 4 October 2018.