Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

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 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.

Reproductive and Developmental Toxcity study (OECD 421, GLP):

Parental NOAEL: 333 mg/kg bw/d

Reproduction NOAEL: 333 mg/kg bw/d

Developmental NOAEL: 333 mg/kg bw/d

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019 to 2020
Reliability:
1 (reliable without restriction)
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.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016.
Deviations:
yes
Remarks:
see report
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch number of test material: PE00206659 (Week 1 and 2) and VE00598644 (Week 3 onward)
- Expiration date of the lot/batch: 18 May 2019 and 06 March 2020 respectively
- Purity test date: 99.69%
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han rats
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 Deutschland, Sulzfeld, Germany
- Age at study initiation: (P) males were 11-12 weeks old and females were 13-14 weeks old.
- Weight at study initiation: (P) Males: between 260 and 313 g; Females: between 203 and 247 g.
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The room in which the animals were kept was documented in the study records.
- Diet (e.g. ad libitum): powder diets were provided ad libitum throughout the study, except during designated procedures. During the acclimatization period, animals had free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): tap water was freely available
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 21°C
- Humidity (%): mean relative humidity of 47 to 72%
- Air changes (per hr): min 10 times/h
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light
IN-LIFE DATES: From: 10 Apr 2019 To: 24 Jun 2019
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
The test item was mixed without the use of a vehicle, directly with the required amount of powder feed. Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used.
Diets were prepared for use at room temperature for a maximum of 10 days. Diets were kept in the freezer (≤-15°C) until use for a maximum of 3 weeks, if not used on the day of preparation.
Any remaining food left after filling the food hoppers may be stored at room temperature for a maximum of 10 days) for supplementing food during the respective food consumption measurement interval.
Details on mating procedure:
FREQUENCY:
Daily, after a minimum of 14 days of treatment. The mating period will consist of a maximum of 14 consecutive days.
PROCEDURE:
Animals will be cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating will be confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day will be designated Day 0 post-coitum. Once mating has occurred, the males and females will be separated. A maximum of 14 days will be allowed for mating, after which females who have not shown evidence of mating will be separated from their males. In case less than 9 females per group have shown evidence of mating, each non-mated female may be re-mated once with a male for a maximum of 7 days (if possible). A male of the same group having previously shown evidence of mating (non-selected male if possible, see section 13) will be used for re-mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
SAMPLE COLLECTION AND ANALYSIS
Diet preparation samples were collected for analysis as indicated below.

Occasion Concentration Homogeneity Stability
Week 1 of treatment All groups + 200 ppm, Groups 2 and 4 + 200 ppm 200 ppm

The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 and 200 ppm preparations were averaged and utilized as the concentration results.
The 200 ppm diet was only used for analytical purposes and was not administered to the animals. QC samples at 200 ppm were included in 5-fold during the analyses to extend the validation range to 200-15000 ppm, i.e. thereby also covering the lower diet concentrations which were used during the lactation phase.

All samples to be analyzed were transferred (at room temperature under normal laboratory light conditions) to the analytical laboratory at the Test Facility.
Residual samples were discarded after completion of the sample analysis.

ANALYTICAL METHOD
Analyses were performed using a validated analytical procedure (Test Facility Study No. 20155390).

CONCENTRATION ANALYSIS
Duplicate sets of samples (approximately 5 g) were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. After acceptance of the analytical results, backup samples were discarded.
Duration of treatment / exposure:
The test item and control item were administered to the appropriate animals by inclusion in the diet ad libitum from Day 1 onwards for a minimum of 29 days. Males were exposed for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females were exposed for 51 to 61 days, i.e. 14 days prior to mating (to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day of scheduled necropsy. Females which failed to deliver were treated for 42-55 days.
Frequency of treatment:
Daily in ad libitum diet
Dose / conc.:
500 ppm
Remarks:
extrapolated to an equivalent of 33 mg/kg bw/d
Dose / conc.:
1 500 ppm
Remarks:
extrapolated to an equivalent of 100 mg/kg bw/d
Dose / conc.:
5 000 ppm
Remarks:
extrapolated to an equivalent of 333 mg/kg bw/d
No. of animals per sex per dose:
10 females and 10 males per dose level
Control animals:
yes, concurrent no treatment
Details on study design:
DOSE SELECTION
The dose levels were selected based on a dose range finding study with dietary administration of Amyl Salicylate in pregnant rats treated from Day 6 to Day 21 post-coitum, inclusive (Test Facility Study No. 20155389) and a 90-day study with dietary administration of Amyl Salicylate in non-pregnant rats (Test Facility Study No. 20155386), and in an attempt to produce graded responses to the test item without interfering with normal nutrition of the animals.

ANIMAL ASSIGNMENT
A total of 40 females was selected at randomization before initiation of the pretest phase. Any selected female classified as not having regular estrous cycles during the pretest phase was replaced before initiation of administration by one of the 8 additional females having regular estrous cycles, if feasible. A total of 40 females with regular estrous cycles continued in the study. The supernumerary females were removed from the study, and their estrous cycle results were kept in the raw data but not reported.
Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.

BLOOD SAMPLING
Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m. (for exceptions, see Appendix 7), from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. After collection all samples were transferred to the appropriate laboratory for analysis.
F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0 females were not fasted overnight.
Blood of F1-animals was collected on PND 4 and PND 14-16, if possible. This was performed in the necropsy room.
On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation, between 7.00 and 10.30 a.m., and samples were pooled to one sample per litter. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single pup.
On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female, if possible). Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure.
Positive control:
no
Parental animals: Observations and examinations:
MORTALITY/MORIBUNDITY CHECKS
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 OBSERVATIONS
Clinical observations were performed once daily, once prior to the first administration and from start of administration onwards, up to the day prior to necropsy. During the dosing period, these observations were performed at no specific time point.
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 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 (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

BODY WEIGHTS
Animals were weighed individually on the first day of administration, and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
A terminal weight was recorded on the day of scheduled necropsy (for exceptions, see Appendix 7).

FOOD CONSUMPTION
Food consumption was 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.
Food spillage was estimated during the study by means of sieving the bedding material, including the enrichments, with a metal sieve (mesh-size 500 μm) at the end of each food consumption period (for exceptions, see Appendix 7).

WATER CONSUMPTION
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.
Oestrous cyclicity (parental animals):
ESTROUS CYCLE DETERMINATION
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females 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 estrous.
Sperm parameters (parental animals):
Reproductive organs collected and examined. Histopathology analysis performed on epididymis and testes.
Litter observations:
MORTALITY/MORIBUNDITY
Pups were observed daily for general health/mortality. The number of live and dead pups was determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

CLINICAL OBSERVATION
Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check were given in the respective report tables.

BODY WEIGHTS
Live pups were weighed individually on PND 1, 4, 7 and 13.

SEX RATIO
Sex was externally determined for all pups on PND 1 and 4.

ANOGENITAL DISTANCE
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.

AREOLA7MIPPLE RETENTION
All male pups in each litter were examined for the number of areola/nipples on PND 13.

CULLING
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.
Postmortem examinations (parental animals):
SCHEDULED EUTHANASIA
Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
Scheduled necropsies were conducted on the following days:
Males (which sired and failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14-16.
Females which failed to deliver: With evidence of mating: Post-coitum Day 26 (No. 56, 59 and 79).
Without evidence of mating: 26 days after the last day of the mating period (No. 63 and 75).
All males surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0-females were not fasted.

NECROPSY
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
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.
The numbers of former implantation sites were recorded for all paired females.
In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

ORGAN WEIGHTS
The organs identified below were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.
Epididymis, Coagulation gland, Parathyroid gland, Prostate gland, Seminal vesicle, Thyroid gland, Testes.

TISSUE COLLECTION/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: Cervix, Epididymis, Coagulation gland, Mammary gland, Parathyroid gland, Pituitary gland, Prostate gland, Seminal vesicle gland, Thyroid gland,Gross lesions/masses, Ovaries, Testes, Uterus, Vagina.

HISTOLOGY
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
All animals: Gross lesions/masses
All animals of Groups 1-4: Epididymis, thyroid gland, ovaries and testes.
Males that failed to sire and females that failed to delivery pups: Tissues identified in "tissue collection/preservation" above.(except animal identification, mammary gland, parathyroid gland and pituitary gland).
All tissues were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.
For the testes of all males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
A peer review on the histopathology data was performed by a second pathologist.

THYROID HORMONE
Blood samples at a target volume of 1.0 mL (F0-animals), 0.5 mL (pooled PND 4 pups) and 1.0 mL (PND 14-16 pups) were collected into tubes without anticoagulant. Blood samples were processed for serum, and serum was analyzed for total Thyroxine (T4).
Measurement of total T4 was conducted for F0-males and PND 14-16 pups.
For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was not performed seeing that no histopathological correlation was found.
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.
Serum samples retained for possible future analysis were maintained by the Test Facility in the freezer (≤-75°C). Under these storage conditions, samples are stable for 6 months. Any remaining sample will be discarded (i.e. 24 Dec 2019).

