3-methoxybutan-1-ol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17/02/2020 - 07/05/2020 Final report 15/09/2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In the Final Decision number: CCH-D-2114453510-59-01/F (2018) issued for 3 methoxybutyl acetate, ECHA identified the need to fulfill information requirements for a Pre-natal developmental toxicity study in a second species (rabbit), oral route.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-methoxybutyl acetate

EC Number:

224-644-9

EC Name:

3-methoxybutyl acetate

Cas Number:

4435-53-4

Molecular formula:

C7H14O3

IUPAC Name:

3-methoxybutyl acetate

Constituent 2

Reference substance name:

butoxyl

IUPAC Name:

butoxyl

Specific details on test material used for the study:

3-methoxybutyl acetate CAS No: 4435-53-4 EC No: 224-644-9
Batch (Lot) Number (Batch A): 120001198625 Expiry date (Batch A): 18 Mar 2020
Batch (Lot) Number (Batch B): 120001247491 Expiry date (Batch B): 14 Aug 2020
Batch (Lot) Number (Batch C): 120001251521 Expiry date (Batch C): 04 Sep 2020
Physical Description: Clear colourless liquid
Purity/Composition: 99.5 - 99.9%
Storage Conditions: At room temperature
Test Facility test item number: 210574/A (Batch A used from 24 Feb 2020 until 29 Feb 2020 and from 07 Mar 2020 until 09 Mar 2020)
210574/B (Batch B used from 01 Mar 2020 until 06 Mar 2020)
210574/C (Batch C used from 10 Mar 2020 onwards)

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

On arrival and following randomization females were housed individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles. The rooms in which the animals were kept was documented in the study records. Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group and animal number. Target temperatures of 17 to 21°C with a relative target humidity of 40 to 70% were maintained.
Restricted access to pelleted diet for rabbits (KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Kliba NAFAG Granovit AG, Kaiseraugst, Swizerland) was provided throughout the study, except during designated procedures.
Municipal tap water was freely available to each animal via water bottles/containers.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

A dose control system was used as additional check to verify the dosing procedure according to Standard Operating Procedures. The oral route of exposure was selected because it is the guideline-preferred route for OECD 414 studies and the oral route was specifically requested by ECHA and this is a possible route of human exposure during manufacture, handling or use of the test item.
The dose levels were selected based on the results of the dose range finder (Test Facility Reference No. 20235788), and in an attempt to produce graded responses to the test item.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20215472) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Details on mating procedure:

The females arrived Day 1-2 post-coitum (Day 0 post-coitum is defined as the day of successful mating). They were 17-19 weeks old and weighed between 2984 and 3951 g at the initiation of dosing.

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 7 to Day 28 post-coitum, inclusive. The dose volume (1.25 mL/kg) for each animal was based on the most recent body weight measurement.

Frequency of treatment:

daily

Duration of test:

The Study Plan or protocol was signed on 14 Feb 2020, and dosing was initiated on
24 Feb 2020. The in-life phase was completed on 20 Mar 2020. The experimental start date was 19 Feb 2020, and the experimental completion date was 07 May 2020.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control group

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low-dose

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Mid-dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High-dose

No. of animals per sex per dose:

22 females

Control animals:

yes

Details on study design:

Animals were individually weighed on Days 7, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum. Food consumption was quantitatively measured daily from Day 3 post-coitum onwards. Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers. Terminal procedures were scheduled on day 29.
A necropsy was conducted for animals that died on study, and specified tissues were saved.
If necessary for humane reasons (i.e. daily food consumption of less than 15 gram (10% of the daily allotment) for more than 7 consecutive days, resulting in a body weight loss of 5-13% compared to Day 7 post-coitum and/or clinical signs such as lean and pale appearance and hunched posture), animals were euthanized as per Test Facility SOPs. These animals were euthanized by intravenous injection of pentobarbital (approx. 1 mL/kg Euthasol® 20%), underwent necropsy, and specified tissues were retained.

Examinations

Maternal examinations:

The in-life procedures, observations, and measurements listed below were performed for parental animals. Animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Clinical observations were performed at least once daily, beginning on Day 7 post-coitum and lasting up to the day prior to necropsy.

Ovaries and uterine content:

Each ovary and uterine horn of all animals was dissected and examined as quickly as possible

Fetal examinations:

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

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. Mean and SD (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix: Group 2 vs. Group 1; Group 3 vs. Group 1; and Group 4 vs. Group 1 when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
Parametric - Datasets with at least 3 groups were compared using Dunnett-test (many-to-one-t-test). Non-Parametric - 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.
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 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.
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.

Indices:

•The number of corpora lutea (Table 6)
•The weight of the (gravid) uterus (not for animals found dead or sacrificed before planned necropsy) (for exceptions, see Tables 2).
•The number of implantation sites (Table 6).
•The number and distribution of live and dead fetuses (Table 6).
•The number and distribution of early and late resorptions (Table 6).

Historical control data:

Yes, included as attachment.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

one rat showed some type of seizure on Day 27 post-coitum. it was incidental in the mid-dose and spontaneous spasms have been reported in NZ rabbits. Another rat showed some discharge from the nose in combination with focal erythema on the snout and vocalizations on a single occasion. this was most likely the results of the gavage procedure. Other findinds include alopecia, wounds/scbas, diarrhoea, piloerection and broken teeth expected for rabbits of this age which are housed and treated under the conditions of the study.

Mortality:

mortality observed, treatment-related

Description (incidence):

