Reaction mass of 2-methylbutyl salicylate and pentyl salicylate

Developmental toxicity / teratogenicity

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

as of June 2018

Deviations:

no

GLP compliance:

yes

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: VE00598644
- Expiration date of the lot/batch: 06 March 2020
- Purity test date: 99.89%

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han rats (females)

Details on test animals or test system 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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 ppm

Remarks:

equivalent to 39 mg/kg bw/d [34 - 43 mg/kg bw/d]

Doses / concentrations 2

Dose / conc.:

1 500 ppm

Remarks:

equivalent to 121 mg/kg bw/d [106 - 131 mg/kg bw/d]

Doses / concentrations 3

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.

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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.

Maternal developmental toxicity

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

Effect levels (maternal animals)

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

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

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.

Effect levels (fetuses)

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

Fetal abnormalities

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

Overall developmental toxicity

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.