Postmortem examinations (offspring):
METHOD OF EUTHANASIA
All pups (PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%).
The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

UNSCHEDULED DEATHS
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally).
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

SCHEDULED EUTHANASIA
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible.
All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
In addition, blood was collected from two pups per litter (see also section 4.10.1), and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin.
Statistics:
STATISTICAL ANALYSIS
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% 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 deemed 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
Group 3 vs. Group 1
Group 4 vs. Group 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).

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.
Reproductive indices:
Mating index (%), Precoital time, Fertility index (%), Gestation index (%), Duration of gestation, Post-implantation survival index (%), Live birth index (%), Percentage live males at First Litter Check (%), Percentage live females at First Litter Check (%), Viability index (%), Lactation index (%).
Offspring viability indices:
see above
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were noted during daily detailed clinical observations.
Hunched posture and scabs were noted in a single Male animal (No. 12) of the 500 ppm group on Days 15-16 and Days 14-17, respectively. These and other clinical signs noted (including scabs in the neck and shoulder area and alopecia) occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be non-adverse and unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable (feeding study)
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):
A statistically significant and test-item related reduced body weight gain was noted in females of the 5000 ppm group during the pre-mating period (up to 0% body weight gain at 5000 ppm compared to 6% in concurrent controls at the end of the pre-mating period) and the first week of the mating period. Body weight gain was in the same range as controls in the mating period following the first week, post-coitum and lactation period. The lower body weight gain during the pre-mating period resulted in a slightly lower body weight of females of the 5000 ppm group compared to controls during mating, post-coitum and the first week of the lactation phase reaching statistical significance in some instances.
As body weight gain normalized to control levels during the course of the study, this was considered not adverse
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of females of the 5000 ppm group was lower (up to 0.68x of controls) compared to levels in the control group during the pre-mating period, post-coitum period and the first week of the lactation period. Changes were not statistically significant. Food consumption returned to control levels between Days 7-13 of the lactation period.
Food consumption before or after correction for body weight for treated males was similar to control levels over the treatment period.
Significant food spillage was observed for all groups during the study, and although sieving of the bedding to recover the spilled powder diet was performed to account for it, it was not deemed sufficient to correct food intake values appropriately as control values were not in the range of historical controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A test-item related decrease in serum levels of T4 in F0-males was noted, reaching statistical significance at the 1500 and 5000 ppm groups (0.77x and 0.40x of controls at 1500 and 5000 ppm, respectively). Values of Group 3 animals were within the historical control range and as such, the test-item related decrease at 1500 ppm was considered not toxicologically relevant. Except for the T4 values of one Group 4 animal, serum levels of all animals of the 5000 ppm group were below the historical control range. Histopathological evaluation of the thyroid did not reveal any correlating alterations.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations.
All 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
Description (incidence and severity):
There were no test item-related microscopic observations.
All 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.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment.
Most females had regular cycles of 4 days. Extended estrous occurred in two females of the control group, an irregular cycle was noted for one animal of the control group, two animals of the 500 ppm group and one animal of the 1500 ppm group. Furthermore, an acyclic estrous cycle was noted for one animal of the 1500 ppm group. Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
MATING INDEX
Mating index was considered not to be affected by treatment.
Except for one female administered 1500 ppm and one female administered 5000 ppm, all females showed evidence of mating.
The mating indices were 100, 100, 90 and 90% for the control, 500, 1500 and 5000 ppm groups, respectively.

PRECOITAL TIME
Precoital time was considered not to be affected by treatment.
All females showed evidence of mating within 5 days, except for one animal of the control group which showed evidence of mating after 9 days.

NUMBER OF IMPLEMENTATION SITE
Number of implantation sites was considered not to be affected by treatment.

FERTILITY INDEX
Fertility index was considered not to be affected by treatment.
The fertility indices were 100, 80, 100 and 89% for the control, 500, 1500 and 5000 ppm groups, respectively.
Two females of the 500 ppm group and one female of the 5000 ppm group were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was considered not to be related to treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 333 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test item related adverse effect observed up to the highest dose
Key result
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
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:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Only a one generation study, no P1 produced.
Remarks on result:
not measured/tested
Critical effects observed:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups in any groups.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
Not applicable
Mortality / viability:
no mortality observed
Description (incidence and severity):
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment. Viability indices were 99, 100, 99 and 99% for the control, 500, 1500 and 5000 ppm groups, respectively.
One pup of the control group, one pup at 1500 ppm and one pup at 5000 ppm were missing on PND 4, 2 and 4, respectively. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Slightly lower body weights were recorded for pups of the 5000 ppm group compared to controls from PND1 onwards (0.89x of controls for males and 0.93x of controls for females on PND1). Based on the minimum magnitude of the effect, the lack of statistical significance, since mean body weights remained within the historical control data and the differences appear to become smaller over time, this was considered not toxicologically relevant.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female 14-16 pups were considered not to be affected by treatment.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
A slightly higher anogenital distance (absolute and normalized for body weight) in male and female pups was noted for pups of the 5000 ppm group compared to controls. Statistical significance was achieved for anogenital distance normalized for body weight only. Absolute anogenital distance values at 5000 ppm were close to the mean of the historical control range . The increased corrected anogenital distance is due to the decreased pup body weights on PND1 at 5000 ppm. As changes in corrected anogenital distance were minimal and values remained within the historical control range and since the direction of the change in both sexes is considered not biologically relevant, this was considered not toxicologically relevant.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Administration up to 5000 ppm had no effect on areola/nipple retention.
For none of the examined male pups nipples were observed at PND 13.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 333 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test item related adverse effect up to the highest dose
Key result
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
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:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Remarks on result:
not measured/tested
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Reproductive results
No reproductive toxicity was observed up to the highest dose level tested (5000 ppm).
No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, and histopathological examination of reproductive organs).
Developmental results
No developmental toxicity was observed up to the highest dose level tested (5000 ppm).
No toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, maternal care, litter size, sex ratio and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).
In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) of Amyl Salicylate were established:
Parental NOAEL: at least 5000 ppm (333 mg/kg)
Reproduction NOAEL: at least 5000 ppm (333 mg/kg)
Developmental NOAEL: at least 5000 ppm (333 mg/kg)
Executive summary:

The objectives of this study were to determine the potential toxic effects of Amyl Salicylate when given via diet for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development.

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The dose levels in this study were selected to be 0, 500, 1500, 5000 ppm (estimated to be corresponding to 33, 100 and 333 mg/kg bw/day), based on the results of a Dose Range Finder in pregnant rats(Test Facility Study No. 20155389) and the results of a 90-day study with dietary administration of Amyl Salicylate in non-pregnant rats (Test Facility Study No. 20155386).

The study design was as follows:

Group No.

Test Item Identification

Dose Level

(ppm)

Dose Level

(mg/kg bw/day)

Number of Animals

Males

Females

1

-

0 (Vehicle)a

0

10

10

2

Amyl Salicylate

500

33

10

10

3

Amyl Salicylate

1500

100

10

10

4

Amyl Salicylate

5000

333

10

10

Id.= identification.

a  Standard powder rodent diet without test item.

During the lactation period, the following dietary concentrations (ppm) were usedto account for increase in food intake from females during the lactation period

Lactation

 

Group 1

Group 2

Group 3

Group 4

Days 1-4

0

336

1009

3364

Days 4-7

0

261

782

2606

Day 7 – end of study

0

211

634

2114


Chemical analyses of dietary preparations were conducted once during the study to assess accuracy, homogeneity and stability.

The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, body weight and food consumption, estrous cycle determination, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormone T4 (PND 14-16 pups)).

Analysis of diet preparations showed that the diets were prepared accurately and homogeneously. Additionally, stability of diets at 200 ppm was confirmed after storage for 12 days at room temperature in open containers and for 21 days in the freezer.

 

The calculation of test item intake during the study was somewhat hampered, since excessive spillage of the diet was observed in females of all groups. Despite measures to prevent the spillage of diet, spillage still occurred. To prevent an overestimation and as normal body weight (gain) at the control, low and mid dose level indicate that the actual diet intake in the current study is similar compared to historical controls, the nominal dose levels of 33, 100 and 333 mg/kg bw/day were used as estimates for test article intake to derive NOAELs. Nevertheless an impact on the overall nutrition of the females, as previously observed in a 90-day study, cannot be excluded for the females treated with 5000 ppm of test item.