Four females were euthanized for animal welfare reasons between Days 17-20 post-coitum. Food consumption appeared to recover to control levels from Day 13 post-coitum onwards. As a result, body weight loss was observed in almost all females at 1000 mg/kg/day (up to 6% for individual animals) between Days 7-9 post-coitum. This body weight loss at 1000 mg/kg/day was followed by reduced or absent body weight gain between Days 9-18 post-coitum in individual females (as a result, four of these females were sacrificed for animal welfare reasons between Day 17-22 post-coitum). Macroscopic observations at necropsy revealed many dark red foci in the forestomach or irregular surface of the forestomach for 4/22 females at 1000 mg/kg/day. This finding was most likely related to the physical-chemical properties of the test item and at the incidence observed, it was considered related to treatment with the test item. It was noted that this finding occurred in 2 females that was prematurely sacrificed due to absent food consumption and body weight loss. Moreover, decreased or absent food consumption was also noted for prolonged periods in the remaining two females with this finding. Although histopathological evaluation of the gastrointestinal tract was not performed to assess adversity of these findings, they were gastrointestinal irritation was likely related to the adverse effects on body weight and food consumption leading to morbidity. In the 1000 mg/kg/day dose group, which started with 22 dams, the breakdown of the excluded dams is as follows: 4 females were sacrificed in extremis, 2 were not pregnant at scheduled necropsy, 2 had an early delivery, and 1 gavage related death occurred As according to the OECD 414 test guideline, a minimum of 16 litters is required for evaluation of developmental data, the 13 litters available at 1000 mg/kg/day were not sufficient for a complete and valid evaluation of developmental data. As such, developmental data of Group 4 were excluded from reporting, to avoid bias and misinterpretation as the data is incomplete. (Table 1 and 3).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, slight body weight loss was observed in almost all females between Days 7-9 post-coitum (mean body weight gain -2% at 1000 mg/kg/day vs. 2% in the concurrent controls and up to -6% for individual females). This body weight loss at 1000 mg/kg/day was followed by reduced or absent body weight gain between Days 9-18 post-coitum in individual females (it should be noted that these females were sacrificed for animal welfare reasons between Day 17-22 post-coitum. In females surviving until scheduled necropsy, body weight gain appeared to recover from Day 9 post-coitum onwards. At the end of the treatment period, body weights and corrected body weight gain were considered to be similar between the high dose group and concurrent control.
No effects on body weights and (corrected) body weight gain were noted in females treated at 100 and 300 mg/kg/day.
Data in Table 2.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Based on GIT irritation with the high dose treatment via gavage, food consumption and body weights were reduced. Females at 1000 mg/kg/day, mean food consumption before or after allowance for body weight was significantly lower compared to control females between Days 7-12 post-coitum (up to 62% lower than concurrent controls for absolute food consumption between Day 8-9 post-coitum), with 7/22 females consuming approximately no food in this interval (vs. 0/22 in the concurrent controls. However, food consumption at 1000 mg/kg/day appeared to recover to control levels from Day 13 post-coitum onwards. Also, a statistically significant increase in food consumption at 100 mg/kg/day between Day 26-27 post-coitum were considered to be unrelated to treatment with the test item as no trend was apparent regarding dose and duration of treatment.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at scheduled necropsy revealed many dark red foci in the forestomach for two females (Nos. 67 and 87) at 1000 mg/kg/day. This finding and a similar finding (irregular surface of the forestomach) were also recorded for two females at 1000 mg/kg/day that were sacrificed early for animal welfare reasons (Nos. 71 and 77). At the incidence observed, it was considered related to treatment with the test item. Other findings that were noted among control and/or treated animals were considered to be of no toxicological significance, since they remained within the range of biological variation for rabbits of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not specified

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

Treated dose groups were none significantly different from control group (see Table 3 and Historical Control data, available under "Any other information on results including tables")

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

None significantly different from control group. Data in Table 3.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

None significantly different from control group.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

The mean percentage of early resorptions was somewhat increased at 100 mg/kg/day (8.0 vs. 4.6 in concurrent controls) and as a result, the mean percentage of total resorptions and postimplantation loss was also increased (total resorptions: 9.3 vs. 6.3%, and postimplantation loss: 9.3 vs. 6.7% at 100 mg/kg/day vs. in concurrent controls, respectively). However, as no apparent dose response relationship was observed, this finding was considered not toxicologically relevant.

The mean percentage pre-implantation loss was increased at 300 mg/kg/day (13.3 vs. 7.9 in concurrent controls) and values were outside the range of the available historical control range3. This higher mean value could mainly be attributed to a single female, No. 66, which had only two implantation sites and 10 corpora lutea, resulting in a pre-implantation loss of 80%. After excluding this female from calculations, the mean pre-implantation loss per litter at 300 mg/kg/day was 9.3%, which is within the available historical control range. Additionally, the number of implantation sites were unaffected by treatment with the test item, and treatment was initiated on Day 7 post-coitum, i.e. after implantation has occurred. As such, this finding was considered not toxicologically relevant. Data in Table 5.

Dead fetuses:

no effects observed

Description (incidence and severity):

Data in Table 4.

Changes in pregnancy duration:

effects observed, non-treatment-related

Description (incidence and severity):

Data in Table 3.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

At scheduled necropsy, all females were pregnant, except for female Nos. 16 (control group), 46 (300 mg/kg/day), 68 and 80 (1000 mg/kg/day). Six females sacrificed for animal welfare reasons preterm were all pregnant at the time of necropsy (6, 23, 71, 77, 85, and 88), except for female No. 77 (1000 mg/kg/day). As such, the number of pregnant females was 21 in the control group, 22 at 100 mg/kg/day, 21 at 300 mg/kg/day and 19 at 1000 mg/kg/day. Three females delivered their litters early and were sacrificed within 24 hours thereafter. Female No. 54 (300 mg/kg/day) had an early delivery on Day 28, female No. 72 (1000 mg/kg/day) on Day 29, and female No. 84 (1000 mg/kg/day) on Day 27 post-coitum. For Female No. 54, a decrease in food consumption was observed from Day 20-21 onwards. This decrease was considered a sign of maternal toxicity which resulted in the early delivery. Excluding non-pregnant females, females with an early delivery, and females that did not survive until scheduled necropsy, there were 20 females in the control, 20 females at 100 mg/kg/day, 18 females at 300 mg/kg/day, and 13 females at 1000 mg/kg/day with viable litters.
Note: As according to the guidelines, a minimum of 16 litters is required for evaluation of developmental data, the 13 litters available at 1000 mg/kg/day were not sufficient for a complete and valid evaluation of developmental data. As such, developmental data of Group 4 were excluded from reporting, to avoid bias and misinterpretation as the data at 1000 mg/kg/day is incomplete. Data in Table 3.

Other effects:

no effects observed

Details on maternal toxic effects:

Note: As according to the guidelines, a minimum of 16 litters is required for evaluation of developmental data, the 13 litters available at 1000 mg/kg/day were not sufficient for a complete and valid evaluation of developmental data. As such, developmental data of Group 4 were excluded from reporting, to avoid bias and misinterpretation as the data at 1000 mg/kg/day is incomplete.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

other: uncheduled deaths in dams treated at 1000 mg/kg/day

Remarks on result:

other:

