Reaction mass of 2-methylbutyl salicylate and pentyl salicylate

Toxicity to reproduction

Administrative data

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.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

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 for cross-reference:

reference to same study

Qualifier:

according to guideline

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. QA statement)

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 or test system 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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

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. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Pentyl salicylate

EC Number:

218-080-2

EC Name:

Pentyl salicylate

Cas Number:

2050-08-0

Molecular formula:

C12H16O3

IUPAC Name:

Pentyl 2-hydroxybenzoate

Constituent 2

Reference substance name:

2-methylbutyl salicylate

EC Number:

256-972-3

EC Name:

2-methylbutyl salicylate

Cas Number:

51115-63-0

Molecular formula:

C12H16O3

IUPAC Name:

2-methylbutyl 2-hydroxybenzoate

Test material form:

liquid

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%

Test animals

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 or test system 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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 ppm

Remarks:

extrapolated to an equivalent of 33 mg/kg bw/d

Doses / concentrations 2

Dose / conc.:

1 500 ppm

Remarks:

extrapolated to an equivalent of 100 mg/kg bw/d

Doses / concentrations 3

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Details on results (P1)

Only a one generation study, no P1 produced.

Effect levels (P1)

Remarks on result:

not measured/tested

Target system / organ toxicity (P1)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

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

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Remarks on result:

not measured/tested

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

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)