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) of Amyl Salicylate were established:

Parental NOAEL:                  at least 5000 ppm (333 mg/kg bw/day)

Reproduction NOAEL:         at least 5000 ppm (333 mg/kg bw/day)

Developmental NOAEL:     at least 5000 ppm (333 mg/kg bw/day)

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
333 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
OECD and GLP study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Screening reproductive and developmental study (OECD 421, GLP):

The objectives of this study were to determine the potential toxic effects of Amyl Salicylate when given via diet for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development.

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The dose levels in this study were selected to be 0, 500, 1500, 5000 ppm (estimated to be corresponding to 33, 100 and 333 mg/kg bw/day), based on the results of a Dose Range Finder in pregnant rats(Test Facility Study No. 20155389) and the results of a 90-day study with dietary administration of Amyl Salicylate in non-pregnant rats (Test Facility Study No. 20155386).

The study design was as follows:

Group No.

Test Item Identification

Dose Level

(ppm)

Dose Level

(mg/kg bw/day)

Number of Animals

Males

Females

1

-

0 (Vehicle)a

0

10

10

2

Amyl Salicylate

500

33

10

10

3

Amyl Salicylate

1500

100

10

10

4

Amyl Salicylate

5000

333

10

10

Id.= identification.

a  Standard powder rodent diet without test item.

During the lactation period, the following dietary concentrations (ppm) were usedto account for increase in food intake from females during the lactation period

Lactation

 

Group 1

Group 2

Group 3

Group 4

Days 1-4

0

336

1009

3364

Days 4-7

0

261

782

2606

Day 7 – end of study

0

211

634

2114


Chemical analyses of dietary preparations were conducted once during the study to assess accuracy, homogeneity and stability.

The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, body weight and food consumption, estrous cycle determination, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormone T4 (PND 14-16 pups)).

Analysis of diet preparations showed that the diets were prepared accurately and homogeneously. Additionally, stability of diets at 200 ppm was confirmed after storage for 12 days at room temperature in open containers and for 21 days in the freezer.

 

The calculation of test item intake during the study was somewhat hampered, since excessive spillage of the diet was observed in females of all groups. Despite measures to prevent the spillage of diet, spillage still occurred. To prevent an overestimation and as normal body weight (gain) at the control, low and mid dose level indicate that the actual diet intake in the current study is similar compared to historical controls, the nominal dose levels of 33, 100 and 333 mg/kg bw/day were used as estimates for test article intake to derive NOAELs. Nevertheless an impact on the overall nutrition of the females, as previously observed in a 90-day study, cannot be excluded for the females treated with 5000 ppm of test item.

In conclusion, based on the absence of test item related adverse effects in this reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) of Amyl Salicylate were established:

Parental NOAEL:                  at least 5000 ppm (333 mg/kg bw/day)

Reproduction NOAEL:         at least 5000 ppm (333 mg/kg bw/day)

Developmental NOAEL:     at least 5000 ppm (333 mg/kg bw/day)

Effects on developmental toxicity

Description of key information

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 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.

Pre-natal Developmental Toxicity study (OECD 414, GLP):

A NOAEL could not be derived with enough certainty with regards to relevance of effects in light of the data available on the substance and the weight of evidence of the anticipated mode of action.

For risk assessment purposes, the NOAEL developmental toxicity was proposed to be set at 121 mg/kg bw/d, based on maternal toxicity. (see further justification below)

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
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. Regarding OECD 414, the final report is not yet available, it is in the draft version, because of that we attached the tables with all findings for your reference (please check Attached Background materials section).
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.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
as of June 2018
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch number of test material: VE00598644
- Expiration date of the lot/batch: 06 March 2020
- Purity test date: 99.89%
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han rats (females)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld
- Age at study initiation: 10-14 weeks old;
- Weight at study initiation: weighed between 183 and 252 g at the initiation of administration;
- Housing: individually in Makrolon Type M3 plastic cages (height 18cm) with standard bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles
- Diet (e.g. ad libitum): powder diets were provided ad libitum (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): tap water freely available
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21
- Humidity (%): 50% to 82%. The values that were outside the targeted range occurred for five days with a maximum of 90% (range 77 to 90%) and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study
- Air changes (per hr): 10 times (at least) /h with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
The test item was mixed without the use of a vehicle, directly with the required amount of powder feed. Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used. The control animals received the standard powder rodent without the test item.

- Rate of preparation of diet and storage temperature: Diets were prepared for use at room temperature for a maximum of 10 days. Diets were kept in the freezer (≤-15°C) until use for a maximum of 3 weeks, 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 (stability was confirmed under Test Facility Study No. 20155390 (Analytical Method Development and Validation Study)) 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:
Chemical analyses of dietary preparations were conducted once (week 1) during the study to assess concentration (all groups) and homogeneity (groups 2 and 4). 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.

All samples to be analyzed were transferred (at room temperature protected from light) to the analytical laboratory at the Test Facility.
Residual samples were discarded after completion of the sample analysis.

ANALYTICAL METHOD
Analyses were performed using a validated analytical procedure (Test Facility Study No. 20155390).

CONCENTRATION ANALYSIS
Duplicate sets of samples (approximately 5 g) for each sampling time point were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ±20% for diet of target concentration. After acceptance of the analytical results, backup samples were discarded.

HOMOGENEITY ANALYSIS
Duplicate sets of samples (approximately 5 g) for each sampling time point were used for homogeneity analysis, the remaining samples were retained at the Test Facility as backup samples. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was 10%. After acceptance of the analytical results, backup samples were discarded.

STABILITY ANALYSIS
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20155390) demonstrated that the test item is stable in diet when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20155390.
Details on mating procedure:
On 20 Aug 2019 and 22 Aug 2019, time-mated female Wistar Han Rats were received from Charles River Laboratories Deutschland, Sulzfeld. The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating). They were 10 14 weeks old and weighed between 183 and 252 g at the initiation of administration.
A health inspection was performed upon receipt of the animals.
Duration of treatment / exposure:
The test item and control item were administered to the appropriate animals by inclusion in the diet ad libitum from Day 6 to Day 21 post-coitum, inclusive.
Frequency of treatment:
Test item was included in the diet provided daily ad libitum.
Duration of test:
From Day 0 or Day 1 post-coitum until Day 21 post-coitum, inclusive.
Dose / conc.:
500 ppm
Remarks:
equivalent to 39 mg/kg bw/d [34 - 43 mg/kg bw/d]
Dose / conc.:
1 500 ppm
Remarks:
equivalent to 121 mg/kg bw/d [106 - 131 mg/kg bw/d]
Dose / conc.:
5 000 ppm
Remarks:
equivalent to 391 mg/kg bw/d [332 - 443 mg/kg bw/d]
No. of animals per sex per dose:
22 females per dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
The dose levels were selected based on a dose range finding study with dietary administration of Amyl Salicylate in pregnant rats treated from Day 6 to Day 21 post-coitum, inclusive (Test Facility Reference No. 20155389, Appendix 7), a full dietary OECD 421 study (Test Facility Study No. 20155387) and a 90-day study with dietary administration of Amyl Salicylate in non-pregnant rats (Test Facility Study No. 20155386), and in an attempt to produce graded responses to the test item without interfering with normal nutrition of the animals.

- Rationale for animal assignment:
On the day of receipt, animals were assigned to groups by a computer-generated random algorithm according to body weights. Each set of females mated on the same date (i.e. 4 sets) was distributed as evenly as possible over the dose groups with body weights within ± 25% of the mean for each set of animals.
Maternal examinations:
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 OBERVATIONS
Clinical observations were performed at least once daily, beginning on Day 2 post-coitum and lasting up to the 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, 2, 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 (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.
Cage debris was examined to detect premature birth.

BODY WEIGHTS
Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION
Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18 21 post-coitum.

WATER CONSUMPTION
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

BLOOD ANALYSIS
Blood of F0-animals was collected on the day of scheduled necropsy. Animals were not fasted overnight. Samples were collected, between 7:00 and 9:00 a.m., from the jugular vein in the animal facility. After collection, samples were transferred to the appropriate laboratory for processing.
- Thyroid Hormone:
Blood samples at a target volume of 1.0 mL were collected into tubes without anticoagulant. Blood samples were processed for serum, and serum was analyzed for Triiodothyronine (T3), Thyroxine (T4), Thyroid-Stimulating Hormone (TSH).
Remnants of serum samples were retained in the freezer (≤-75°C) for possible future analysis by the Test Facility. Under these storage conditions, samples are stable for 6 months. Any remaining sample will be discarded upon finalization.

SCHEDULED EUTHANASIA
Animals surviving until scheduled euthanasia were euthanized by an oxygen/carbon dioxide procedure. No terminal body weight was recorded.

NECROPSY
All animals were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No organs (except for the gravid uterus and thyroid gland) were weighed.
Each ovary and uterine horn of all animals were dissected and examined as quickly as possible to determine:
• The number of corpora lutea.
• The weight of the (gravid) uterus.
• The number of implantation sites.
• The number and distribution of live and dead fetuses.
• The number and distribution of embryo-fetal deaths.
• The sex of each fetus based on the anogenital distance.
In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
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 thyroid gland and gravid uterus were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of one of the organs of a pair may be taken and entered as a tissue comment. Organ to body weight ratio (using the body weight on Day 21 post-coitum) were calculated for thyroid glands only.