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Data in Table 4 and 5.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Data in Table 5.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Data in Table 4.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Data in Table 4 and 5.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were observed in two fetuses each of all groups. In Group 3, Fetus No. A048-13 had a distended abdomen that also occurred in control fetus A009-08 and at visceral examination, both these fetuses had multiple causal cardiovascular anomalies. The other 300 mg/kg/day fetus (A062-01) had a limb with malrotated forepaw and an absent digit with matching skeletal findings. The malformed Group 2 fetuses had a short tail (A034-12) and spina bifida (A042-02) and the second malformed control fetus (A012-09) had malrotated hind limbs. Skeletal examination substantiated the tail and vertebral column findings, but the hind limbs finding had no correlating skeletal abnormality.
The single occurrence and group distribution of all above malformations does not suggest a test item-relationship, and therefore these were considered to be chance findings.
No external variations were observed in this study. Data in Table 6 and 9.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on skeletal morphology following treatment up to 300 mg/kg/day.
Besides the anomalies associated with external malformations in fetuses described in a previous paragraph, three skeletal malformations occurred. A vertebral anomaly with or without associated rib anomaly was noted in Fetus Nos. A018-02 (control group), A025-04, A034-12 (100 mg/kg/day) and A058-05 (300 mg/kg/day). Of these fetuses, A034-12 (with a short tail externally) also had costal cartilage anomaly. The remaining malformation was a skull anomaly that was observed in the fetus with spina bifida (A042-02) at 100 mg/kg/day. The single occurrence and group distribution of these malformations does not suggest a test item-relationship, and therefore they were considered chance findings.
The following skeletal variations were considered test item-related: The number of fetuses with an ossification site near the 7th cervical vertebra was increased at 300 mg/kg/day. Eight fetuses from four litters were affected, while none occurred in the control group and one at 100 mg/kg/day. The increase was statistically significant and although the mean litter incidence at 300 mg/kg/day (4.8% per litter) remained below the historical control maximum value (5.5% per litter), it was above the 95th percentile-value (4.4% per litter). It should be noted that the overall percent per litter with skeletal variations was almost identical to controls. At 100 mg/kg/day, the occurrence of a single fetus with 7th cervical ossification sites was considered a chance finding.
In addition, three cases of 7th cervical full ribs occurred in three 300 mg/kg/day litters that had fetuses with 7th cervical ossification sites. The incidence of this finding was just above the historical control maximum value (1.9% vs. 1.8% per litter). Although the increase was not statistically significant when compared to control fetuses, it was considered treatment related in combination with the 7th cervical ossification sites that occurred.
Noteworthy, the incidences of caudal shift of pelvic girdle (13.7% per litter) and 13th full ribs (34.7% per litter) at 300 mg/kg/day were slightly lower compared to the control values
(30.5% and 55.3% per litter, respectively). However, as values remained within the available historical control range, these findings were considered not test item-related.
Other skeletal variations that occurred were considered not test item-related as they occurred infrequently, in control fetuses only and/or at frequencies that were within the range of available historical control data. Data in Table 8 and 9.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on visceral morphology following treatment up to 300 mg/kg/day.
Besides the cardiovascular malformations of the control and 300 mg/kg/day fetus with a distended abdomen (A009-08 and A048-13, with large heart and absent right atrioventricular valve; and large heart, ventricular septum defect and dilated aortic arch, respectively), there were five fetuses at 300 mg/kg/day that were viscerally affected. Two of these also had cardiovascular malformations, namely A048-07 (narrow pulmonary trunk and dilated aortic arch) and A066-01 (interrupted aortic arch and ventricular septum defect). Thus, three fetuses from two litters at 300 mg/kg/day (as A048-07 and -13 are littermates) had cardiovascular malformations. The presence of cardiovascular malformations in two littermates suggests a familiar cause and as each individual anomaly previously occurred in control fetuses, these were considered unrelated to treatment with the test item.
Other viscerally malformed fetuses at 300 mg/kg/day had abnormal lobation of the lung (A053-06), a large cyst (A059-04) or abnormal liver lobation (A062-01, the fetus with two limb malformations). Due to the single occurrence and/or occurrence in historical control fetuses, these were considered spontaneous in origin.
All visceral variations noted were considered unrelated to treatment as they occurred in the absence of a dose-related trend, infrequently, in control fetuses only and/or at frequencies that were within the range of available historical control data. Data in Table 7 and 9.

Other effects:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: on the absence of any test-related effects on fetal observations.

Overall developmental toxicity

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

Treatment related:

no

Any other information on results incl. tables

Table 1. Breakdown of excluded dams

Female No.	Group (mg/kg/day)	Reason for exclusion
6	Control		Euthanized in extremis Day 22 post-coitum
16	Control	Not pregnant at scheduled necropsy
23	100		Euthanized in extremis Day 22 post-coitum
28	100		Inadvertently sacrificed on Day 28 instead of Day 29
46	300	Not pregnant at scheduled necropsy
50	300		Inadvertently sacrificed on Day 28 instead of Day 29
51	300		On day 18, likely as the result of a misgavage.
54	300		Early delivery on day 28 and sacrificed within 24 hours
68	1000	Not pregnant at scheduled necropsy
71	1000		Euthanized in extremis Day 18 post-coitum
72	1000		Early delivery on Day 29 post-coitum and sacrificed within 24 hours
77	1000	No pregnant	Euthanized in extremis Day 17 post-coitum
80	1000	Not pregnant at scheduled necropsy
84	1000		Early delivery on Day 27 post-coitum and sacrificed within 24 hours
85	1000		Euthanized in extremis Day 20 post-coitum
86	1000		Found dead on Day 20 Post-coitum, shortly after dosing
88	1000		Euthanized in extremis Day 18 post-coitum
In summary, excluding non-pregnant females, females with an early delivery, and females that did not survive until scheduled necropsy, there were 20 females in the control, 20 females at 100 mg/kg/day, 18 females at 300 mg/kg/day, and 13 females at 1000 mg/kg/day with viable litters.  Note: As according to the guidelines, a minimum of 16 litters is required for evaluation of developmental data, the 13 litters available at 1000 mg/kg/day were not sufficient for a complete and valid evaluation of developmental data. As such, developmental data of Group 4 were excluded from reporting, to avoid bias and misinterpretation as the data at 1000 mg/kg/day is incomplete.

 

Table 2. Body Weight, Body Weight Gain, Gravid Uterine Weight

		Body Weights (gr)				Body Weight Gain (%)
Post Coitum		Group 1 (Control)	Group 2	Group 3	Group 4	Group 1 (Control)	Group 2	Group 3	Group 4
Day 0	Mean S.D. N	3499 259.3 21	3568 247.7 22	3614 256.3 21	3526 309.1 19	-	-	-	-
Day 7	Mean S.D. N	3481 215.3 21	3518 216.4 22	3536 224.0 21	3520 246.4 19	0 0.0 21	0 0.0 22	0 0.0 21	0 0.0 19
Day 9	Mean S.D. N	3543 212.3 21	3584 215.2 22	3595 223.9 21	3433 213.4 19	2 0.8 21	2 1.2 22	2 1.1 21	-2** 2.1 19
Day 12	Mean S.D. N	3586 212.7 21	3630 211.6 22	3641 230.2 21	3447 218.0 19	3 1.2 21	3 2.0 22	3 2.3 21	-2** 3.2 19
Day 15	Mean S.D. N	3639 224.9 21	3661 247.9 22	3712 250.8 21	3520 228.2 19	5 2.6 21	4 4.2 22	5 2.9 21	0** 3.7 19
Day 18	Mean S.D. N	3659 246.9 21	3701 239.7 22	3737 239.4 21	3530 260.7 19	5 3.0 21	5 4.0 22	6 3.0 21	0** 5.5 19
Day 21	Mean S.D. N	3680 252.0 21	3729 257.7 22	3790 219.8 20	3624 265.3 15	6 3.3 21	6 4.2 22	7 2.6 20	3 3.3 15
Day 24	Mean S.D. N	3726 252.6 20	3794 239.7 21	3833 239.3 20	3698 275.2 15	7 2.8 20	8 3.9 21	8 3.1 20	5 3.6 15
Day 27	Mean S.D. N	3770 254.0 20	3868 246.1 21	3907 230.8 20	3751 259.0 14	9 3.6 20	10 4.1 21	10 3.8 20	6 4.1 14
Day 29	Mean S.D. N	3806 231.4 20	3914 239.6 20	3946 234.1 18	3780 260.9 13	10 4.1 20	11 4.1 20	12 3.8 18	7 5.1 13
Weight of uterus	Mean S.D. N	-	-	-	-	531.4 97.8 20	499.1 113.4 20	531.0 123.8 18	447.1 75.3 13
Corrected body weight Gain for gravid uterine weight <1> in gram (g)	Mean S.D. N					-197.3 129.7 20	-126.0 138.4 20	-125.8 113.9 18	-192.8 131.9 13
Corrected Body Weight Gain for gravid uterine weight <2> in percent (%)	Mean S.D. N	-	-	-	-	-5.6 3.6 20	-3.5 3.8 20	-3.5 3.1 18	-5.5 3.7 13

(-) = Not applicable

<1> : (Weight on Day of Section) - (Weight on Day 7 P.C.) - (Weight Uterus)

<2> : Corrected Weight Gain in percent of Weight on Day 7 P.C.

