Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019 to 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Following ECHA communication (CCH-C-2114522615-53-01/F) we are updating our dossier by including the study reports as in our view the robust study summaries (of OECD 408, 421 and 414) well reflected the obtained outcome of the reports already withing the first submission.
Following ECHA decision (CC(CCH-D-2114371485-43-01/F) on Reaction mass of 2-methylbutyl salicylate and pentyl salicylate, EC No 911-280-7, it was requested to conduct additional toxicological studies:
The Sub-chronic toxicity study (90-day), oral route (Annex IX, Section 8.6.2.; test method: EU B.26./OECD TG 408) in rats with the registered substance; the Screening study for reproductive/developmental toxicity (Annex VIII, Sections 8.6.1 and 8.7.1.; test method: OECD TG 421) in rats, oral route, and the Pre-natal developmental toxicity study (Annex IX, Section 8.7.2.; test method: EU B.31./OECD 414) in a first species (rat or rabbit), oral route.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
as of 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
as of 2008 (EC No 440/2008)
Deviations:
no
Principles of method if other than guideline:
Following ECHA decision (CC(CCH-D-2114371485-43-01/F) on Reaction mass of 2-methylbutyl salicylate and pentyl salicylate, EC No 911-280-7, it was requested to conduct additional toxicological studies:
The Sub-chronic toxicity study (90-day), oral route (Annex IX, Section 8.6.2.; test method: EU B.26./OECD TG 408) in rats with the registered substance; the Screening study for reproductive/developmental toxicity (Annex VIII, Sections 8.6.1 and 8.7.1.; test method: OECD TG 421) in rats, oral route, and the Pre-natal developmental toxicity study (Annex IX, Section 8.7.2.; test method: EU B.31./OECD 414) in a first species (rat or rabbit), oral route.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han) from Charles River Deutschland, Sulzfeld, Germany
Details on species / strain selection:
The rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6 weeks old
- Weight at study initiation (at arrival): 107 to 187 g
- Housing: up to 5 animals in Makrolon Type IV (height 18cm) cages with standard bedding
- Diet (e.g. ad libitum): The test item was administered by inclusion in the diet to the appropriate animals ad libitum from Day 1 for a minimum of 90 days, up to and including the day before scheduled necropsy.
- Water (e.g. ad libitum): community tap water freely available
- Acclimation period: 7 days under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 20
- Humidity (%): 51 to 60
- Air changes (per hr): 10 times/h
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The oral route of exposure was chosen to comply with regulatory requirements and as historically, this route has been used extensively for studies of this nature, although the most likely route of human exposure during manufacture, handling or use of the test item is the dermal route.
The dietary inclusion was selected, in an attempt to simulate a slow and constant release of the test item during the day as it seemed more relevant to the actual exposure of humans as opposed to a gavage once per day.
Vehicle:
unchanged (no vehicle)
Remarks:
directly added in the diet powder
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): every 4 days (Food hoppers were shaken on a daily basis to divide any sawdust equally over the diet in order to facilitate food consumption; During motor activity measurements, animals did not have access to food for a maximum of 2 hours)
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) were used
- Storage temperature of food: Diets were prepared freshly for use at room temperature for a maximum of 10 days. Diets were kept in the freezer (≤-15°C) until use, if not used on the day of preparation. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 10 days for supplementing food during the respective food consumption measurement interval.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diet preparation samples were collected for analysis as indicated in the following table:

Occasion Concentration Homogeneity Stability
Week 1 All groups Groups 2 and 4 Low and high
Week 6 All groups Groups 2 and 4 Low and high
Week 13 All groups Groups 2 and 4 not applicable

The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results.
The food batches that were used in week 1, 6 and 13 were sampled directly after preparation.
To determine stability of diets over a larger time range, two additional trial diets were prepared in Week 1.