HISTOPATHOLOGY
Representative samples of thyroid gland were collected from all animals and preserved in 10% neutral buffered formalin.
Thyroid glands of all animals of Group 1 and 4 were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.
Tissues were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.
A peer review on the histopathology data was performed by a second pathologist.
Ovaries and uterine content:
Each ovary and uterine horn of all animals were dissected and examined as quickly as possible to determine:
• The number of corpora lutea.
• The weight of the (gravid) uterus.
• The number of implantation sites.
• The number and distribution of live and dead fetuses.
• The number and distribution of embryo-fetal deaths.
• The sex of each fetus based on the anogenital distance.
In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
Fetal examinations:
EUTHANASIA
Live fetuses were euthanized by administration of sodium pentobarbital into the oral cavity using a small metal feeding tube.

EXAMINATIONS
Litters of females surviving to scheduled necropsy, or that delivered on the day of scheduled necropsy, were subjected to detailed external, visceral and skeletal examinations, as described in the following Sections.
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

EXTERNAL EXAMINATIONS
Each viable fetus was sexed, examined in detail to detect macroscopic visible abnormalities and their weight was determined.
The anogenital distance (AGD) was measured for all viable fetuses. The AGD was normalized to the cube root of the fetal body weight.
For late resorptions (A072-09), a gross external examination was performed.

VISCERAL EXAMINATIONS
The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses (live and dead) in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe ref 1. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar ref 2.
The heads were removed from this one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination using the Wilson Sectioning technique ref 3. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin.
As visceral malformations were suspected for fetuses (A078-10, A080-04, A085-06), selected for skeletal examination, these fetuses were also subjected to visceral examination.
All carcasses, including the carcasses without heads, were eviscerated, labeled and fixed in 96% aqueous ethanol for subsequent examination of skeletons.

SKELETAL EXAMINATION
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson ref 4.
Subsequently, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads). As skeletal malformations were suspected for fetuses (A078-09, A078-12, A080-02, A080-08, A085-09, A088-01), selected for visceral examination, these fetuses were also subjected to skeletal examination.
All specimens were archived in glycerin with bronopol as preservative.
A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% 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 deemed appropriate) were reported whenever possible. Inferential statistics were performed (matrix below) when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
Pairwise comparisons:
Gp 2 vs. Gp 1
Gp 3 vs. Gp 1
Gp 4 vs. Gp 1

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 were compared using a Steel-test (many-to-one rank test).
Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution were compared using the Mann Whitney test.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and postimplantation loss.
Indices:
MATERNAL VARIABLES
Body Weight Gain: Calculated against the body weight on Day 6 post-coitum.
Corrected Body Weight Gain: Body weight on Day 21 post-coitum minus the body weight on Day 6 post-coitum and the weight of gravid uterus.
Relative Food Consumption: Calculated against the body weight for scheduled intervals.
Test item intake: Calculated as concentration of test item in diet (ppm) against relative food consumption.
Organ Weight Relative to Body Weight: Calculated against the body weight on Day 21 post-coitum.

REPRO/DEVELOPMENTAL VAIABLES
For each group, the following calculations were performed:
Preimplantation loss (%): (number of corpora lutea - number of implantation sites) / number of corpora lutea x 100
Postimplantation loss (%): (number of implantation sites - number of live fetuses) / number of implantation sites x 100

The fetal developmental findings were summarized by:
1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and
2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:
Viable fetuses affected/litter (%): number of viable fetuses affected/litter / number of viable fetuses/litter x 100
Historical control data:
available in appendix
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Piloerection was observed for 1/22 females treated at 1500 ppm during the first days of treatment, which was considered to be non-toxicological relevant as it only affected one female, and for 10/22 females treated at 5000 ppm for various days during treatment. In addition, hunched posture was observed on few days for 5/22 females treated at 5000 ppm. As both findings at 5000ppm were not observed in the concurrent control animals and/or were only observed in multiple animals of the high dose group, this was considered to be test item-related. Considering the nature of clinical signs this was regarded to be non-adverse.
Dermal irritation (if dermal study):
not examined
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):
At 5000 ppm, mean body weight was lower compared to controls from Day 9 post-coitum onwards, reaching statistical significance on Day 21 post-coitum only (7% lower compared to controls).
In addition, body weight gain was statistically significantly decreased compared to controls from Day 9 post-coitum onwards (between 21 - 60% lower than controls, p ≤ 0.01).
Body weight gain corrected for gravid uterus weight was statistically significantly lower at 5000 ppm compared to controls (37% lower, p ≤ 0.01) and was just below the lower limit of the historical control data .

The effect on (corrected) body weight gain was considered to be adverse, based on the magnitude of change and without correlating effects on food intake.

Mean body weights, body weight gain and weight gain corrected for gravid uterus of animals treated at 500 and 1500 ppm remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 5000 ppm, food consumption was statistically significantly lower (17%, p ≤ 0.01) compared to controls on Days 6-9 post-coitum and was statistically significantly higher (14%, p ≤ 0.05) compared to control on Days 15-18 post coitum. A normal food consumption was observed for the remaining periods, and as such, the variations observed were considered not adverse.
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded at 500 and 1500 ppm.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Note 1: In total, four females were not gravid (No. 28 at 500 ppm, No. 53 at 1500 ppm and Nos 69 and 73 at 5000 ppm) and were therefore excluded from the data tables.
Note 2: Possible adversity of these effects could not be assessed within this type of study.

At 5000 ppm, all individual Total T3 and Total T4 values (n=20) were below the lower limit of quantification (LLOQ) of 40.0 ng/dL and 1.00 µg/dL, respectively (values were reported as LLOQ/2 (i.e. 20 ng/dL and 0.5 µg/dL, respectively)).
TSH levels were slightly below the concurrent control levels, with 12/20 females below the lower limit of the historical control range and 1/20 females above the upper limit of the historical control range. Mean TSH concentration remained within the historical control data .

At 1500 ppm, a statistically significantly decrease in Total T3 and Total T4 was observed compared to concurrent controls (0.75x (p ≤ 0.05) and 0.61x (p ≤ 0.01) of controls, respectively), with 10/21 and 6/21 females showing serum levels below the LLOQ, respectively. Mean values were below the lower limit of the historical control data.
TSH values were comparable to concurrent controls and remained within the available historical control data.

At 500 ppm, TSH and Total T3 levels were increased, but remained within the available historical control data.
Total T4 levels were comparable to concurrent controls, with 2/20 females below the lower limit of quantification. Mean Total T4 at this dose level remained within the available historical control data.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Thyroid weights and thyroid:body weight ratios of treated animals were comparable to those of control animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with the test item.
Incidental findings were noted in individual animals treated at 500 ppm only and included right-sided enlargement of the thyroid gland (No. 34), reddish discoloration of the thymus (No. 31) and watery fluid in the uterus (No. 28, non-pregnant)
These findings are occasionally seen among rats used in these types of study and in the absence of a dose-response, they were considered changes of no toxicological significance. The presence of the watery fluid is related to a stage in the estrous cycle and is a normal finding for non-pregnant females.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations in the thyroid gland.
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
Description (incidence and severity):
There were no test item-related microscopic observations in the thyroid gland.
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.
Number of abortions:
no effects observed
Description (incidence and severity):
Not indicated in the report
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were comparable and in the range of normal biological variation.
At 5000 ppm, a higher number of postimplantation loss was observed (15.9% at 5000 ppm compared to 7.0% in controls); this was mainly caused by increased number of early resorptions. Although statistical significance was not reached, this number was above the higher limit of the available historical control data (HCD: P5-P95: 1.9-10.1%) and was considered to be adverse.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Not indicated in the report
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
At 5000 ppm, a higher number of postimplantation loss was observed (15.9% at 5000 ppm compared to 7.0% in controls); this was mainly caused by increased number of early resorptions. Although statistical significance was not reached, this number was above the higher limit of the available historical control data (HCD: P5-P95: 1.9-10.1%) and was considered to be adverse.
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
At 5000 ppm, the mean number of viable fetuses was lower (9.6 versus 10.5 in the control group) but remained within the historical control range. The relative number of viable fetuses was below the lower limit of the historical control data (84.1% at 5000 ppm; HCD: P5-P95: 90.0-98.2%), although no statistical significance was reached, probably due to the high standard deviation observed. The lower relative number of viable fetuses was considered to be adverse.