 

Table 3. Summary of Maternal Survival and Pregnancy Status

Dose group	Group 1		Group 2		Group 3		Group 4
	No.	%	No.	%	No.	%	No.	%
Females on study	22	-	22	-	22	-	22	-
Aborted/delivered	0	0.0	0	0.0	1	4.5	2	9.1
Deaths	0	0.0	0	0.0	1	4.5	1	4.5
Aborted	0	0.0	0	0.0	0	0.0	0	0.0
Nongravid	0	0.0	0	0.0	0	0.0	0	0.0
Gravid	0	0.0	0	0.0	1	100.0	1	100.0
Euthanized	1	4.5	2	9.1	1	4.5	4	18.2
Nongravid	0	0.0	0	0.0	0	0.0	1	25.0
Gravid	1	100.0	2	100.0	1	100.0	3	75.0
At scheduled necropsy	21	95.5	20	90.9	19	86.4	15	68.2
Nongravid	1	4.8	0	0.0	1	5.3	2	13.3
Gravid	20	95.2	20	100.0	18	94.7	13	86.7
With resorption only	0	0.0	0	0.0	0	0.0	0	0.0
With viable fetuses	20	100.0	20	100.0	18	100.0	13	100.0
Total females gravid	21	95.5	22	100.0	21	95.5	19	86.4

Group 1= 0 mg/kg; 2= 100 mg/kg; 3= 300 mg/kg; 4= 1000 mg/kg

Historical Control: No. of Animals that Aborted or Delivered 6

(-) = Not applicable

 

Table 4. Summary of fetal data at scheduled necropsy

Group		Sex		Viable fetuses	Dead fetuses	Resorptions		Post Implantation loss	Implantation sites	Corpora lutea	Pre Implantation Loss	Fetal weight (gr)	No. of gravid females
		M	F			Early	Late
1	Total Mean S.D.	103 5.2 1.95	98 4.9 1.48	201 10.1 1.70	1 0.1 0.22	10 0.5 1.00	4 0.2 0.41	15 0.8 0.97	216 10.8 1.70	235 11.8 1.68	19 0.9 1.00	- 36.7 4.15	20
2	Total Mean S.D.	97 4.9 1.73	87 4.4 2.35	184 9.2 2.93	0 0.0 0.00	16 0.8 1.54	3 0.2 0.49	19 1.0 1.57	203 10.2 2.62	223 11.2 2.68	20 1.0 1.26	- 38.2 4.99	20
3	Total Mean S.D.	77 4.3 2.30	96 5.3 2.06	173 9.6 2.48	0 0.0 0.00	9 0.5 0.51	3 0.2 0.51	12 0.7 0.69	185 10.3 2.56	212 11.8 1.59	27 1.5 1.82	- 38.7 5.81	18
4	Excluded from reporting, as insufficient litters were available as scheduled necropsy

Group 1= 0 mg/kg; 2= 100 mg/kg; 3= 300 mg/kg; 4= 1000 mg/kg

None significantly different from control group

(-) = not applicable

Mean number of viable fetuses, mean number of implantation sites, mean number of corpora lutea, fetal weights compared using Dunnett’s Test.

 

 

Table 5. Summary of fetal data at scheduled necropsy [% per litter]

Group	0 mg/kg	100 mg/kg	300 mg/kg	1000 mg/kg
Corpora lutea Mean S.D. N	11.8 1.68 20	11.2 2.68 20	11.8 1.59 18	NA
Implantation sites Mean S.D. N	10.8 1.70 20	10.2 2.62 20	10.3 2.56 18	NA
Viable fetuses Mean S.D. N	93.3 9.24 20	90.7 15.00 20	93.7 6.34 18	NA
Dead fetuses Mean S.D. N	0.4 1.59 20	0.0 0.00 20	0.0 0.00 18	NA
Early resorptions Mean S.D. N	4.6 9.56 20	8.0 14.63 20	4.9 5.22 18	NA
Late resorptions Mean S.D. N	1.8 3.62 20	1.3 4.13 20	1.4 4.56 18	NA
Total resorptions Mean S.D. N	6.3 9.36 20	9.3 15.00 20	6.3 6.34 18	NA
Pre-implantation loss Mean S.D. N	7.9 8.30 20	8.6 10.71 20	13.3 17.89 18	NA
Post-implantation loss Mean S.D. N	6.7 9.24 20	9.3 15.00 20	6.3 6.34 18	NA
Males Mean S.D. N	50.5 14.86 20	54.2 16.37 20	43.9 18.98 18	NA
Females Mean S.D. N	49.5 14.86 20	45.8 16.37 20	56.1 18.98 18	NA
Male fetal weights (g) Mean S.D. N	37.3 4.29 20	38.5 5.75 20	39.3 6.74 18	NA
Female fetal weights (g) Mean S.D. N	36.1 4.85 20	38.2 5.52 20	38.5 5.75 18	NA
Combined fetal weights (g) Mean S.D. N	36.7 4.15 20	38.2 4.99 20	38.7 5.81 18	NA

Group 1= 0 mg/kg; 2= 100 mg/kg; 3= 300 mg/kg; 4= 1000 mg/kg

Proportional (%) data compared using the Mann-Whitney Test

Corpora lutea, implantation sites, fetal weights compared using Dunnett’s Test

None significantly different from control group

Group 4 (NA) = Not available, data were excluded from reporting, as insufficient litters were available at scheduled necropsy.

 

Table 6. Summary of fetuses and litters with examined external malformations in absolute number and percent per litters.

	FETUSES				LITTERS
Dose group	1	2	3	4	1	2	3	4
Number of litters examined externally	-	-	-	-	20	20	18	NA
Number of fetuses examined externally	201	184	173	NA	-	-	-	-
Abdomen – distended Mean (%) S.D.	1	0	1	NA	1 0.5 2.03	0 0.0 0.00	1 0.5 2.14	NA
Limb(s) – malrotated Mean (%) S.D.	1	0	0	NA	1 0.6 2.48	0 0.0 0.00	0 0.0 0.00	NA
Short Tail Mean (%) S.D.	0	1	0	NA	0 0.0 0.00	1 0.3 1.32	0 0.0 0.00	NA
Limb(s) – multiple malformations Mean (%) S.D.	0	0	1	NA	0   0.0 0.00	0   0.0 0.00	1   0.5 2.14	NA
Trunk – spine bifida Mean (%) S.D.	0	1	0	NA	0 0.0 0.00	1 0.5 2.03	0 0.0 0.00	NA

Group 1= 0 mg/kg; 2= 100 mg/kg; 3= 300 mg/kg; 4= 1000 mg/kg

Group 4 (NA) = Not available, data were excluded from reporting, as insufficient litters were available at scheduled necropsy.

(-) = Not applicable

NA = Not available

None significantly different from control group.