All samples to be analysed were transferred (at room temperature protected from light) to the analytical laboratory at the Test Facility.
To check cleaning procedures, a diet residue test was performed after the diet preparation of week 6 using a validated analytical procedure (Test Facility Study No.20155390).
Residual samples were discarded after completion of the sample analysis.
Duration of treatment / exposure:
The test item was mixed in the diet.
Frequency of treatment:
Daily administration of the test item in the diet
Doses / concentrationsopen allclose all
Dose / conc.:
750 ppm
Remarks:
equiv to 55 mg/kg bw/d (males)
equiv to 67 mg/kg bw/d (females)
Dose / conc.:
3 750 ppm
Remarks:
equiv to 281 mg/kg bw/d (males)
equiv to 329 mg/kg bw/d (females)
Dose / conc.:
7 500 ppm
Remarks:
equiv to 569 mg/kg bw/d (males)
equiv to 607 mg/kg bw/d (females)
No. of animals per sex per dose:
10 females and 10 males in each dose group
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels were selected based on results of a dose range finder 28-day repeated dose toxicity study with dietary exposure of Amyl Salicylate in rats provided by the Sponsor, and in an attempt to produce graded responses to the test item. In the 28-day study, exposure to 1000 mg/kg/day was well tolerated, although effects on body weight and food consumption were noted throughout the exposure period. Decreased food consumption was expected to be a consequence of unpalatability, therefore 500 mg/kg/day was selected as high dose for this 90-day study. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation and should not interfere with nutrition. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Positive control:
not performed

Examinations

Observations and examinations performed and frequency:
MORTALITY/MORIBUNDITY
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

CLINICAL OBSERVATION
Performed once prior to first administration and at least once daily from start of administration onwards, up to de day prior to necropsy.
The time of onset, grade and duration of any observed sign was recorded.
Signs were graded for severity and the maximum grade was predefined at 1, 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (i.e. maximum grade 1) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.
Clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment.

BODY WEIGHTS
Animals were weighed individually weekly, starting on Day 1. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION
Food consumption was quantitatively measured weekly starting on Day 1 and continuing weekly throughout the Dosing Period.

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

OPHTALMIC EXAMINATIONS
The eyes were examined using an ophthalmoscope after application of a mydriatic agent (Tropicol 5 mg/mL solution, THEA Pharma, Wetteren, Belgium) during Pretreatment in all study animals (including spare animals), and at the end of the Dosing Period in Week 13 in all Group 1 and 4 study animals.

FUNCTIONAL TESTS
Functional tests were performed the first 5 animals per sex per group during Week 12-13. These tests were performed after completion of clinical observations (including arena observation, if applicable).
The following tests were performed: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength (recorded as the mean of three measurements per animal), Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA).

CLINICAL PATHOLOGY
Blood was collected at the end of treatment between 7.00 and 10.30 a.m. from the retro-orbital sinus under anesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) of animals that were fasted (overnight with a maximum of 24 hours, water was available). After collection all samples were transferred the appropriate laboratory for analysis (haematology, coagulation and clinical chemistry).
Sacrifice and pathology:
SCHEDULED EUTHANASIA
Animals were weighed, and euthanized using isoflurane, followed by exsanguination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy.

NECFROPSY
Animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

ORGAN WEIGHTS
The organs identified in the below were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratio (using the terminal body weight) were calculated.

Organs Weighed at Necropsy:
Brain, Cervix, Epididymises, Adrenal Glands, Prostate Gland, Seminal vesicle Glands, Thyroid Gland, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus

TISSUE COLLECTION AND PRESERVATION
Representative samples of the tissues identified below were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated.

Tissue Collection and Preservation:
Animal identification, Aorta artery, Nasopharynx, Femur, Bone marrow, Sternum, Brain, Cervix, Epididymis, Esophagus, Eye, Adrenal gland, Clitoral gland, Harderian gland, lacrimal gland, Mammary gland, Parathyroid gland, Pituitary gland, Preputial gland, Prostate, Salivary gland, Seminal vesicle gland, Thyroid gland, Gross lesions/masses, Gut-associated lymphoid tissue, Heart, Kidney, Large intestine (Cecum, Colon, Rectum), Larynx, Liver, Lung, Mandibular lymph node, Mesenteric lymph node, Skeletal muscle, Optic nerve, Sciatic nerve, Ovary, Pancreas, Skin, Small intestine (duodenum, ileum, jejunum), Spinal cord, Spleen, Stomach, Testis, Thymus, Tongue, Trachea, Urinary bladder, Uterus, Vagina.