No test item-related effects on litter size of the 500 and 1500 ppm groups were observed.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
121 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
pre and post implantation loss
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The fetal weights (male, female and combined) at 5000 ppm were statistically significantly lower (25% for all three) compared to controls. In addition, all mean values were below the historical control range of the Test Facility (HCD: P5-P95: 5.2-5.5 gram, 4.9-5.3 gram and 5.0-5.4 gram for males, females and combined weights, respectively). This delay in development was observed in the presence of maternal toxicity demonstrated by a concurrent body weight and (corrected-)body weight gain decrease.
Based on the magnitude of change, these lower fetal body weights were considered an adverse effect of treatment with the test item.

Mean fetal weights at 500 and 1500 ppm were unaffected by treatment with the test item.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was considered unaffected by treatment up to 5000 ppm.
The statistically significant difference observed at the 500, 1500 and 5000 ppm groups are caused by the slightly higher percentage of females in the control group, whereas in the treated groups a slightly higher percentage of males was observed. All values are within the normal range and are therefore considered to be unrelated to treatment with the test item.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
At 5000 ppm, the mean number of viable fetuses was lower (9.6 versus 10.5 in the control group) but remained within the historical control range. The relative number of viable fetuses was below the lower limit of the historical control data (84.1% at 5000 ppm; HCD: P5-P95: 90.0-98.2%), although no statistical significance was reached, probably due to the high standard deviation observed. The lower relative number of viable fetuses was considered to be adverse.

No test item-related effects on litter size of the 500 and 1500 ppm groups were observed.
Changes in postnatal survival:
not examined
External malformations:
effects observed, treatment-related
Description (incidence and severity):
A test item-related increase of fetuses with body wall closure effects was observed at 5000 ppm. In this dose-group, spina bifida, omphalocele and craniorachischisis occurred in respectively 10 (4), 2 (1) and 1 (1) fetuses (litters), whereas none were noted in fetuses of Groups 2 and 3, nor in the control group. In addition, values were above the maximum values of the historical control data (omphalocele) or were not reported in the historical control data (spina bifida and craniorachischisis).
Therefore, all three malformations were considered related to treatment and adverse. This delay in development was observed in the presence of maternal toxicity demonstrated by a concurrent body weight and (corrected-)body weight gain decrease.

One other external malformation occurred in this study; Group 3 fetus A045-09 had anal atresia. As this occurred singly at the mid-dose level and was previously observed in historical control fetuses, it was considered to be unrelated to treatment with the test item.
External variations were not seen in any group.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
A high incidence of adverse test item-related axial skeletal malformations was observed at 5000 ppm and in a singleton fetus at 1500 ppm.

At 5000 ppm, malformations causing this high incidence were: vertebral anomaly with or without associated rib anomaly, sternum anomaly, costal cartilage anomaly and sternoschisis . The vertebral anomaly observed in the cervical region was mainly characterized by an additional vertebra in the cervical column. All abovementioned malformations are strongly influencing each other. For instance, an extra vertebra in the cervical column with two full ribs and costal cartilages connecting to the sternum can influence the connecting position of other costal cartilages and hence total sternum anatomy. Because these were interrelated, all these malformations were considered test item-related.

At 1500 ppm, two fetuses (A062-13 and A066-09) were observed with a vertebral anomaly. The incidence at 1500 ppm was below the historical control upper value, although an anomaly of the specific affected cervical arche was not recorded in the historical control data. In addition, clear similarities with the vertebral anomalies of fetuses at 5000 ppm were observed for one of these fetuses (A062-13; see paragraphs below), and therefore a test item relationship cannot be fully excluded.
The findings for fetus A066-09 were considered chance findings as these were not in line with the findings in the lumbar region of the vertebral column observed in the eight fetuses at 5000 ppm examined in full detail.
At 1500 ppm, fetus A062-13 had a malformation in the cervical region with rib anomaly variations, consisting of an absent and small cervical 5th arch in addition to a full rib and ossification site in the last (7th) cervical vertebra. These findings were in line with the combined description of variations and malformations affecting the vertebral column, ribs and/or sternum of eight litters at 5000 ppm that were fully evaluated in detail. The reasoning for this was given below:
• In six of the eight litters examined in full detail at 5000 ppm, the primary finding in 21/24 fetuses was the presence of an 8th cervical vertebrae, with a rib on that anomalous 8th vertebra. An additional full rib was observed on the 7th cervical vertebra for 1500 ppm fetus A062 13. Although the occurrence of a 7th cervical full rib can be observed in control fetuses of the historical control data as well (2.4 % per litter basis at 1500 ppm compared to HCD: mean [min-max]; 6.3 [0.0-13.1]); and the additional full rib occurred on the 7th cervical vertebra as opposed to the 8th cervical vertebra at 5000 ppm, , it could not be fully excluded that this finding may be related to treatment with the test item,as the findings for A062-13 were in line with the increased incidence of additional full cervical ribs at 5000 ppm
• Another pre-dominant finding at 5000 ppm (16/24 fetuses) was one or more absent and/or small cervical arches. Notably in 4/16 high dose fetuses in which the cervical arches were affected, this concerned the 5th cervical arch, the same arch which was affected in 1500 ppm fetus A062-13. Additionally, in 10/16 fetuses, abnormalities were observed in adjacent cervical arches (i.e. #4 and #6). Noteworthy, an anomaly of the 5th arch was previously not observed in the historical control data.
• At 5000 ppm, malformations affecting the cervical region did occur in absence of malformations elsewhere in the vertebral column, but to a much lesser extent (9 fetuses with only the cervical region affected vs 55 fetuses with the cervical region being affected in combination with other regions.
In summary, given the similarities with the test item-related effects observed at 5000 ppm, it cannot be fully excluded that the vertebral anomaly with associated rib anomaly in this single fetus at 1500 ppm was test item-related and therefore adverse.


The litter incidence of the observation “bent limb bones” (currently classified as a malformation) was 0.0%, 2.5%, 2.1% and 11.8% at 0, 500, 1500 and 5000 ppm, respectively. The incidence at the high dose was statistically significantly increased compared to concurrent controls and was considered to be related to treatment with the test item. In addition, incidences at 500 and 1500 ppm were near the historical control maximum value of 2.3% per litter. Bent limb bones did not occur in control fetuses and given the high number of affected fetuses in the high dose group, the “bent limb bones” observation was also considered potentially test item-related at 500 and 1500 ppm. However, since this finding is known to be reversible after birth ref 5-6, this was considered non-adverse.

Other malformations that occurred in this study were a skull anomaly in Fetus No. A032-02 (500 ppm) and brachydactyly in control fetus A010-08.
These were considered chance findings due to single occurrence at low dose or control levels.

Skeletal variations occurred at a statistically significantly increased incidence at 5000 ppm compared to the control value. Mean litter incidences of total variations were 78.1%, 70.2%, 73.1% and 96.1% per litter at 0, 500, 1500 and 5000 ppm, respectively.

Taking into account the malformations affecting the vertebral column, ribs and sternum, the following variations that reached statistical significance at 5000 ppm were considered test item-related and adverse: 14th rudimentary and full ribs, caudal shift of pelvic girdle, malaligned and fused sternebrae, and supernumerary sternebra. Additionally, non-statistically significant increases in following parameter were observed at 5000 ppm, namely: reduced vertebral arches, unossified vertebral centra, branched sternebrae and extra sternal ossification sites. Because these occurred in regions, in which also test item-related malformations occurred and virtually none was observed in any other group, a relationship with treatment with the test item could not be excluded.

In addition, two fetuses at 5000 ppm (A067-3 and A083-9) had a skeletal variation consisting of ossification site(s) at the 7th cervical vertebra. As this variation was also located in the (cervical) vertebral column region and occurred at higher incidences in other dose groups, it could be regarded as background finding. However, in view of all test item-related malformations and variations, it was considered to be related to treatment with the test item in this study.

The (statistically significant) increased incidences of skeletal variations consisting of bent ribs, bent scapulae, reduced ossification of the skull, unossified metacarpals and/or metatarsals, reduced ossification of vertebral centra and unossified sternebra nos. #5 and/or #6 at 5000 ppm were considered to be related to the low fetal weights in this group.
All other skeletal variations that occurred in the control, 500 and 1500 ppm groups occurred in the absence of a dose-related incidence trend, occurred infrequently and/or at frequencies that were within the range of available historical control data. Therefore, they were not considered test item-related.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
A test item-related increase of the number of fetuses with hemorrhagic stomach contents, large kidneys and malpositioned testes was observed at 5000 ppm. These respective malformations were observed in 8 (4), 5 (2), 5 (3) fetuses (litters) of this group, whereas none were noted in Groups 2 and 3, nor in the control group.
Therefore, all these malformations were considered adverse and related to treatment with the test item.