 

Table 7. Summary of fetuses and litters with examined visceral malformations in absolute number and percent per litters.

	FETUSES				LITTERS
Dose group	1	2	3	4	1	2	3	4
Number of litters examined externally					20	20	18	NA
Number of fetuses examined externally	201	184	173	NA	-	-	-	-
Pulmonary trunk - narrow Mean (%) S.D.	0	0	1	NA	0 0.0 0.00	0 0.0 0.00	1 0.5 2.14	NA
Aortic arch - dilated Mean (%) S.D.	0	0	2	NA	0 0.0 0.00	0 0.0 0.00	1 1.0 4.29	NA
Heart large Mean (%) S.D.	1	0	1	NA	1 0.5 2.03	0 0.0 0.00	1 0.5 2.14	NA
Ventricular septum defect Mean (%) S.D.	0	0	2	NA	0 0.0 0.00	0 0.0 0.00	2 3.3 11.85	NA
Lung – abnormal lobation Mean (%) S.D.	0	0	1	NA	0 0.0 0.00	0 0.0 0.00	1 0.6 2.36	NA
Atrioventricular valve - absent Mean (%) S.D.	1	0	0	NA	1 0.5 2.03	0 0.0 0.00	0 0.0 0.00	NA
Viscera – cyst(s) Mean (%) S.D.	0	0	1	NA	0 0.0 0.00	0 0.0 0.00	1 0.5 1.96	NA
Liver abnormal lobation Mean (%) S.D.	0	0	1	NA	0 0.0 0.00	0 0.0 0.00	1 0.5 2.14	NA
Aortic arch - interrupted Mean (%) S.D.	0	0	1	NA	0 0.0 0.00	0 0.0 0.00	1 2.8 11.79	NA

Group 1= 0 mg/kg; 2= 100 mg/kg; 3= 300 mg/kg; 4= 1000 mg/kg

Group 4 (NA) = Not available, data were excluded from reporting, as insufficient litters were available at scheduled necropsy.

(-) = Not applicable

NA = Not available

None significantly different from control group.

 

Table 8. Summary of fetuses and litters with examined skeletal malformations in absolute number and percent per litters.

	FETUSES				LITTERS
Dose group	1	2	3	4	1	2	3	4
Number of litters examined skeletally	-	-	-	-	20	20	18	NA
Number of fetuses examined skeletally	201	184	173	NA	-	-	-	-
Vertebral anomaly with/without associated rib anomaly Mean (%) S.D.	1	2	1	NA	1   0.6 2.48	2   1.0 3.39	1   0.6 2.36	NA
Costal cartilage anomaly Mean (%) S.D.	0	1	0	NA	0 0.0 0.00	1 0.3 1.32	0 0.0 0.00	NA
Skull anomaly Mean (%) S.D.	0	1	0	NA	0 0.0 0.00	1 0.5 2.03	0 0.0 0.00	NA

Group 1= 0 mg/kg; 2= 100 mg/kg; 3= 300 mg/kg; 4= 1000 mg/kg

Group 4 (NA) = Not available, data were excluded from reporting, as insufficient litters were available at scheduled necropsy.

(-) = Not applicable

NA = Not available

None significantly different from control group.

 

Table 9. Summary of fetuses and litters with malformations in absolute number and proportion of malformations percentage per litters.

	FETUSES				LITTERS
Dose group	1	2	3	4	1	2	3	4
Number of litters examined skeletally	-	-	-	-	20	20	18	NA
Number of fetuses examined skeletally	201	184	173	NA	-	-	-	-
Total number with external malformations Mean (%) S.D.	2	2	2	NA	2   1.0 3.13	2   0.7 2.36	2   1.0 2.94	NA
Total number with soft tissue malformations Mean (%) S.D.	1	0	6	NA	1   0.5 2.03	0   0.0 0.00	5   5.3 12.31	NA
Total number with skeletal malformations Mean (%) S.D.	1	3	1	NA	1   0.6 2.48	3   1.5 3.83	1   0.6 2.36	NA
Combined total malformations Mean S.D.	3	3	7	NA	3   1.6 3.84	3   1.5 3.83	6   5.9 12.28	NA

Group 1 = 0 mg/kg; 2= 100 mg/kg; 3= 300 mg/kg; 4= 1000 mg/kg

Group 4 (NA) = Not available, data were excluded from reporting, as insufficient litters were available at scheduled necropsy.

(-) = Not applicable

NA = Not available

None significantly different from control group.

 

HISTORICAL DATA IN RABBITS PROVIDED BY CRL - DEN BOSCH

 

Historical Control Data Albino, New Zealand White Rabbit Study Date Range: 2015 – 2019		Mean of Study Means
Endpoint	Total	Mean	SD	Median	Min	Max	P5	P95
No of Studies	34
Total No. of Animals in the Control Group	749
No. of Animals that Died	1
No. of Animals that were Euthanized	8
No. of Animals that Aborted or Delivered	6
Percent Pregnant		91.2	6.78	93.2	77.3	100.0	77.3	100.0
No. of Animals Examined at Laparohysterectomy	734
No. Nongravid	66
No. Gravid	668
No. with Only Resorptions	0
No. of Dams with Live Fetuses	668
Mean No. Viable Fetuses/Dam		9.2	0.66	9.2	7.9	10.4	8.1	10.3
Total No. Viable Fetuses	6122
Viable Fetuses (%/Litter)		94.1	2.51	94.8	86.6	97.5	87.9	97.0
Mean No. Postimplantation Loss/Dam		0.6	0.25	0.6	0.2	1.3	0.3	1.2
Total No. Postimplantation Losses	392
Postimplantation Loss (%/Litter)		5.9	2.48	5.2	2.5	13.4	3.0	12.1
Dead Fetuses (%/Litter)		0.2	0.44	0.0	0.0	1.5	0.0	1.5
Early Resorptions (%/Litter)		3.8	1.87	3.5	0.6	8.0	1.2	7.8
Late Resorptions (%/Litter)		1.9	1.16	1.7	0.0	4.3	0.2	4.2
Mean No. Implantations/Dam		9.8	0.67	9.8	8.2	10.8	8.5	10.7
Mean No. Corpora Lutea/Dam		10.3	0.61	10.3	8.9	11.3	9.3	11.2
Mean No. Preimplantation Loss/Dam		0.5	0.30	0.6	0.0	1.3	0.1	1.2
Total No. Preimplantation Losses	365
Preimplantation Loss (%/Litter)		5.3	3.06	5.5	0.0	14.3	0.9	11.8
Total No. Male Fetuses	3015
Total No. Female Fetuses	3107
% Males/Litter		49.5	4.16	48.9	41.2	59.9	43.2	59.0
% Female/Litter		50.5	4.16	51.1	40.1	58.8	41.0	56.9
Mean Fetal Body Weight (g)		39.1	1.59	38.9	36.3	42.7	36.7	42.3
Mean Male Body Weight (g)		39.4	1.60	38.8	36.2	43.1	36.7	42.7
Mean Female Body Weight (g)		38.6	1.59	38.7	36.1	42.5	36.2	42.1
Mean Male Placenta Weight (g) ^1		5.0	0.53	5.0	4.7	5.4	@	@
Mean Female Placenta Weight (g) ^1		4.8	0.59	4.8	4.4	5.3	@	@

 

 ¹ Based on 2 datasets            @ Insufficient number of data for calculation.