HISTOLOGY
Tissues identified for collection and preservation (except animal identification, clitoral gland, femur, lacrimal gland, nasopharynx, preputial gland and skeletal muscle) were embedded in paraffin (Klinipath, Duiven, The Netherlands), sectioned, mounted on glass slides, and stained with hematoxylin and eosin (Klinipath, Duiven, The Netherlands).

HISTOPATHOLOGY
All tissues as defined under "Histology" were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated and reported.
A peer review on the histopathology data was performed by a second pathologist.
Other examinations:
none
Statistics:
CONSTRUCTED VARIABLES:
Body Weight Gains: Calculated against the body weight on Day 1.
Food Consumption: Calculated between at least each scheduled interval.
Test Item Intake: Calculated as concentration of test item in diet against relative food consumption.
Organ Weight Relative to Body Weight: Calculated against the Terminal body weight.

DESCRIPTIVE STATISTICAL ANALYSIS
Means, SD (or % coef. of variation or standard error, when appropriate), ratio, %, numbers, and/or incidences were reported as appropriate by dataset.

STATISTICS
All statistical tests was conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Group 2 vs. Group 1
Gp 3 vs. Gp 1
Gp 4 vs. G 1

PARAMETRIC
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

NON-PARAMETRIC
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).

INCIDENCE
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

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No test item-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight gain of all animals treated at 750 and 3750 ppm remained in the same range as controls over the study period.
From Day 6 onwards, statistically significant decreased body weight and body weight gain were observed in males (0.86x and 0.71x control on Day 90, respectively) and females (0.90x and 0.71x control on Day 90, respectively) at 7500 ppm. These test item-related effects were considered to be adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight at 750 and 3750 ppm was similar to the control level over the study period.
In males and females at 7500 ppm, food consumption and relative food consumption was slightly decreased over Days 1-6, when compared to controls.
This result was considered to be a response related to the taste of the test item rather than a sign of toxicity. Normal values for food consumption and relative food consumption were noted during the remainder of the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
no effect reported
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The nature and incidence of ophthalmology findings noted during the Pretreatment Period and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related effects on hematology parameters were seen in animals at 750 ppm.

A statistically significant, but slightly decreased lymphocyte counts in males at 3750 and 7500 ppm, white blood cell count in males at 7500 ppm and red blood cell count in females at 7500 ppm were observed. In addition, a statistically significant, but slightly increased reticulocyte counts in males and females were observed at 7500 ppm. All these changes were considered to be test item-related and not adverse at the severities noted and in the absence of degenerative changes.

Other statistically significant changes consisted of increases in RDW (red cell distribution width) and MCH (mean corpuscular haemoglobin) levels, and the slight decrease in haemoglobin levels and haematocrit in females at 7500 ppm and higher MCV (mean corpuscular volume) in females at 3750 and 7500 ppm. These changes occurred within the range considered normal for rats of this age and strain and were therefore considered not to be related to the test item.

Any other statistically significant changes in hematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Coagulation parameters of treated males were considered not to have been affected by treatment.
The statistically significant decreased PT (prothrombin time) and higher activated partial thromboplastin time of females treated at 750 and 7500 ppm was not considered to be test item related because of the absence of a treatment-related distribution.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item-related effects on clinical chemistry parameters were seen in animals at 750 ppm.

A statistically significant increased (ALP) alkaline phosphatase activity were observed in males at 7500 ppm. Furthermore, a statistically significant decreased total bilirubin levels were observed in females at 3750 ppm and males and females at 7500 ppm. These changes were considered to be test item-related and not adverse, as they were not supported by clinical observations, were slight in nature and had no supportive morphological correlates in examined tissues.

In addition, increased Inorg. Phos (inorganic phosphate) and potassium levels and decreased cholesterol and total protein levels in males and/or females were observed at 7500 ppm. In addition, decreased glucose levels in males were observed at 3750 and 7500 ppm. These minor statistically significant differences were, although considered to be test item related, to be of no toxicological relevance due to the minimal magnitude of change.