Other malformations that occurred in this study were dilated aortic arch and narrow pulmonary trunk (both in Fetus No. A044-01 (500 ppm) and Fetus No. A080-02 (5000 ppm)) and internal hydrocephaly (Fetus No. A085-07).
The single occurrence, group distribution and occurrence in historical control fetuses does not indicate a treatment relationship and therefore these were considered chance findings.

Of the visceral variations, the incidence of convoluted ureters and dilated ureters (5.8% and 7.9% per litter, respectively) at 5000 ppm was above the upper limit of the historical control data (P95: 4.2% and 2.3% per litter, respectively). Control fetus A009-02 was observed with these two variations as well. It is expected that these variations at the high dose are reversible and resemble a general development delay based on the low fetal body weights at this dose level.
Therefore, both ureter variations were considered to be an indirect, non-adverse effect of the test item.

Other variations occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data.
Other effects:
no effects observed
Description (incidence and severity):
FETAL ANOGENITAL DISTANCE
There were no toxicologically relevant effects on (corrected) fetal anogenital distance (both sexes) noted after treatment up to 5000 ppm.
Key result
Dose descriptor:
NOAEL
Effect level:
121 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
external malformations
skeletal malformations
visceral malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
external: cranium
external: umbilicus
skeletal: sternum
skeletal: rib
skeletal: vertebra
visceral/soft tissue: gastrointestinal tract
visceral/soft tissue: male reproductive system
other: visceral/soft tissue: kidney
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
391 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Developmental effects occured together with maternal toxicity, but the relationship between the two could not be examined within the frame of the protocol of this study.

Conclusions:
In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for Amyl Salicylate was established as being 1500 ppm (corresponding with an actual test item intake of 121 mg/kg/day), based on the observed effects on (corrected) body weight gain.
The repro/developmental NOAEL for Amyl Salicylate is proposed to be set at 1500 ppm (corresponding to approximately 121 mg/kg/d) based on developmental effects observed in this study at 5000ppm consisting of an increased postimplantation loss (early resorption), in utero-growth retardation and increased visceral and skeletal malformations. At 1500 ppm, only one singleton fetus in 213 fetuses available for examination (0,46%) at this dose presented a cervical malformation. This is considered a possible chance finding unrelated to treatment, since no other developmental effect was observed in any other fetuses or dams at this dose level. Besides, a GLP OECD 421 Reproduction/Developmental Toxicity Screening dietary study was conducted with this test item at the same dose levels of 500, 1500 and 5000 ppm (equivalent to 33, 100 and 333 mg/kg/d (Charles River Laboratories Test Facility Study No. 20155387)) showing no reproductive or developmental effects up to the highest dose, either in the dams or pups. Consequently, the NOAEL for reproductive and developmental effects in this OECD 421 study was estimated to be above the highest dose tested of 333 mg/kg/d. Despite the high incidence of bent bones observed in the fetus in the treatment of 5000 ppm in the present OECD 414 study, pups in the OECD 421 study subjected to postnatal day (PND) 21 examination were found not to have any of these effects.
Finally, when considering ADME leading to a potentially toxic metabolite of Amyl salicylate, the weight of evidence is demonstrating an inconsistency in the dose level at which adverse developmental effects are seen in this study. (see endpoint summary of chapter 7.8 Toxicity to reproduction)
Executive summary:

The objectives of this study were to determine the potential of Amyl Salicylate to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given via diet to time-mated female Wistar Han rats from Day 6 to 21 post-coitum, inclusive.The dose levels in this study were selected to be 0, 500, 1500, 5000 ppm (calculated average dose equivalent of 39, 121 and 391 mg test item/kg body weight/day, respectively) , based on the results of the dose range finder.

Chemical analyses of dietary preparations were conducted once during the study to assess accuracy, homogeneity.

The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, test item intake, thyroid hormone levels (Total T3, Total T4, TSH), gross necropsy findings, organ weights (gravid uterus and thyroid gland), uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and postimplantation loss.

In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations.

Dietary analyses confirmed that test item diets were prepared accurately and homogenously.

In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for Amyl Salicylate was established as being 1500 ppm (corresponding with an actual test item intake of 121 mg/kg/day), based on the observed effects on (corrected) body weight gain. The repro/developmental NOAEL for Amyl Salicylate is proposed to be set at 1500 ppm (corresponding to approximately 121 mg/kg/d) based on developmental effects observed in this study at 5000ppm consisting of an increased postimplantation loss (early resorption),in utero-growth retardation and increased visceral and skeletal malformations.At 1500 ppm, only one singleton fetus in 213 fetuses available for examination (0,46%) at this dose presented a cervical malformation. This is considered a possible chance finding unrelated to treatment, since no other developmental effect was observed in any other fetuses or dams at this dose level. Besides, a GLP OECD 421 Reproduction/Developmental Toxicity Screening dietary study was conducted with this test item at the same dose levels of 500, 1500 and 5000 ppm (equivalent to 33, 100 and 333 mg/kg/d (Charles River Laboratories Test Facility Study No. 20155387)) showing no reproductive or developmental effects up to the highest dose, either in the dams or pups. Consequently, the NOAEL for reproductive and developmental effects in this OECD 421 study was estimated to be above the highest dose tested of 333 mg/kg/d.

Endpoint:
developmental toxicity
Remarks:
Screening reproductive/developmental study
Type of information:
experimental study
Adequacy of study:
supporting 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.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD 421, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch number of test material: PE00206659 (Week 1 and 2) and VE00598644 (Week 3 onward)
- Expiration date of the lot/batch: 18 May 2019 and 06 March 2020 respectively
- Purity test date: 99.69%
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han rats
Details on test animals and environmental conditions:
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.
TEST ANIMALS
- Source:
Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: (P)
males were 11-12 weeks old and females were 13-14 weeks old.
- Weight at study initiation: (P) Males: between 260 and 313 g; Females: between 203 and 247 g.
- Housing:
On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The room in which the animals were kept was documented in the study records.
- Diet (e.g. ad libitum):
powder diets were provided ad libitum throughout the study, except during designated procedures. During the acclimatization period, animals had free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum):
tap water was freely available
- Acclimation period:
7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C):
19 to 21°C
- Humidity (%):
mean relative humidity of 47 to 72%
- Air changes (per hr):
min 10 times/h
- Photoperiod (hrs dark / hrs light):
12 hrs dark / 12 hrs light
IN-LIFE DATES: From: 10 Apr 2019 To: 24 Jun 2019
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
The test item was mixed without the use of a vehicle, directly with the required amount of powder feed. Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used.
Diets were prepared for use at room temperature for a maximum of 10 days. Diets were kept in the freezer (≤-15°C) until use for a maximum of 3 weeks, if not used on the day of preparation.
Any remaining food left after filling the food hoppers may be 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:
SAMPLE COLLECTION AND ANALYSIS
Diet preparation samples were collected for analysis as indicated below.

Occasion Concentration Homogeneity Stability
Week 1 of treatment All groups + 200 ppm, Groups 2 and 4 + 200 ppm 200 ppm

The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 and 200 ppm preparations were averaged and utilized as the concentration results.
The 200 ppm diet was only used for analytical purposes and was not administered to the animals. QC samples at 200 ppm were included in 5-fold during the analyses to extend the validation range to 200-15000 ppm, i.e. thereby also covering the lower diet concentrations which were used during the lactation phase.

All samples to be analyzed were transferred (at room temperature under normal laboratory light conditions) to the analytical laboratory at the Test Facility.
Residual samples were discarded after completion of the sample analysis.

ANALYTICAL METHOD
Analyses were performed using a validated analytical procedure (Test Facility Study No. 20155390).

CONCENTRATION ANALYSIS
Duplicate sets of samples (approximately 5 g) were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. After acceptance of the analytical results, backup samples were discarded.
Details on mating procedure:
FREQUENCY:
Daily, after a minimum of 14 days of treatment. The mating period will consist of a maximum of 14 consecutive days.
PROCEDURE:
Animals will be cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating will be confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day will be designated Day 0 post-coitum. Once mating has occurred, the males and females will be separated. A maximum of 14 days will be allowed for mating, after which females who have not shown evidence of mating will be separated from their males. In case less than 9 females per group have shown evidence of mating, each non-mated female may be re-mated once with a male for a maximum of 7 days (if possible). A male of the same group having previously shown evidence of mating (non-selected male if possible, see section 13) will be used for re-mating.
Duration of treatment / exposure:
The test item and control item were administered to the appropriate animals by inclusion in the diet ad libitum from Day 1 onwards for a minimum of 29 days. Males were exposed for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females were exposed for 51 to 61 days, i.e. 14 days prior to mating (to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day of scheduled necropsy. Females which failed to deliver were treated for 42-55 days.
Frequency of treatment:
Daily in ad libitum diet
Duration of test:
The test item and control item were administered to the appropriate animals by inclusion in the diet ad libitum from Day 1 onwards for a minimum of 29 days. Males were exposed for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females were exposed for 51 to 61 days, i.e. 14 days prior to mating (to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day of scheduled necropsy. Females which failed to deliver were treated for 42-55 days.
Dose / conc.:
500 ppm
Remarks:
extrapolated to an equivalent of 33 mg/kg bw/d
Dose / conc.:
1 500 ppm
Remarks:
extrapolated to an equivalent of 100 mg/kg bw/d
Dose / conc.:
5 000 ppm
Remarks:
extrapolated to an equivalent of 333 mg/kg bw/d
No. of animals per sex per dose:
10 females and 10 males per dose level
Control animals:
yes, plain diet
Details on study design:
DOSE SELECTION
The dose levels were selected based on a dose range finding study with dietary administration of Amyl Salicylate in pregnant rats treated from Day 6 to Day 21 post-coitum, inclusive (Test Facility Study No. 20155389) and a 90-day study with dietary administration of Amyl Salicylate in non-pregnant rats (Test Facility Study No. 20155386), and in an attempt to produce graded responses to the test item without interfering with normal nutrition of the animals.