 

 

Historical Control Data Albino, New Zealand White Rabbit
No. of Studies	34
Total No. Fetuses/Litters Examined Externally	6122	668
Total No. Fetuses/Litters Examined Viscerally	6122	668
Total No. Fetuses/Litters Examined Skeletally	6122	668
	Mean of Study Means							Summary Incidence
	(% Per Litter Basis)							(Total No. Affected)
MALFORMATIONS	Mean	SD	Median	Min	Max	P5	P95	Fetuses	Litters
TOTAL EXTERNAL MALFORMATIONS								32	29
TOTAL VISCERAL MALFORMATIONS								85	73
TOTAL SKELETAL MALFORMATIONS								61	56
TOTAL MALFORMATIONS								162	130
EXTERNAL
Abdomen- Distended	0.0	0.14	0.0	0.0	0.6	0.0	0.5	3	3
Anencephaly	0.0	0.07	0.0	0.0	0.4	0.0	0.1	1	1
Anus- Atresia	0.0	0.07	0.0	0.0	0.4	0.0	0.1	1	1
Brachydactyly	0.0	0.15	0.0	0.0	0.7	0.0	0.6	2	2
Carpal and/or Tarsal Flexure	0.2	0.34	0.0	0.0	1.1	0.0	1.1	12	9
Cleft Lip	0.0	0.07	0.0	0.0	0.4	0.0	0.1	1	1
Cleft Palate	0.0	0.20	0.0	0.0	1.1	0.0	0.7	4	2
Eye(s)- Open	0.0	0.07	0.0	0.0	0.4	0.0	0.1	1	1
Grossly Malformed	0.0	0.09	0.0	0.0	0.5	0.0	0.1	1	1
Head- Domed	0.0	0.07	0.0	0.0	0.4	0.0	0.1	1	1
Limb(s)- Large	0.0	0.09	0.0	0.0	0.5	0.0	0.1	1	1
Limb(s)- Malrotated	0.0	0.07	0.0	0.0	0.4	0.0	0.1	1	1
Limb(s)- Short	0.0	0.12	0.0	0.0	0.7	0.0	0.2	1	1
Meningocele	0.0	0.10	0.0	0.0	0.6	0.0	0.2	1	1
Omphalocele	0.2	0.59	0.0	0.0	3.0	0.0	2.0	8	8
Tail- Absent. Short or Filamentous	0.0	0.10	0.0	0.0	0.4	0.0	0.4	2	2

 

 

Historical Control Data Albino, New Zealand White Rabbit
	Mean of Study Means							Summary Incidence
	(% Per Litter Basis)							(Total No. Affected)
MALFORMATIONS	Mean	SD	Median	Min	Max	P5	P95	Fetuses	Litters
VISCERAL
Abdomen- Ascites	0.0	0.09	0.0	0.0	0.5	0.0	0.1	1	1
Aorta- Overriding	0.0	0.12	0.0	0.0	0.7	0.0	0.2	2	1
Aortic Arch- Dilated	0.1	0.24	0.0	0.0	0.9	0.0	0.8	9	7
Aortic Arch- Interrupted	0.0	0.14	0.0	0.0	0.6	0.0	0.6	2	2
Aortic Arch- Narrow	0.0	0.12	0.0	0.0	0.5	0.0	0.5	2	2
Aortic Arch- Retroesophageal	0.0	0.07	0.0	0.0	0.4	0.0	0.1	1	1
Atrial Septum- Defect	0.0	0.13	0.0	0.0	0.6	0.0	0.5	3	2
Atrioventricular Valve- Absent	0.0	0.14	0.0	0.0	0.6	0.0	0.5	3	3
Atrium- Large	0.0	0.14	0.0	0.0	0.6	0.0	0.5	3	3
Diaphragm- Hernia	0.0	0.10	0.0	0.0	0.6	0.0	0.2	1	1
Eye(s)- Absent And/Or Small	0.0	0.15	0.0	0.0	0.8	0.0	0.4	3	2
Eye(s)- Hemorrhagic	0.0	0.10	0.0	0.0	0.6	0.0	0.2	1	1
Eye(s)- Retina Folded	0.0	0.22	0.0	0.0	1.3	0.0	0.3	1	1
Fistula	0.0	0.07	0.0	0.0	0.4	0.0	0.1	1	1
Gallbladder- Contents	0.0	0.09	0.0	0.0	0.5	0.0	0.1	1	1
Heart- Large	0.0	0.13	0.0	0.0	0.5	0.0	0.4	3	3
Hydrocephaly- Internal	0.1	0.20	0.0	0.0	0.8	0.0	0.7	4	3
Intestine- Large	0.0	0.10	0.0	0.0	0.6	0.0	0.2	1	1
Kidney(s)- Malpositioned	0.1	0.24	0.0	0.0	0.9	0.0	0.8	6	6
Liver- Abnormal Lobation	0.1	0.47	0.0	0.0	2.7	0.0	1.1	5	3
Liver- Anomaly	0.0	0.10	0.0	0.0	0.6	0.0	0.2	1	1
Liver- Cyst	0.0	0.09	0.0	0.0	0.5	0.0	0.1	1	1
Lung- Abnormal Lobation	0.1	0.17	0.0	0.0	0.7	0.0	0.6	3	3
Lung- Cyst	0.1	0.29	0.0	0.0	1.6	0.0	0.8	3	3
Pulmonary Trunk- Atretic	0.0	0.12	0.0	0.0	0.6	0.0	0.5	3	2
Pulmonary Trunk- Narrow	0.0	0.17	0.0	0.0	0.9	0.0	0.6	3	3
Subclavian (Right)- Originating from the Pulmonary Trunk	0.0	0.12	0.0	0.0	0.7	0.0	0.2	1	1
Testis- Malpositioned	0.3	0.59	0.0	0.0	2.4	0.0	2.1	20	16
Tetralogy of Fallot	0.1	0.22	0.0	0.0	0.8	0.0	0.7	7	7
Urinary Bladder- Dilated	0.0	0.07	0.0	0.0	0.4	0.0	0.1	1	1
Urinary Tract Content- Anomaly	0.0	0.10	0.0	0.0	0.6	0.0	0.2	1	1
Ventricular Septum- Defect	0.1	0.18	0.0	0.0	0.5	0.0	0.5	6	6

 

 

 

Historical Control Data Albino, New Zealand White Rabbit
	Mean of Study Means							Summary Incidence
	(% Per Litter Basis)							(Total No. Affected)
MALFORMATIONS	Mean	SD	Median	Min	Max	P5	P95	Fetuses	Litters
SKELETAL
Caudal Vertebral Anomaly	0.0	0.15	0.0	0.0	0.7	0.0	0.6	2	2
Costal Cartilage Anomaly	0.0	0.16	0.0	0.0	0.6	0.0	0.6	3	3
Limb Bones- Bent	0.0	0.07	0.0	0.0	0.4	0.0	0.1	1	1
Rib Anomaly	0.0	0.14	0.0	0.0	0.6	0.0	0.6	2	2
Rib(s)- Bent	0.0	0.17	0.0	0.0	1.0	0.0	0.3	2	2
Sternal Anomaly	0.1	0.43	0.0	0.0	2.3	0.0	1.2	5	4
Vertebral Anomaly With or Without Associated Rib Anomaly	0.5	0.57	0.2	0.0	1.7	0.0	1.6	28	28
Vertebral Centra Anomaly	0.0	0.13	0.0	0.0	0.5	0.0	0.4	3	3