Any other statistically significant changes in clinical chemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.

The statistically significant decreased fore grip strength observed in males at 3750 ppm and in males and females at 7500 ppm was considered not to represent an adverse effect on neurobehaviour. This test item-related result was 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.

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, which is normal behaviour.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item-related effects on organ weights were seen in animals at 750 and 3750 ppm.

Test item-related lower thymus weights (absolute) were noted in the 7500 ppm group males and females. It is considered non adverse at the severities noted and in the absence of degenerative changes.

There were no other test item-related organ weight changes. Some organ weight differences were statistically significant when compared to the control group but were considered to be the result of a test item-related effect on final body weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related effects on macroscopic findings were seen in animals at 750 and 3750 ppm.

A test item-related reduced thymus size was present a single (1/10) female treated at 7500 ppm. This correlated with the reduced thymus weight and with the microscopic lymphoid depletion. This findings are in line with the decrease observed in the body weight of males and are considered non adverse at the severities noted and in the absence of degenerative changes.

The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
In the thymus, test item-related lymphoid depletion was present in males and females treated at 7500 ppm up to slight degree. This correlated with the lower thymus weight and with the macroscopic reduced in size in a single female. These findings are often correlated with a stress in the animals caused by a body weight loss.

In the spleen, a test item-related increased incidence and severity of extramedullary hematopoiesis was present in females treated at 7500 ppm up to moderate degree and as such non-adverse. The minimal severity recorded in the remaining dose groups, including controls, was considered to be within background limits and therefore not toxicological relevant.

In the bone marrow (sternum), increased adipocytes were present at increased incidence and severity in males treated at 7500 ppm up to slight degree. The minimal severity recorded in the remaining dose groups, including controls, was considered to be within background limits and therefore not toxicological relevant.

The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
281 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
329 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Effect level:
3 750 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: 3750 ppm (corresponding to an actual test article intake of 281 and 329 mg/kg/day for males and females, respectively)

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The administration of Amyl Salicylate by dietary route was well tolerated in rats at levels of 3750 ppm.
Effects on body weight (gain) were observed at 7500 ppm that were reflective of an undernutrition that occurred during the first days of test item diet administration.
Based on these results, the No Observed Adverse Effect Level (NOAEL) for Amyl Salicylate was established as being 3750 ppm (corresponding to an actual test article intake of 281 and 329 mg/kg/day for males and females, respectively).
Executive summary:

The objective of this study was to determine the potential toxicity of Amyl Salicylate, when given via diet for 90 days to Wistar Han rats. In addition, a No Observed Adverse Effect Level (NOAEL) was established.

The study design was as follows:

Table1: Experimental Design

Group No.

Test Item Id.

Dose Level

(ppm)

mg test item/kg body weight

Number of Animals

Males

Females

1

Control diet

0 (diet)

0

10

10

2

Amyl Salicylate

750

55 (males)

67 (females)

10

10

3

Amyl Salicylate

3750

281 (males)

329 (females)

10

10

4

Amyl Salicylate

7500

569 (males)

607 (females)

10

10

Id.= identification.

Chemical analyses of dietary preparations were conducted three times during the study to assess accuracy and homogeneity, moreover, stability was established during the study.

The following parameters and end points were evaluated in this study: clinical signs, body weights, food consumption, ophthalmology, functional tests, clinical pathology parameters (hematology, coagulation and clinical chemistry), gross necropsy findings, organ weights, and histopathologic examinations.

Dietary analyses confirmed that diets were prepared accurately and homogenously. The diets were stable for at least 25 days in the freezer (≤ -15°C) and at least 13 days at room temperature.

In conclusion, administration of Amyl Salicylate by dietary administration was well tolerated in rats at levels of 3750 ppm. Effects on body weight (gain) were observed at 7500ppm that were reflective of an undernutrition that occurred during the first days of test item diet administration. Based on these results, the No Observed Adverse Effect Level (NOAEL) for Amyl Salicylate was established as being 3750 ppm (corresponding to an actual test article intake of 281 and 329 mg/kg/day for males and females, respectively),since no adverse effect was observed.