ANIMAL ASSIGNMENT
A total of 40 females was selected at randomization before initiation of the pretest phase. Any selected female classified as not having regular estrous cycles during the pretest phase was replaced before initiation of administration by one of the 8 additional females having regular estrous cycles, if feasible. A total of 40 females with regular estrous cycles continued in the study. The supernumerary females were removed from the study, and their estrous cycle results were kept in the raw data but not reported.
Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.

BLOOD SAMPLING
Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m. (for exceptions, see Appendix 7), from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. After collection all samples were transferred to the appropriate laboratory for analysis.
F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0 females were not fasted overnight.
Blood of F1-animals was collected on PND 4 and PND 14-16, if possible. This was performed in the necropsy room.
On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation, between 7.00 and 10.30 a.m., and samples were pooled to one sample per litter. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single pup.
On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female, if possible). Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure.
Maternal examinations:
MORTALITY/MORIBUNDITY CHECKS
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 OBSERVATIONS
Clinical observations were performed once daily, once prior to the first administration and from start of administration onwards, up to the day prior to necropsy. During the dosing period, these observations were performed at no specific time point.
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 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 (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

BODY WEIGHTS
Animals were weighed individually on the first day of administration, and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
A terminal weight was recorded on the day of scheduled necropsy (for exceptions, see Appendix 7).

FOOD CONSUMPTION
Food consumption was 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.
Food spillage was estimated during the study by means of sieving the bedding material, including the enrichments, with a metal sieve (mesh-size 500 μm) at the end of each food consumption period.

WATER CONSUMPTION
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.
Ovaries and uterine content:
not applicable
Fetal examinations:
MORTALITY/MORIBUNDITY
Pups were observed daily for general health/mortality. The number of live and dead pups was determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

CLINICAL OBSERVATION
Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check were given in the respective report tables.

BODY WEIGHTS
Live pups were weighed individually on PND 1, 4, 7 and 13.

SEX RATIO
Sex was externally determined for all pups on PND 1 and 4.

ANOGENITAL DISTANCE
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.

AREOLA7MIPPLE RETENTION
All male pups in each litter were examined for the number of areola/nipples on PND 13.

CULLING
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

METHOD OF EUTHANASIA
All pups (PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%).
The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

UNSCHEDULED DEATHS
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally).
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

SCHEDULED EUTHANASIA
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible.
All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
In addition, blood was collected from two pups per litter (see also section 4.10.1), and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin.
Statistics:
STATISTICAL ANALYSIS
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% 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 deemed 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
Group 3 vs. Group 1
Group 4 vs. Group 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).

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.
Indices:
Mating index (%), Precoital time, Fertility index (%), Gestation index (%), Duration of gestation, Post-implantation survival index (%), Live birth index (%), Percentage live males at First Litter Check (%), Percentage live females at First Litter Check (%), Viability index (%), Lactation index (%).
Historical control data:
See report
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were noted during daily detailed clinical observations.
Hunched posture and scabs were noted in a single Male animal (No. 12) of the 500 ppm group on Days 15-16 and Days 14-17, respectively. These and other clinical signs noted (including scabs in the neck and shoulder area and alopecia) occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be non-adverse and unrelated to treatment.
Dermal irritation (if dermal study):
not examined
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):
A statistically significant and test-item related reduced body weight gain was noted in females of the 5000 ppm group during the pre-mating period (up to 0% body weight gain at 5000 ppm compared to 6% in concurrent controls at the end of the pre-mating period) and the first week of the mating period. Body weight gain was in the same range as controls in the mating period following the first week, post-coitum and lactation period. The lower body weight gain during the pre-mating period resulted in a slightly lower body weight of females of the 5000 ppm group compared to controls during mating, post-coitum and the first week of the lactation phase reaching statistical significance in some instances.
As body weight gain normalized to control levels during the course of the study, this was considered not adverse
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of females of the 5000 ppm group was lower (up to 0.68x of controls) compared to levels in the control group during the pre-mating period, post-coitum period and the first week of the lactation period. Changes were not statistically significant. Food consumption returned to control levels between Days 7-13 of the lactation period.
Food consumption before or after correction for body weight for treated males was similar to control levels over the treatment period.
Significant food spillage was observed for all groups during the study, and although sieving of the bedding to recover the spilled powder diet was performed to account for it, it was not deemed sufficient to correct food intake values appropriately as control values were not in the range of historical controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A test-item related decrease in serum levels of T4 in F0-males was noted, reaching statistical significance at the 1500 and 5000 ppm groups (0.77x and 0.40x of controls at 1500 and 5000 ppm, respectively). Values of Group 3 animals were within the historical control range and as such, the test-item related decrease at 1500 ppm was considered not toxicologically relevant. Except for the T4 values of one Group 4 animal, serum levels of all animals of the 5000 ppm group were below the historical control range. Histopathological evaluation of the thyroid did not reveal any correlating alterations.
Urinalysis findings:
not examined
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 were no test item-related alterations in organ weights (Epididymis, coagulation gland, parathyroid gland, prostate, seminal vesicle, thyroid, testes).
Statistically significant changes between relative seminal vesicle weights of animals treated at 500 ppm and control animals were considered not to be a sign of toxicity in the absence of a dose-related trend.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations.
All 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):
There were no test item-related microscopic observations.
All 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
Description (incidence and severity):
There were no test item-related microscopic observations.
All 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.
Number of abortions:
no effects observed
Description (incidence and severity):
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
The total number of offspring born compared to the total number of uterine implantations was slightly lower in the 5000 ppm group.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 94, 93, 93 and 88% for the control, 500, 1500 and 5000 ppm groups, respectively.
As all indices remained within the historical control range , this was considered not toxicologically relevant.
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment.
Live birth indices (number of live offspring on PND 1 as percentage of total number of offspring born) were 99, 100, 100 and100% for control, 500, 1500 and 5000 ppm groups, respectively.
One pup of the control group (Pup No. 4, litter 49) was found dead at first litter check. No toxicological relevance was attributed to this dead pup since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Gestation index and duration of gestation were considered not to be affected by treatment.
Except for one female of the control group and one female of the 500 ppm group, all pregnant females had live offspring. The gestation indices were 90, 88, 100 and 100% for the control, 500, 1500 and 5000 ppm groups, respectively.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The failed pregnancies of female Nos. 48 and 60, were considered to be unrelated to treatment due to the incidental occurrence and lack of a dose-related trend.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 333 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Slightly lower body weights were recorded for pups of the 5000 ppm group compared to controls from PND1 onwards (0.89x of controls for males and 0.93x of controls for females on PND1). Based on the minimum magnitude of the effect, the lack of statistical significance, since mean body weights remained within the historical control data and the differences appear to become smaller over time, this was considered not toxicologically relevant.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment.
Changes in sex ratio:
effects observed, treatment-related
Description (incidence and severity):
Sex ratio was statistically significantly altered by treatment at 5000 ppm (% of males / females 60 / 40 at 5000 ppm versus 44 / 56 in concurrent controls).
At 5000 ppm, there were 4/8 litters with a sex ratio skewed towards males, 1/8 litter with a sex ratio skewed towards females and 3/8 litters with an approximate equal ratio of males/females.
As the sex ratio of 60 / 40 at 5000 ppm was within normal range, this was considered not toxicologically relevant.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No toxicologically relevant effect on litter size was observed.
Litter size was slightly lower in the 5000 ppm group compared to controls. Live litter sizes were 11.3, 10.6, 10.7 and 9.6 living pups/litter for the control, 500, 1500 and 5000 ppm groups, respectively. As the change was minimal, values did not reach statistical significance and values were well within the historical control range this was considered not toxicologically relevant.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
LIVE BIRTH INDEX
The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment.
Live birth indices (number of live offspring on PND 1 as percentage of total number of offspring born) were 99, 100, 100 and100% for control, 500, 1500 and 5000 ppm groups, respectively.
One pup of the control group (Pup No. 4, litter 49) was found dead at first litter check. No toxicological relevance was attributed to this dead pup since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