 

 

Historical Control Data Albino. New Zealand White Rabbit
	Mean of Study Means							Summary Incidence
	(% Per Litter Basis)							(Total No. Affected)
VARIATIONS	Mean	SD	Median	Min	Max	P5	P95	Fetuses	Litters
EXTERNAL
None Observed
VISCERAL
Adrenal- Malpositioned	0.0	0.13	0.0	0.0	0.6	0.0	0.5	2	2
Adrenal- Supernumeray	0.0	0.15	0.0	0.0	0.9	0.0	0.2	1	1
Aortic Arch- Supernumerary Artery	0.4	0.58	0.4	0.0	2.2	0.0	2.1	29	28
Carotid (Left)- Originating from Brachiocephalic Trunk	1.9	1.38	1.8	0.0	5.3	0.0	5.0	120	80
Eye(s)- Hemorrhagic	0.0	0.10	0.0	0.0	0.6	0.0	0.2	1	1
Gallbladder- Absent or Small	1.1	0.79	0.9	0.0	3.1	0.0	2.6	64	58
Gallbladder- Bilobed	0.1	0.22	0.0	0.0	0.8	0.0	0.7	4	4
Kidney(s)- Discolored	0.0	0.09	0.0	0.0	0.5	0.0	0.1	1	1
Liver- Appendix	0.1	0.25	0.0	0.0	1.1	0.0	1.0	5	5
Liver- Cyst	0.1	0.23	0.0	0.0	0.8	0.0	0.7	8	7
Liver- Discolored	0.1	0.20	0.0	0.0	0.8	0.0	0.7	4	4
Liver- Small Supernumerary Lobe(s)	0.2	0.54	0.0	0.0	2.7	0.0	1.9	13	11
Lung- Absent Accessory Lobe	1.1	1.06	1.0	0.0	5.3	0.0	3.4	66	52
Lung- Discolored	0.0	0.11	0.0	0.0	0.6	0.0	0.4	2	2
Ovary- Cyst	0.5	0.60	0.2	0.0	2.0	0.0	1.9	30	26
Renal Papilla(e)- Absent and/or Small	0.2	0.37	0.0	0.0	1.6	0.0	1.2	13	11
Spleen- Constricted	0.2	0.59	0.0	0.0	3.1	0.0	1.8	13	11
Spleen- Pale	0.1	0.25	0.0	0.0	1.3	0.0	0.9	4	3
Spleen- Supernumerary	0.0	0.16	0.0	0.0	0.7	0.0	0.6	2	2
Subclavian (Right)- Retroesophageal	0.3	0.52	0.0	0.0	1.9	0.0	1.5	20	17
Testis- Cyst(s)	0.1	0.24	0.0	0.0	1.1	0.0	0.8	5	5
Thymus- Discolored	0.0	0.14	0.0	0.0	0.8	0.0	0.2	1	1
Thymus- Partially Undescended Horn(s)	0.3	0.56	0.0	0.0	2.1	0.0	1.9	18	16
Thyroid(s)- Large	0.0	0.09	0.0	0.0	0.5	0.0	0.1	1	1
Ureter(s)- Convoluted	0.0	0.08	0.0	0.0	0.4	0.0	0.3	2	2
Ureter(s)- Dilated	0.1	0.15	0.0	0.0	0.5	0.0	0.5	4	4
Ureter(s)- Retrocaval	2.3	1.19	2.3	0.0	4.1	0.4	4.0	132	99
Viscera- Attachment(s)	0.1	0.24	0.0	0.0	1.1	0.0	0.8	5	5
Viscera- Cyst(s)	0.0	0.09	0.0	0.0	0.5	0.0	0.1	1	1

 

 

 

Historical Control Data Albino. New Zealand White Rabbit
	Mean of Study Means							Summary Incidence
	(% Per Litter Basis)							(Total No. Affected)
VARIATIONS	Mean	SD	Median	Min	Max	P5	P95	Fetuses	Litters
SKELETAL
7th Cervical Full Rib(s)	0.1	0.34	0.0	0.0	1.8	0.0	0.9	5	4
7th Cervical Ossification Site(s)	1.1	1.40	0.8	0.0	5.5	0.0	4.4	64	43
13th Full Rib(s)	45.8	9.27	46.7	23.5	63.6	26.7	60.4	2795	605
13th Rudimentary Rib(s)	9.0	2.98	9.7	2.5	13.7	3.5	13.5	548	357
14th Rudimentary Rib(s)	0.0	0.14	0.0	0.0	0.8	0.0	0.2	1	1
Caudal Vertebral Anomaly	0.5	0.50	0.5	0.0	1.7	0.0	1.6	34	33
Hyoid Arch(es) Bent	0.2	0.37	0.0	0.0	1.3	0.0	1.2	11	11
Hyoid Body and/or Arches Unossified	0.7	0.74	0.6	0.0	3.2	0.0	2.5	53	44
Metacarpal(s) and/or Metatarsal(s) Unossified	5.2	2.03	4.8	1.5	10.1	2.2	8.8	358	165
Pelvic Girdle- Caudal Shift	15.2	5.01	15.0	5.5	25.4	6.1	25.1	936	368
Pelvic Girdle- Cranial Shift	0.2	0.42	0.0	0.0	1.9	0.0	1.3	8	8
Pubis- Unossified	0.3	0.37	0.0	0.0	1.2	0.0	1.0	20	18
Rib(s)- Bent	0.0	0.09	0.0	0.0	0.5	0.0	0.1	1	1
Rib(s)- Nodulated	0.2	0.29	0.0	0.0	1.0	0.0	0.9	9	9
Rib(s)- Short	0.0	0.11	0.0	0.0	0.5	0.0	0.4	2	2
Skeleton- Generally Reduced in Ossification	0.0	0.07	0.0	0.0	0.4	0.0	0.1	1	1
Skull Bones- Fused	0.0	0.07	0.0	0.0	0.4	0.0	0.1	1	1
Skull Bone(s)- Split	0.0	0.09	0.0	0.0	0.5	0.0	0.1	1	1
Skull Bone- Unossified Line	0.3	0.36	0.0	0.0	1.0	0.0	1.0	15	14
Skull- Reduced Ossification	0.1	0.26	0.0	0.0	0.8	0.0	0.8	9	7
Skull- Supernumerary Site	0.7	0.81	0.5	0.0	3.6	0.0	2.6	38	34
Sternebra(e) #1,#2,#3 and/or #4 Unossified	0.0	0.07	0.0	0.0	0.4	0.0	0.1	1	1
Sternebra(e) #5 and/or #6 Unossified	23.7	6.38	24.5	11.8	38.9	13.5	35.7	1468	466
Sternebra(e)- Branched	0.2	0.40	0.0	0.0	1.5	0.0	1.3	10	10
Sternebra(e)- Fused	1.0	0.91	0.9	0.0	3.4	0.0	3.3	60	53
Sternebra(e)- Malaligned ^1	7.4	4.04	7.5	2.1	18.9	2.1	18.2	288	182
Sternebra(e)- Malaligned (Slight or Moderate) ^2	8.5	5.32	8.7	0.0	16.6	@	@	172	95
Sternebra(e)- Malaligned (Severe) ^2	0.0	0.14	0.0	0.0	0.5	@	@	1	1
Sternebra(e)- Wide	0.0	0.09	0.0	0.0	0.5	0.0	0.1	1	1

 

¹ Based on 22 datasets

² Based on 12 datasets

@ Insufficient number of data for calculation.