VIABILITY INDEX
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment. Viability indices were 99, 100, 99 and 99% for the control, 500, 1500 and 5000 ppm groups, respectively.
One pup of the control group, one pup at 1500 ppm and one pup at 5000 ppm were missing on PND 4, 2 and 4, respectively. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

LACTATION INDEX
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment.
No pups were found dead/missing between lactation Days 5 and 13, resulting in a lactation index of 100% for all groups.
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 333 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test item related adverse effects observed up to the highest dose
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Reproductive results
No reproductive toxicity was observed up to the highest dose level tested (5000 ppm).
No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, and histopathological examination of reproductive organs).
Developmental results
No developmental toxicity was observed up to the highest dose level tested (5000 ppm).
No toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, maternal care, litter size, sex ratio and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).
In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) of Amyl Salicylate were established:
Parental NOAEL: at least 5000 ppm (333 mg/kg)
Reproduction NOAEL: at least 5000 ppm (333 mg/kg)
Developmental NOAEL: at least 5000 ppm (333 mg/kg)
Executive summary:

The objectives of this study were todetermine thepotential toxic effects of Amyl Salicylate when given via diet for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development.

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The dose levels in this study were selected to be 0, 500, 1500, 5000 ppm(estimated to be corresponding to 33, 100 and 333 mg/kg bw/day), based on the results of a Dose Range Finderin pregnant rats(Test Facility Study No. 20155389) and the results of a90-day study with dietary administration of Amyl Salicylate in non-pregnant rats(Test Facility Study No. 20155386, for details see section4.7.2).

The study design was as follows:

Group No.

Test Item Identification

Dose Level

(ppm)

Dose Level

(mg/kg bw/day)

Number of Animals

Males

Females

1

-

0 (Vehicle)a

0

10

10

2

Amyl Salicylate

500

33

10

10

3

Amyl Salicylate

1500

100

10

10

4

Amyl Salicylate

5000

333

10

10

Id.= identification.

a  Standard powder rodent diet without test item.

During the lactation period, the following dietary concentrations (ppm) were usedto account for increase in food intake from females during the lactation period

Lactation

 

Group 1

Group 2

Group 3

Group 4

Days 1-4

0

336

1009

3364

Days 4-7

0

261

782

2606

Day 7 – end of study

0

211

634

2114


Chemical analyses of dietary preparations were conducted once during the study to assess accuracy, homogeneity and stability.

The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, body weight and food consumption, estrous cycle determination, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormone T4 (PND 14-16 pups)).

Analysis of diet preparations showed that the diets were prepared accurately and homogeneously. Additionally, stability of diets at 200 ppm was confirmed after storage for 12 days at room temperature in open containers and for 21 days in the freezer.

 

The calculation of test item intake during the study was somewhat hampered, sinceexcessivespillage of the diet was observed in females of all groups. Despite measures to prevent the spillage of diet, spillage still occurred. To prevent an overestimation and as normal body weight (gain) at the control, low and mid dose level indicate that the actual diet intake in the current study is similar compared to historical controls, the nominal dose levels of 33, 100 and 333 mg/kg bw/day were used as estimates for test article intake to derive NOAELs. Nevertheless an impact on the overall nutrition of the females, as previously observed in a 90-day study, cannot be excluded for the females treated with 5000 ppm of test item.

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) of Amyl Salicylate were established:

Parental NOAEL:                  at least 5000 ppm (333 mg/kg bw/day)

Reproduction NOAEL:         at least 5000 ppm (333 mg/kg bw/day)

Developmental NOAEL:     at least 5000 ppm (333 mg/kg bw/day)

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
121 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
OECD and GLP study
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Pre-natal developmental toxicity study (OECD 414, GLP):

The objectives of this study were to determine the potential of Amyl Salicylateto induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given via diet to time-mated female Wistar Han rats from Day 6 to 21 post-coitum, inclusive.The dose levels in this study were selected to be 0, 500, 1500, 5000 ppm (calculated average dose equivalent of 39, 121 and 391 mg test item/kg body weight/day, respectively) , based on the results of the dose range finder.

Chemical analyses of dietary preparations were conducted once during the study to assess accuracy, homogeneity.

The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, test item intake, thyroid hormone levels (Total T3, Total T4, TSH), gross necropsy findings, organ weights (gravid uterus and thyroid gland), uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and postimplantation loss.

In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations.

Dietary analyses confirmed that test item diets were prepared accurately and homogenously.

In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for Amyl Salicylate was established as being 1500 ppm (corresponding with an actual test item intake of 121 mg/kg/day), based on the observed effects on (corrected) body weight gain.

The reproductive/developmental NOAEL for Amyl Salicylate is proposed to be set at 1500 ppm (corresponding to approximately 121 mg/kg/d) based on the unequivocally test item-related developmental effects observed in this study at 5000ppm consisting of an increased postimplantation loss (early resorption), in utero-growth retardation and increased visceral and skeletal malformations.At 1500 ppm, only one singleton fetus in 213 fetuses available for examination (0,46%) at this dose presented a cervical malformation. This is considered a possible chance finding unrelated to treatment, since no other developmental effect was observed in any other fetuses or dams at this dose level. Besides, a GLP OECD 421 Reproduction/Developmental Toxicity Screening dietary study was conducted with this test item at the same dose levels of 500, 1500 and 5000 ppm (equivalent to 33, 100 and 333 mg/kg/d (Charles River Laboratories Test Facility Study No. 20155387)) showing no reproductive or developmental effects up to the highest dose, either in the dams or pups. Consequently, the NOAEL for reproductive and developmental effects in this OECD 421 study was estimated to be above the highest dose tested of 333 mg/kg/d.

Toxicity to reproduction: other studies

Description of key information

No other study available

Mode of Action Analysis / Human Relevance Framework

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 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.

Within the standard protocols of the above mentioned requested studies, we are unable to characterise with enough certainty and precision the mode of action of Amyl Salicylate.

Further data on the impact on pup development of maternal toxicity showed by compromised nutrition, in terms of extent, magnitude and time of occurence during the critical stage of development; and further data on the in vivo ADME of Amyl salicylate following oral absorption are necessary to enable clarification as to the potential mode of action of Amyl Salicylate.

Justification for classification or non-classification

In all newly generated in vivo studies (OECD 408, OECD 421 and OECD 414) performed following an ECHA decision, we could observe that a prolonged exposure to higher doses of Amyl salicylate resulted in a consistent compromised nutrition in both male and female rats. The interference of the treatment with normal nutrition was demonstrated with variable magnitude among the studies by a significant decrease in body weight, body weight gain and/or food consumption.

In the OECD 421, the compromised nutrition (body weight gain) occured at the top dose of 5000ppm, mainly at the very beginning of the study (mating period) and then normalised during the post-coitum and lactation periods. In this study, the overal impact on nutrition was not deemed of sufficient magnitude throughout the study to be considered adverse up to the top dose of 5000ppm, and as such the NOAEL for maternal toxicity was found to be at least 5000ppm (estimated to be 333mg/kg bw/d). Concurrently, no effects on reproductive functions or the development of the pups was found in this study and the NOAEL repro/developmental was found to be at least 5000ppm (estimated to be 333mg/kg bw/d) as well.

The findings of the OECD 414 were extremely inconsistent with the findings of the OECD 421 just described. In the OECD 414 study, the compromised nutrition occured at the top dose of 5000ppm (equivalent to 391mg/kgbw/d) from day 9 post-coitum and persisted until the end of the study (day 21 post-coitum). Here, the compromised nutrition was of sufficient magnitude to be considered adverse. As a consequence, the NOAEL for maternal toxicity was found to be the mid-dose of 1500ppm (eq. 121mg/kgbw/d). Concurrently, unequivocally test item related reproductive and developmental effects (external, skeletal and visceral malformations together with increased post-implantation loss) were observed on the pups at the top dose of 5000ppm. At the mid-dose of 1500ppm only one singleton fetuse was found with a cervical malformation that could not be fully concluded as being treatment related or not.

The overall conflicting information and inconsistent outcomes observed between the OECD 421 and the OECD 414 at equivalent doses of Amyl salicylate raise significant concerns on the reliability of the dataset when we try to interpret the weight of evidence on Amyl salicylate.

Moreover, the OECD guideline 414 do not include sufficient systemic toxicity endpoints to fully characterise the extent and potential consequences of the maternal toxicity that occurred during this study.

Overall, the studies available on Amyl Salicylate contained conflicting data and are not sufficient to properly conclude on the impact of maternal toxicity on the development of pups and as such, the need or not for a classification according to the (EC) No 1272/2008 Regulation (CLP).