 

 

Historical Control Data Albino. New Zealand White Rabbit
	Mean of Study Means							Summary Incidence
	(% Per Litter Basis)							(Total No. Affected)
VARIATIONS	Mean	SD	Median	Min	Max	P5	P95	Fetuses	Litters
SKELETAL (continued)
Sternum- Supernumerary Ossification Site	0.2	0.44	0.0	0.0	1.5	0.0	1.4	13	11
Sternum- Supernumerary Sternebra	0.0	0.17	0.0	0.0	0.9	0.0	0.5	2	2
Tarsal(s)- Unossified	0.9	0.53	1.1	0.0	2.1	0.0	1.7	64	52
Vertebra- Supernumerary Site	0.0	0.15	0.0	0.0	0.8	0.0	0.5	2	2
Vertebral Arces- Reduced Ossification	0.0	0.07	0.0	0.0	0.4	0.0	0.1	1	1
Vertebral Centra- Anomaly	0.0	0.07	0.0	0.0	0.4	0.0	0.1	1	1
Vertebral Centra- Reduced Ossification	0.3	0.53	0.0	0.0	2.4	0.0	1.7	15	14

 

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results in this prenatal developmental toxicity study (i.e. adverse effects on body weight and food consumption and unscheduled deaths in dams treated at 1000 mg/kg/day) the maternal No Observed Adverse Effect Level (NOAEL) for
3-methoxybutyl acetate was established as being 300 mg/kg/day.
Due to the absence of any test item-related effects on fetal observations, the developmental No Observed Adverse Effect Level (NOAEL) for 3-methoxybutyl acetate was established as being at least 300 mg/kg/day.

Executive summary:

The objectives of this study were to determine the potential of 3-methoxybutyl acetate (Source Substance) 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 orally by gavage to time-mated female New Zealand White rabbits from Day 7 to 28 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels in this study were selected to be 0, 100, 300, 1000 mg/kg/day, based on the results of the dose range finder.

In the main rabbit developmental toxicity study, dams treated with 1000 mg/kg/day of 3‑methoxybutyl acetate (Source Substance) resulted in a significant level of gastrointestinal tract irritation and subsequent decreased food consumption and body weigh decreased. Four females that were euthanized for animal welfare reasons between Days 17-20 post-coitum due to decreased and/or absent food consumption. All these females had a severely reduced food consumption for at least 7 consecutive days and a significant body weight loss (5-13% of their initial weights) during the treatment period and were sacrificed according to the Laboratory animal welfare guidelines. Clinical signs of toxicity noted in a single early sacrificed animal included piloerection, lean appearance, hunched posture, moribundity, and pale appearance.

Overall, mean food consumption was significantly lower in females at 1000 mg/kg/day between Days 7-12 post-coitum compared to controls. Food consumption appeared to recover to control levels from Day 13 post-coitum onwards. As a result, body weight loss was observed in almost all females at 1000 mg/kg/day (up to 6% for individual animals) between Days 7-9 post-coitum. This body weight loss at 1000 mg/kg/day was followed by reduced or absent body weight gain between Days 9-18 post-coitum in individual females (as a result, four of these females were sacrificed for animal welfare reasons between Day 17-22 post-coitum). In females surviving until scheduled necropsy, body weight gain appeared to recover from Day 9 post-coitum onwards. Despite the apparent recovery, given the size of the observed effects on body weight gain and food consumption resulting in sacrifice of four high-dose dams, these effects were considered adverse.

Macroscopic observations at necropsy revealed many dark red foci in the forestomach or irregular surface of the forestomach for 4/22 females at 1000 mg/kg/day, likely reflecting a site-of-contact irritant response. This finding was most likely related to the physical-chemical properties of 3‑methoxybutyl acetate and at the incidence observed, it was considered related to treatment with the 3‑methoxybutyl acetate . It was noted that this finding occurred in 2 females that were prematurely sacrificed due to absent food consumption and body weight loss. Moreover, decreased or absent food consumption was also noted for prolonged periods in the remaining two females with this finding. Although histopathological evaluation of the gastrointestinal tract was not performed to assess adversity of these findings, they were likely related to the adverse effects on body weight and food consumption leading to morbidity. No 3‑methoxybutyl acetate (Source Substance) related effects were seen at treatment up to 300 mg/kg/day. The number of pregnant females was 21 in the control group, 22 at 100 mg/kg/day, 21 at 300 mg/kg/day and 19 at 1000 mg/kg/day. Excluding non-pregnant females, females showing an early delivery and females that did not survive until scheduled necropsy, there were 20 females in the control group, 20 females at 100 mg/kg/day, 18 females at 300 mg/kg/day, and 13 females at 1000 mg/kg/day with viable litters for developmental evaluation. As according to the OECD 414 test guideline, a minimum of 16 litters is required for evaluation of developmental data, the 13 litters available at 1000 mg/kg/day were not sufficient for a complete and valid evaluation of developmental data. The number of corpora lutea, implantation sites, viable or dead fetuses, early or late resorptions, and pre- and postimplantation loss were considered not affected by treatment up to 300 mg/kg/day.

At 300 mg/kg/day 3‑methoxybutyl acetate related skeletal variations were noted: The litter incidence of an ossification site near the 7th cervical vertebra was statistically significantly increased compared with the control group, although this specific finding was within the historical control range and the percent per litter with total variations was almost identical to controls. In addition, three cases of 7th cervical full ribs were observed in this study that occurred in three 300 mg/kg/day litters that had fetuses with 7th cervical ossification sites. As these skeletal findings only consisted of variations, they were considered not adverse. No 3‑methoxybutyl acetate related toxicologically significant changes were noted in any of the remaining developmental parameters investigated in this study, up to the highest dose level evaluated (300 mg/kg/day) (i.e. litter size, sex ratio, fetal body weights, external, visceral and skeletal malformations, and external and visceral developmental variations).

Based on the results in this prenatal developmental toxicity study (i.e. adverse effects on body weight and food consumption and unscheduled deaths in dams treated at 1000 mg/kg/day) the maternal No Observed Adverse Effect Level (NOAEL) for 3‑methoxybutyl acetate (Source Substance) was established as being 300 mg/kg/day. Due to the absence of any 3‑methoxybutyl acetate related effects on development and fetal morphology, the developmental No Observed Adverse Effect Level (NOAEL) for 3‑methoxybutyl acetate was established as being at least 300 mg/kg/day. Applying the read across framework based on Annex XI, section 1.5 the results from the rabbit developmental toxicity study with 3-methoxybutyl acetate (CRL, 2020b) fulfill the information requirement for a one species  developmental toxicity study with 3-methoxybutan-1-ol.